Medicine › Radiology, Nuclear Medicine and Imaging

Monoclonal and Polyclonal Antibodies Research

Works Count: 177398

Wikipedia

Description

This cluster of papers focuses on the development, engineering, and applications of therapeutic antibodies, including their interaction with Fc receptors, monoclonal antibody therapy for cancer, antibody engineering techniques such as nanobodies and bispecific antibodies, the impact of glycosylation on antibody function, and antibody pharmacokinetics.

Keywords

Therapeutic Antibodies; Fc Receptors; Monoclonal Antibodies; Antibody Engineering; Immunoglobulin G; Nanobodies; Antibody Pharmacokinetics; Bispecific Antibodies; Glycosylation; Fc? Receptors

Rapid Isolation of Antigens from Cells with A Staphylococcal Protein A-Antibody Adsorbent: Parameters of the Interaction of Antibody-Antigen Complexes with Protein A

1975-12-01

Steven Kessler

The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a … The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.

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Antibodies, a laboratory manual

1989-01-01

Ed Harlow , David P. Lane

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

1985-12-01

Gérard I. Evan , George K. Lewis , Gary Ramsay +1 more

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate … Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.

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Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes.

1972-07-01

Eva Engvall , Peter Perlmann

In the DNP system, the specificity of the reaction was assessed by inhibition with hapten. The reaction of immune serum against DNP with DNP-protein, adsorbed to the tubes, was completely … In the DNP system, the specificity of the reaction was assessed by inhibition with hapten. The reaction of immune serum against DNP with DNP-protein, adsorbed to the tubes, was completely inhibited by hapten in solution.

Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules

1991-05-01

Kirsten Falk , Olaf Rötzschke , Stefan Stevanovié +2 more

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On the size of the active site in proteases. I. Papain

1967-04-01

Israël Schechter , Arieh Berger

Genetics of rheumatoid arthritis contributes to biology and drug discovery

2013-12-24

Yukinori Okada , Di Wu , Gosia Trynka +7 more

Purification of Mouse Immunoglobulin Heavy‐Chain Messenger RNAs from Total Myeloma Tumor RNA

1980-06-01

Charles Auffray , François Rougeon

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from … A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. γ2a and α H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 ± 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56000 (γ2a) and 60000 (α) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25000 (λ) and 27000 (к). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (γ2a), 62% (α), for H-chain mRNAs and 60% (к), for L-chain mRNAs.

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Drug Discovery: A Historical Perspective

2000-03-17

Jürgen Drews

Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more to the progress of medicine during the past century than any other scientific … Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more to the progress of medicine during the past century than any other scientific factor. The advent of molecular biology and, in particular, of genomic sciences is having a deep impact on drug discovery. Recombinant proteins and monoclonal antibodies have greatly enriched our therapeutic armamentarium. Genome sciences, combined with bioinformatic tools, allow us to dissect the genetic basis of multifactorial diseases and to determine the most suitable points of attack for future medicines, thereby increasing the number of treatment options. The dramatic increase in the complexity of drug research is enforcing changes in the institutional basis of this interdisciplinary endeavor. The biotech industry is establishing itself as the discovery arm of the pharmaceutical industry. In bridging the gap between academia and large pharmaceutical companies, the biotech firms have been effective instruments of technology transfer.

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells

1984-05-01

A Ullrich , Lisa M. Coussens , Joel S. Hayflick +7 more

Immunochemical quantitation of antigens by single radial immunodiffusion

1965-09-01

G Mancini , A. O. Carbonara , J.F. Heremans

Humanization of an anti-p185HER2 antibody for human cancer therapy.

1992-05-15

Paul Carter , Leonard G. Presta , C Gorman +7 more

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human … The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

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Searching for Peptide Ligands with an Epitope Library

1990-07-27

Jamie K. Scott , George P. Smith

Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast … Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface. The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's. Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates.

Phage antibodies: filamentous phage displaying antibody variable domains

1990-12-01

John McCafferty , Andrew D. Griffiths , Greg Winter +1 more

Efficient isolation of genes by using antibody probes.

1983-03-01

R A Young , Ryan Davis

A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method … A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.

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Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133

1989-11-01

Gustavo González , Marc Montminy

Molecular and Genetic Properties of Tumors Associated with Local Immune Cytolytic Activity

2015-01-01

Michael S. Rooney , Sachet A. Shukla , Catherine J. Wu +2 more

How the genomic landscape of a tumor shapes and is shaped by anti-tumor immunity has not been systematically explored. Using large-scale genomic data sets of solid tissue tumor biopsies, we … How the genomic landscape of a tumor shapes and is shaped by anti-tumor immunity has not been systematically explored. Using large-scale genomic data sets of solid tissue tumor biopsies, we quantified the cytolytic activity of the local immune infiltrate and identified associated properties across 18 tumor types. The number of predicted MHC Class I-associated neoantigens was correlated with cytolytic activity and was lower than expected in colorectal and other tumors, suggesting immune-mediated elimination. We identified recurrently mutated genes that showed positive association with cytolytic activity, including beta-2-microglobulin (B2M), HLA-A, -B and -C and Caspase 8 (CASP8), highlighting loss of antigen presentation and blockade of extrinsic apoptosis as key strategies of resistance to cytolytic activity. Genetic amplifications were also associated with high cytolytic activity, including immunosuppressive factors such as PDL1/2 and ALOX12B/15B. Our genetic findings thus provide evidence for immunoediting in tumors and uncover mechanisms of tumor-intrinsic resistance to cytolytic activity.

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Monoclonal Antibody to 5-Bromo- and 5-Iododeoxyuridine: A New Reagent for Detection of DNA Replication

1982-10-29

Howard G. Gratzner

Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen … Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

Signal transduction by receptors with tyrosine kinase activity

1990-04-01

Axel Ullrich , Joseph Schlessinger

A short amino acid sequence able to specify nuclear location

1984-12-01

Daniel Kalderon , Bruce Roberts , William D. Richardson +1 more

Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.

1982-06-01

Elizabeth A. Grimm , A Mazumder , H Z Zhang +1 more

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural … Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

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Antibody therapy of cancer

2012-03-22

Andrew M. Scott , Jedd D. Wolchok , Lloyd J. Old

Xenogeneic Monoclonal Antibodies to Mouse Lymphoid Differentiation Antigens*

1979-10-01

Jeffrey A. Ledbetter , Leonard A. Herzenberg

FcRn: the neonatal Fc receptor comes of age

2007-08-17

Derry C. Roopenian , Shreeram Akilesh

Fcγ receptors as regulators of immune responses

2007-12-07

Falk Nimmerjahn , Jeffrey V. Ravetch

Predominant Autoantibody Production by Early Human B Cell Precursors

2003-08-19

Hedda Wardemann , Sergey Yurasov , Anne Schaefer +3 more

During B lymphocyte development, antibodies are assembled by random gene segment reassortment to produce a vast number of specificities. A potential disadvantage of this process is that some of the … During B lymphocyte development, antibodies are assembled by random gene segment reassortment to produce a vast number of specificities. A potential disadvantage of this process is that some of the antibodies produced are self-reactive. We determined the prevalence of self-reactive antibody formation and its regulation in human B cells. A majority (55 to 75%) of all antibodies expressed by early immature B cells displayed self-reactivity, including polyreactive and anti-nuclear specificities. Most of these autoantibodies were removed from the population at two discrete checkpoints during B cell development. Inefficient checkpoint regulation would lead to substantial increases in circulating autoantibodies.

Protein therapeutics: a summary and pharmacological classification

2007-12-21

Benjamin Leader , Quentin Baca , David E. Golan

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.

1989-12-01

David Fiorentino , M W Bond , Timothy R. Mosmann

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF … A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.

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A dot-immunobinding assay for monoclonal and other antibodies

1982-01-01

Richard Hawkes , Evelyn Niday , Julian Gordon

Derivation of specific antibody‐producing tissue culture and tumor lines by cell fusion

1976-07-01

Georges Köhler , César Milstein

Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable … Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice. Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8-azaguanine-resistant clone (X63-Ag8) of MOPC 21 myeloma. Over 50% of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10% of the hybrid lines exhibit anti-SRBC activity. The high proportion of antibody-producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production. In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1-Ag4-1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains. Three anti-SRBC (probably of the mu, gamma2b and gamma1 classes, respectively) and two anti-2,4,6-trinitrophenyl (of the mu class) antibody-producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma gamma and K chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.

A Radioimmunoassay Using a Monoclonal Antibody to Monitor the Course of Epithelial Ovarian Cancer

1983-10-13

Robert C. Bast , Thomas L. Klug , Elena St. John +7 more

The murine monoclonal antibody OC 125 reacts with an antigen (CA 125) common to most nonmucinous epithelial ovarian carcinomas. An assay has been developed to detect CA 125 in serum. … The murine monoclonal antibody OC 125 reacts with an antigen (CA 125) common to most nonmucinous epithelial ovarian carcinomas. An assay has been developed to detect CA 125 in serum. By this assay, only 1 per cent of 888 apparently healthy persons and 6 per cent of 143 patients with nonmalignant disease had serum CA 125 levels above 35 U per milliliter. In contrast, 83 of 101 patients (82 per cent) with surgically demonstrated ovarian carcinoma had elevated levels of antigen. In 38 patients with epithelial ovarian carcinoma monitored on 2 to 18 occasions during 2 to 60 months, antigen levels ranged from less than 1 to more than 8000 U per milliliter. Rising or falling levels of CA 125 correlated with progression or regression of disease in 42 of 45 instances (93 per cent). Determination of CA 125 levels may aid in monitoring the response to treatment in patients with epithelial ovarian cancer. (N Engl J Med 1983; 309:883–7.)

Naturally occurring antibodies devoid of light chains

1993-06-01

C. Hamers‐Casterman , T. Atarhouch , Serge Muyldermans +5 more

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

1979-09-01

Harry Towbin , T. Staehelin , J. Gordon

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. … A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

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Solvent-Accessible Surfaces of Proteins and Nucleic Acids

1983-08-19

Michael L. Connolly

A method is presented for analytically calculating a smooth, three-dimensional contour about a molecule. The molecular surface envelope may be drawn on either color raster computer displays or real-time vector … A method is presented for analytically calculating a smooth, three-dimensional contour about a molecule. The molecular surface envelope may be drawn on either color raster computer displays or real-time vector computer graphics systems. Molecular areas and volumes may be computed analytically from this surface representation. Unlike most previous computer graphics representations of molecules, which imitate wire models or space-filling plastic spheres, this surface shows only the atoms that are accessible to solvent. This analytical method extends the earlier dot surface numerical algorithm, which has been applied in enzymology, rational drug design, immunology, and understanding DNA base sequence recognition.

LOCALIZATION OF ANTIGEN IN TISSUE CELLS

1950-01-01

Albert H. Coons , Melvin H. Kaplan

Improvements in a method for the specific microscopic localization of antigen in tissue cells are described. This method employs antibody labelled with fluorescein isocyanate as a histochemical stain, the specific … Improvements in a method for the specific microscopic localization of antigen in tissue cells are described. This method employs antibody labelled with fluorescein isocyanate as a histochemical stain, the specific antigen-antibody precipitate being made visible under the fluorescence microscope. Two isomeric series derived from nitrofluorescein are described.

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Efficacy of B-Cell–Targeted Therapy with Rituximab in Patients with Rheumatoid Arthritis

2004-06-16

Jonathan Edwards , L Szczepański , Jacek Szechiński +5 more

An open-label study indicated that selective depletion of B cells with the use of rituximab led to sustained clinical improvements for patients with rheumatoid arthritis. To confirm these observations, we … An open-label study indicated that selective depletion of B cells with the use of rituximab led to sustained clinical improvements for patients with rheumatoid arthritis. To confirm these observations, we conducted a randomized, double-blind, controlled study.We randomly assigned 161 patients who had active rheumatoid arthritis despite treatment with methotrexate to receive one of four treatments: oral methotrexate (> or =10 mg per week) (control); rituximab (1000 mg on days 1 and 15); rituximab plus cyclophosphamide (750 mg on days 3 and 17); or rituximab plus methotrexate. Responses defined according to the criteria of the American College of Rheumatology (ACR) and the European League against Rheumatism (EULAR) were assessed at week 24 (primary analyses) and week 48 (exploratory analyses).At week 24, the proportion of patients with 50 percent improvement in disease symptoms according to the ACR criteria, the primary end point, was significantly greater with the rituximab-methotrexate combination (43 percent, P=0.005) and the rituximab-cyclophosphamide combination (41 percent, P=0.005) than with methotrexate alone (13 percent). In all groups treated with rituximab, a significantly higher proportion of patients had a 20 percent improvement in disease symptoms according to the ACR criteria (65 to 76 percent vs. 38 percent, P< or =0.025) or had EULAR responses (83 to 85 percent vs. 50 percent, P< or =0.004). All ACR responses were maintained at week 48 in the rituximab-methotrexate group. The majority of adverse events occurred with the first rituximab infusion: at 24 weeks, serious infections occurred in one patient (2.5 percent) in the control group and in four patients (3.3 percent) in the rituximab groups. Peripheral-blood immunoglobulin concentrations remained within normal ranges.In patients with active rheumatoid arthritis despite methotrexate treatment, a single course of two infusions of rituximab, alone or in combination with either cyclophosphamide or continued methotrexate, provided significant improvement in disease symptoms at both weeks 24 and 48.

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NEW EMBO MEMBERS' REVIEW: The ErbB signaling network: receptor heterodimerization in development and cancer

2000-07-03

Monilola A. Olayioye

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Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

1984-02-01

J L Cordell , Brunangelo Falini , Wendy N. Erber +6 more

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal … A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").

Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.

1984-10-01

Johannes Gerdes , Hilmar Lemke , H. Baisch +3 more

The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is … The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.

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A New Mouse Myeloma Cell Line that Has Lost Immunoglobulin Expression but Permits the Construction of Antibody-Secreting Hybrid Cell Lines

1979-10-01

John F. Kearney , Andreas Radbruch , B Liesegang +1 more

Abstract We have isolated a subclone of the mouse myeloma cell line P3-X63-Ag8 that does not express immunoglobulin heavy or light chains. This clone X63-Ag8.653 can be used for efficient … Abstract We have isolated a subclone of the mouse myeloma cell line P3-X63-Ag8 that does not express immunoglobulin heavy or light chains. This clone X63-Ag8.653 can be used for efficient fusion with antibody-forming cells to obtain hybrid cell lines producing pure monoclonal antibodies. Screening of hybrid cell lines for specificity and immunoglobulin classes was done with a modified enzyme-linked immunosorbent assay.

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Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets

2000-04-01

Raphael Clynes , Terri L. Towers , Leonard G. Presta +1 more

Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation

1983-01-15

Johannes Gerdes , Ulrich Schwab , Hilmar Lemke +1 more

Abstract The production of a mouse monoclonal antibody, Ki‐67, is described. The Ki‐67 antibody recognized a nuclear antigen present in proliferating cells, but absent in resting cells. Immunostainings with Ki‐67 … Abstract The production of a mouse monoclonal antibody, Ki‐67, is described. The Ki‐67 antibody recognized a nuclear antigen present in proliferating cells, but absent in resting cells. Immunostainings with Ki‐67 revealed nuclear reactivity in cells of germinal centres of cortical follicles, cortical thymocytes, neck cells of gastrointestinal mucosa, un‐differentiated spermatogonia and cells of a number of human cell lines. The Ki‐67 antibody did not react with cells known to be in a resting stage, such as lymphocytes, monocytes, parietal cells and Paneth's cells of gastrointestinal mucosa, hepatocytes, renal cells, mature sperm cells, brain cells, etc. Expression of the antigen recognized by Ki‐67 could be induced in peripheral blood lymphocytes after stimulation with phytohaemagglutinin, whereas it disappeared from HL‐60 cells stimulated with phorbol esters to differentiate into mature macrophages in a resting stage. These findings suggest that Ki‐67 is directed against a nuclear antigen associated with cell proliferation. A first series of immunostainings of tumour biopsies indicated that Ki‐67 may be a potent tool for easy and quick evaluation of the proportion of proliferating cells in a tumour.

Nanobodies: Natural Single-Domain Antibodies

2013-03-15

Serge Muyldermans

Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and … Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

IgG Subclasses and Allotypes: From Structure to Effector Functions

2014-10-20

Gestur Vidarsson , Gillian Dekkers , Theo Rispens

Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant … Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review.

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Diffusion-In-Gel Methods for Immunological Analysis II (Part 1 of 4)

1962-01-01

Örjan Ouchterlony

Somatic generation of antibody diversity

1983-04-01

Susumu Tonegawa

Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries.

2004-06-01

André Frenzel , Jonas Kügler , Saskia Helmsing +4 more

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Antibodies: A laboratory manual

1989-10-01

J.L.

Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20

1994-01-15

ME Reff , K Carner , KS Chambers +6 more

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The impact of Fc glycosylation on IgG susceptibility to hinge region chemical reduction: implications for the development of immunoassays

2025-06-25

Vanessa Susini , Silvia Ursino , Chiara Sanguinetti +3 more | Biochemistry and Biophysics Reports

Profiling Hinge Plasticity in Intact Monoclonal Antibodies for Antigen Recognition

2025-06-25

Arnab Bhattacharya , Sudhir Sinha , Rozaleen Dash +2 more | Biochemistry

Monoclonal antibodies (mAbs) are multidomain glycosylated proteins that mediate antigen binding, among other protein-protein interactions, which makes them successful therapeutics. However, in many cases, adverse physicochemical properties can affect their … Monoclonal antibodies (mAbs) are multidomain glycosylated proteins that mediate antigen binding, among other protein-protein interactions, which makes them successful therapeutics. However, in many cases, adverse physicochemical properties can affect their antigen-binding abilities, compromising therapeutic potential. Hence, structural characterization of these biotherapeutics is strongly desired to predict and possibly redesign better variants. The presence of glycosylation deters the use of isotope labeling, which is required for the structure determination of these intact proteins (MW ∼150 kDa) by Nuclear Magnetic Resonance Spectroscopy (NMR). In this work, NMR-based structural fingerprinting of three therapeutic mAbs at natural abundance was performed. The robustness of the mAb fingerprints was demonstrated by comparing them with two Fc-fusion proteins. A peptide-based assignment methodology was adopted for these intact proteins, which identified the presence of the flexible hinge segment in mAbs in solution. The plasticity of the hinge was demonstrated by the changes in fingerprints in the presence of the cognate antigen and nonantigen. The methodology underlines the importance of the hinge in antigen recognition beyond the canonical role of Complementarity-Determining Regions (CDRs). This methodology lays the dynamic basis of antibody function in solution.

Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

2025-06-25

Sara Linciano , Ylenia Mazzocato , Zhanna Romanyuk +7 more | Nature Communications

Developing drug-like single-domain antibodies (VHH) from in vitro libraries

2025-06-25

M. Frank Erasmus , André A. Teixeira , Esteban Molina +7 more | mAbs

Here, we describe a new VHH library for therapeutic discovery which optimizes humanness, stability, affinity, diversity, developability, and facile purification using protein A in the absence of an Fc domain. … Here, we describe a new VHH library for therapeutic discovery which optimizes humanness, stability, affinity, diversity, developability, and facile purification using protein A in the absence of an Fc domain. Four therapeutic humanized VHHs were used as scaffolds, into which we inserted human HCDR1s, HCDR2s and HCDR3s. The HCDR1 and HCDR2 sequences were derived from human VH3 family next-generation sequencing datasets informatically purged of sequence liabilities, synthesized as array-based oligonucleotides, cloned as single CDR libraries into each of the parental scaffolds and filtered for protein A binding by yeast display to ensure correct folding and display. After filtering, the CDR1 and CDR2 libraries were combined with amplified human HCDR3 from human CD19+ IgM+ B cells. This library was further improved by eliminating long consecutive stretches of tyrosines in CDR3 and enriching for CDR1-2 diversity with elevated tolerance to high temperatures. A broad diversity of high affinity (100 pM-10 nM), developable binders was directly isolated, with developability evaluated for most assays using the isolated VHHs, rather than fused to Fc, which is customary. This represents the first systematic developability assessment of isolated VHH molecules.

Fragment-Based Immune Cell Engager Antibodies in Treatment of Cancer, Infectious and Autoimmune Diseases: Lessons and Insights from Clinical and Translational Studies

2025-06-24

Ge Yang , Mohammad Massumi | Antibodies

Since the advent of recombinant DNA technologies and leading up to the clinical approval of T cell engager blinatumomab, the modular design of therapeutic antibodies has enabled the fusion of … Since the advent of recombinant DNA technologies and leading up to the clinical approval of T cell engager blinatumomab, the modular design of therapeutic antibodies has enabled the fusion of antibody fragments with proteins of various functionalities. This has resulted in an expansive array of possible mechanisms of action and has given birth to fragment-based antibodies (fbAbs) with immune cell engager modalities. In searchable databases, the preclinical development of these antibodies has shown promise; however, clinical outcomes and restructuring efforts involving these agents have produced mixed results and uncertainties. Amid budgetary cuts in both academia and industry, critical planning and evaluation of drug R&D would be more essential than ever before. While many reviews have provided outstanding summaries of preclinical phase fbAbs and cataloged relevant clinical trials, to date, very few of the articles in searchable databases have comprehensively reviewed the details of clinical outcomes along with the underlying reasons or potential explanations for the success and failures of these fbAb drug products. To fill the gap, in this review, we seek to provide the readers with clinically driven insights, accompanied by translational and mechanistic studies, on the current landscape of fragment-based immune cell engager antibodies in treating cancer, infectious, and autoimmune diseases.

Depletion-restitution therapy of autoimmune rheumatic diseases. Part 2. Perspectives on bispecific antibodies

2025-06-24

А. L. Maslyanskiy , Д. А. Дибров , А. М. Лила +4 more | Modern Rheumatology Journal

One of the most promising approaches to depletion-restitution therapy is the development and use of drugs based on bispecific monoclonal antibodies (bsAbs). Therapeutic bsAbs are genetically engineered biological products (biologics) … One of the most promising approaches to depletion-restitution therapy is the development and use of drugs based on bispecific monoclonal antibodies (bsAbs). Therapeutic bsAbs are genetically engineered biological products (biologics) based on immunoglobulin molecules capable of simultaneously binding multiple antigens, making them a promising platform for novel drugs. A specific type of such agent, which incorporates at least two antigen-binding (Fab) fragments within a single immunoglobulin molecule – one targeting a specific cell-surface receptor and the other binding and activating to the CD3ε domain of CD3 molecule of the T-cell receptor complex – has been termed a bispecific T-cell engager (BiTE).Currently, BiTE molecules that engage effector cells of the humoral immune system are the most clinically advanced subclass of bsAbs. Their ability to deplete target cells in peripheral blood and tissues has been clearly demonstrated in the treatment of resistant hematological malignancies such as B-cell precursor acute lymphoblastic leukemia, various lymphoproliferative disorders, and plasma cell dyscrasias. Recent years have seen attempts to repurpose bsAbs for the treatment of refractory, prognostically unfavorable forms of systemic autoimmune rheumatic diseases (SARDs), supported by theoretical rationale, experimental evidence, and parallels with successful CAR-T cell therapy.Beyond BiTEs, the bsAb platform also enables development of biologics with extended pharmacokinetics, multi-cytokine targeting potential for synergistic suppression of inflammation, and checkpoint-directed modulation of targeted cell functional activity.Advantages such as standardized manufacturing, off-the-shelf availability, predictable pharmacokinetics (with a known and limited half-life), flexible dosing regimens enabling slow escalation of the dose, the possibility of individualizing treatment duration and dosing frequency, the feasibility of repeated treatment cycles, the option to discontinue therapy in case of adverse events, and the significantly lower cost of short low-dose treatment cycles compared to CAR-T cell therapy – all these make bsAb-based strategies a highly attractive priority for next-generation depletion-restitution therapies for SARDs.

Iodoacetyl Tandem Mass Tag-Based Site-Specific Free Thiol Analysis (TMT-SiFTA) of Monoclonal Antibodies

2025-06-24

David Bramhall , Jieqiang Zhong , Yimeng Zhao +4 more | Analytical Chemistry

Monoclonal antibodies (mAbs) consist of four polypeptide chains that are covalently linked by disulfide bonds formed between cysteine residues. However, low levels of free thiols derived from under-processed/reduced cysteines are … Monoclonal antibodies (mAbs) consist of four polypeptide chains that are covalently linked by disulfide bonds formed between cysteine residues. However, low levels of free thiols derived from under-processed/reduced cysteines are commonly present. Because free thiols may increase the risk of undesired immunogenicity in mAb therapeutics, monitoring free thiol occupancies during drug development is crucial to ensure drug quality and safety. Multiple assays have been established to identify and quantify the free thiols of proteins; these assays generally involve spectroscopy-based or mass spectrometry (MS)-based methods. Spectroscopy-based methods may be limited by their sensitivity and the lack of site-specific information. In contrast, MS-based workflows have been developed to address these problems; however, free thiol quantitation mostly relies on peak integration at the full MS level, which is vulnerable to signal variability and suppression. Herein, we describe an iodoacetyl tandem mass tag-based site-specific free thiol analysis (TMT-SiFTA) method incorporating downstream parallel reaction monitoring analysis, thus enabling sensitive and robust characterization of low-abundance free thiols. TMT-SiFTA was validated with the mAb standard NISTmAb and achieved the detection and quantitation of free thiols as low as ∼0.1% for individual cysteine residues. The developed workflow was also successfully applied to one in-house mAb and seven commercialized mAbs. The findings revealed consistent patterns across various molecules: cysteine residues participating in interchain disulfide bonds displayed low free thiol percentages (below 0.6%), whereas those involved in intrachain disulfide bonds exhibited higher percentages (up to 11%). TMT-SiFTA provides a powerful tool for free thiol analysis to support the development of therapeutic mAbs development.

Cancer Therapeutic Antibodies

2025-06-24

Zahra Salimi | Undergraduate Research in Natural and Clinical Science and Technology (URNCST) Journal

Cancer consistently ranks among the top causes of death worldwide. Traditional therapies include surgery, chemotherapy, and radiation. However, many patients either do not respond to or develop specific treatment resistance. … Cancer consistently ranks among the top causes of death worldwide. Traditional therapies include surgery, chemotherapy, and radiation. However, many patients either do not respond to or develop specific treatment resistance. Given the advent of genome, transcriptome, and proteome technologies, personalized medicine has gained tremendous recognition as a therapeutic field. Antibodies are Y-shaped proteins produced by activated B immune cells, featuring two substrate recognition sites. They identify and bind to invading pathogens, such as bacteria, viruses, and toxins to help prevent and eliminate infections. The concept and potential of therapeutic monoclonal antibodies (mAbs) in cancer was put forth by Paul Ehrlich over a century ago. A breakthrough by Köhler and Milstein (Cambridge, 1975) followed the development of hybridoma technology, allowing mass production of specific antigen-induced mAbs. MAbs exert their effect through various mechanisms to combat malignancies. Depending on their design, mAbs bind to specific cognate cell-surface receptors to modulate (insert = cell) growth, apoptosis, and immune recognition. MAbs targeting various cancer types have received clinical approval. For instance, rituximab binds CD20 expressed on B cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), leading to immune-mediated target cell destruction. A class of mAbs known as immune checkpoint inhibitors (ICIs), activate the body's natural immune response against tumor cells by blocking their suppression. Notable ICI include pembrolizumab and nivolumab, which specifically target the programmed cell death receptor-1 (PD-1). Unlike traditional therapies which often cause damage to healthy cells, mAbs are intrinsically target-specific, reducing or eliminating harmful side effects. As such, they have emerged to provide more effective and less toxic options for cancer patients.

Genetically Engineered Bacteria‐Derived Hydrogel Orchestrates T Cell Spatial Dynamics for Triple‐Negative Breast Cancer Immunotherapy

2025-06-23

Guoqing Jia , Qian Li , Mingtan Tang +4 more | Advanced Functional Materials

Abstract Impaired spatial dynamics of T cells represent a major challenge to effective immunotherapy in triple‐negative breast cancer (TNBC). Here, a genetically engineered, bacteria‐derived hydrogel is presented that precisely orchestrates … Abstract Impaired spatial dynamics of T cells represent a major challenge to effective immunotherapy in triple‐negative breast cancer (TNBC). Here, a genetically engineered, bacteria‐derived hydrogel is presented that precisely orchestrates T cell activation, trafficking, and tumor cell engagement. This hydrogel is formed in situ from bacterial outer membrane vesicles (OMV) engineered to display TNBC‐specific antigen α‐lactalbumin, chemokine CXCL10, and CXCR4 ectodomain (CXCR4E). The α‐lactalbumin antigen within OMV is internalized by dendritic cells, promoting robust T cell activation and systemic mobilization. Upon exposure to matrix metalloproteinase‐2 in tumor, the hydrogel enables spatiotemporally controlled release of CXCL10 and CXCR4E, facilitating T cell infiltration and overcoming CXCL12‐mediated contact resistance. In vivo, this strategy establishes a durable immune niche, enhances anti‐tumor T cell responses, and achieves significant tumor suppression with long‐term immune memory. The findings demonstrate the potential of programmable, bacteria‐derived hydrogels as a versatile platform for overcoming immune barriers and advancing immunotherapy in TNBC.

COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale

2025-06-22

Hanfeng Lin , Bin Yang , Lang Ding +6 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent … Abstract Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine k inact and K I values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using the BTK inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and novel off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target BTK. We further demonstrate that COOKIE-Pro is compatible with streamlined cysteine activity-based protein profiling (SLC-ABPP) datasets, enabling efficient conversion of competition ratios to meaningful kinetic parameters. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful new tool for optimizing the potency and selectivity of covalent drugs during preclinical development.

Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis

2025-06-21

Naoya Iwamoto , Saya Ohno , Kensuke Nakamura +7 more | Bioconjugate Chemistry

Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine … Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine substitutions that enable the chemical synthesis of d-monobodies via native chemical ligation. In this study, the structure-activity relationship of monobody scaffold variants was investigated to identify more suitable positions for cysteine modifications. Several monobody variants with different cysteine substitution patterns and additional cysteine-selective modifications were designed and synthesized. Comprehensive functional analysis of the synthetic monobody derivatives led to the identification of a favorable monobody scaffold with potent target binding and high thermal stability. The optimized monobody scaffold with a cysteine cross-linker was used to develop d-monobody with additional functional groups.

Revisión: Infliximab, desarrollo y aplicación terapéutica

2025-06-20

Mildred Angélica Sauce Guevara , Erika Palacios Rosas , Alicia Martínez‐Romero | Revista Ciencia y Salud Integrando conocimientos

Infliximab es un anticuerpo monoclonal quimérico de tipo IgG que ha revolucionado el tratamiento de diversas enfermedades inflamatorias. Su mecanismo de acción se basa en la inhibición del factor de … Infliximab es un anticuerpo monoclonal quimérico de tipo IgG que ha revolucionado el tratamiento de diversas enfermedades inflamatorias. Su mecanismo de acción se basa en la inhibición del factor de necrosis tumoral alfa (TNF-α), una citocina clave en la respuesta inflamatoria del organismo. Debido a su capacidad para modular la inflamación, este medicamento ha sido aprobado para el tratamiento de patologías como la artritis reumatoide, la enfermedad de Crohn, la colitis ulcerativa y la psoriasis, mejorando significativamente la calidad de vida de los pacientes. La producción de infliximab implica técnicas biotecnológicas avanzadas que incluyen la generación de hibridomas murinos. Su administración es intravenosa y sigue protocolos de dosificación específicos según la enfermedad tratada. Esta revisión proporciona un análisis conciso de diferentes aspectos relacionados con su producción, propiedades farmacológicas y farmacocinéticas, así como su impacto en el manejo de estas enfermedades. El objetivo es ofrecer una perspectiva actualizada sobre el desarrollo y la aplicación clínica de este anticuerpo monoclonal quimérico.

Antibody treatment of hepatocellular carcinoma: a review of current and emerging approaches

2025-06-20

Sherif A. El‐Kafrawy , Mostafa S. Elkafrawy , Esam I. Azhar +2 more | Frontiers in Immunology

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths worldwide, underscoring the urgent need for innovative therapeutic strategies. Antibody-based therapies have emerged as a transformative approach, offering specificity and … Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths worldwide, underscoring the urgent need for innovative therapeutic strategies. Antibody-based therapies have emerged as a transformative approach, offering specificity and the potential to overcome the limitations of traditional treatments. This comprehensive review evaluates the current and emerging applications of antibody therapies in HCC, including monoclonal antibodies (mAbs), bispecific antibodies, and antibody-drug conjugates (ADCs). It explores their mechanisms of action, such as immune modulation, angiogenesis inhibition, and targeted cytotoxicity. Key advancements include the integration of immune checkpoint inhibitors (ICIs) like PD-1/PD-L1 and CTLA-4 inhibitors into clinical practice and the development of bispecific antibodies and ADCs targeting tumor-specific antigens like glypican-3. While these therapies have shown promise in improving patient outcomes, challenges such as tumor heterogeneity, resistance mechanisms, and immune-related adverse events persist. This review highlights recent clinical trial data, identifies areas for future research, and emphasizes the potential of combining antibody therapies with other modalities to enhance efficacy and overcome therapeutic barriers. By addressing these challenges and leveraging advancements in antibody engineering and biomarker discovery, antibody-based therapies hold significant promise for revolutionizing the treatment paradigm for HCC.

Mass Spectrometry-Driven Epitope Mapping: Application of Diethylpyrocarbonate Covalent Labeling for the Immunotherapeutic Target Programmed Cell Death Protein 1

2025-06-20

Parawan Ramanandana , Phumrapee Pianpaktr , Jiradej Makjaroen +7 more | Journal of the American Society for Mass Spectrometry

Monoclonal antibodies (mAbs) have revolutionized immuno-oncology, with anti-programmed cell death protein 1 (PD1) mAbs emerging as key therapeutic agents in cancer treatment. This study presents the development and application of … Monoclonal antibodies (mAbs) have revolutionized immuno-oncology, with anti-programmed cell death protein 1 (PD1) mAbs emerging as key therapeutic agents in cancer treatment. This study presents the development and application of diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) for detailed epitope mapping of anti-PD1 mAbs on PD1. By using DEPC CL-MS, we aimed to identify precise antibody binding sites on PD1 and benchmark its effectiveness against traditional X-ray crystallography. DEPC CL-MS offers high sensitivity and specificity while requiring less sample preparation and shorter analysis times, typically taking days or less, instead of months. PD1 was individually incubated with either nivolumab or a novel anti-human PD1 mAb (CU-MAB), followed by DEPC labeling, to assess DEPC modification extents under both binding and nonbinding conditions using bottom-up LC-MS/MS. Significant changes in DEPC modification at residues S27, S60, S62, S127, and K131 indicated binding sites and conformational shifts upon antibody interaction. These findings showed strong alignment with crystallography (PD1/nivolumab) and AlphaFold structural predictions (PD1/nivolumab and PD1/CU-MAB), highlighting the value of in-solution CL-MS for confirming AlphaFold predictions. This study underscores DEPC CL-MS as an efficient tool for epitope mapping, offering actionable insights into PD1-antibody interactions to drive therapeutic antibody development.

Implications of Anaphylaxis Following mRNA-LNP Vaccines: It Is Urgent to Eliminate PEG and Find Alternatives

2025-06-19

Jinxing Song , Dihan Su , Hongbing Wu +1 more | Pharmaceutics

The mRNA vaccine has protected humans from the Coronavirus disease 2019 (COVID-19) and has taken the lead in reversing the epidemic efficiently. However, the Centre of Disease Control (CDC) reported … The mRNA vaccine has protected humans from the Coronavirus disease 2019 (COVID-19) and has taken the lead in reversing the epidemic efficiently. However, the Centre of Disease Control (CDC) reported and raised the alarm of allergic or acute inflammatory adverse reactions after vaccination with mRNA-LNP vaccines. Meanwhile, the US Food and Drug Administration (FDA) has added four black-box warnings in the instructions for mRNA-LNP vaccines. Numerous studies have proven that the observance of side effects after vaccination is indeed positively correlated to the level of anti-PEG antibodies (IgM or IgG), which are enhanced by PEGylated preparations like LNP vaccine and environmental exposure. After literature research and review in the past two decades, it was found that the many clinical trial failures (BIND-014, RB006 fell in phase II) of PEG modified delivery system or PEGylated drug were related to the high expression of anti-PEG IgM and IgG. In the background of shooting multiple mRNA-LNP vaccines in billions of people around the world in the past three years, the level of anti-PEG antibodies in the population may have significantly increased, which brings potential risks for PEG-modified drug development and clinical safety. This review summarizes the experience of using mRNA-LNP vaccines from the mechanism of the anti-PEG antibodies generation, detection methods, clinical failure cases of PEG-containing products, harm analysis of abuse of PEGylation, and alternatives. In light of the increasing prevalence of anti-PEG antibodies in the population and the need to avoid secondary injuries, this review article holds greater significance by offering insights for drug developers. It suggests avoiding the use of PEG excipients when designing PEGylated drugs or PEG-modified nano-formulations and provides references for strategies such as utilizing PEG-free or alternative excipients.

Bispecific Antibodies in Solid Tumors: Advances and Challenges

2025-06-18

Khine Shan , Saba Musleh Ud Din , Shivani Dalal +5 more | International Journal of Molecular Sciences

Bispecific antibodies (BsAbs) have shown potential in cancer treatment and have become a rapidly growing field in cancer immunotherapy. Unlike monoclonal antibodies with two identical binding sites, BsAbs simultaneously bind … Bispecific antibodies (BsAbs) have shown potential in cancer treatment and have become a rapidly growing field in cancer immunotherapy. Unlike monoclonal antibodies with two identical binding sites, BsAbs simultaneously bind two distinct epitopes on the same or different antigens, allowing for a range of mechanisms of action, including engaging immune cells to kill cancer cells and blocking signaling pathways. Despite regulatory approvals for hematological malignancies in the last decade, their clinical success in solid malignancies has been lacking until recently. There are currently five BsAbs approved by the FDA in the United States for solid tumors-amivantamab, tarlatamab, tebentafusp, zanidatamab and zenocutuzumab-and two BsAbs approved in China-cadonilimab and ivonescimab. Currently, several BsAbs are under clinical development for solid tumors, but are mostly in early phase I and II trials. This review provides an overview of the basic mechanism of action of BsAbs, current FDA-approved BsAbs, and current BsAbs under clinical development, their challenges in clinical use, the management of toxicities, and future directions.

Characterization and functional evaluation of JS207, a novel bispecific antibody against human PD-1 and VEGFA

2025-06-18

Shihua Lin , Min Eui Hong , Jilei Zhang +7 more | Frontiers in Immunology

Introduction Cancer immunotherapy has been revolutionized by targeting PD-1 to restore antitumor T-cell activity and blocking VEGF to attenuate immunosuppressive tumor angiogenesis. While combining PD-1 and VEGF inhibition has shown … Introduction Cancer immunotherapy has been revolutionized by targeting PD-1 to restore antitumor T-cell activity and blocking VEGF to attenuate immunosuppressive tumor angiogenesis. While combining PD-1 and VEGF inhibition has shown promise in enhancing antitumor responses, co-administration of two or more monoclonal antibodies face several challenges, including distinct pharmacokinetics, complex dosing, and toxicity. A bispecific antibody (BsAb) targeting both PD-1 and VEGF pathways could overcome these limitations by enabling simultaneous, localized blockades of PD-1 and VEGF signaling within the tumor microenvironment (TME) as both PD-1 and VEGF are usually co-expressed in the TME. Methods Here, we describe the in vitro characterization, functional and preclinical evaluation of JS207, a novel BsAb targeting PD-1 and VEGFA with high antigen binding affinity. JS207 matched or surpassed the activity of benchmarks antibodies in several in vitro binding assessments, T cell activation, VEGF signaling inhibition, cytokines (IL-2 and IFN-γ) release. Results JS207 showed significant anti-tumor efficacy in mouse MC38 colon cancer model and A375 melanoma tumor model. Investigation into the mechanism of action revealed that VEGFA could significantly promote JS207’s antigen binding activity, T cell activation potency, and internalization of cell surface PD-1. In vivo results demonstrated that JS207 was well-tolerated and presented remarkable anti-tumor efficacy. In addition, JS207 showed enhanced thermal stability as evidenced by retained potency under heat stress, a critical factor for CMC (Chemistry, manufacturing and control) manufacture, storage and drug shelf life. Conclusion JS207 is a promising therapeutic candidate that may address unmet clinical needs in cancer immunotherapy.

Artificial intelligence in therapeutic antibody design: Advances and future prospects

2025-06-18

Su‐Jin Park , W.H. Jeong , Yubeen Kim +2 more | Current Opinion in Structural Biology

Optimised nanobody-based quenchbodies for enhanced protein detection

2025-06-18

Jordan H. Cater , Nehad Elsalamouny , Ghada H. Mansour +7 more | Communications Biology

Nucleotide context models outperform protein language models for predicting antibody affinity maturation

2025-06-18

Mackenzie M. Johnson , Kevin Sung , Hugh K. Haddox +6 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract Antibodies play a crucial role in adaptive immunity. They develop as B cell receptors (BCRs): membrane-bound forms of antibodies that are expressed on the surfaces of B cells. BCRs … Abstract Antibodies play a crucial role in adaptive immunity. They develop as B cell receptors (BCRs): membrane-bound forms of antibodies that are expressed on the surfaces of B cells. BCRs are refined through affinity maturation, a process of somatic hypermutation (SHM) and natural selection, to improve binding to an antigen. Computational models of affinity maturation have developed from two main perspectives: molecular evolution and language modeling. The molecular evolution perspective focuses on nucleotide sequence context to describe mutation and selection; the language modeling perspective involves learning patterns from large data sets of protein sequences. In this paper, we compared models from both perspectives on their ability to predict the course of antibody affinity maturation along phylogenetic trees of BCR sequences. This included models of SHM, models of SHM combined with an estimate of selection, and protein language models. We evaluated these models for large human BCR repertoire data sets, as well as an antigen-specific mouse experiment with a pre-rearranged cognate naive antibody. We demonstrated that precise modeling of SHM, which requires the nucleotide context, provides a substantial amount of predictive power for predicting the course of affinity maturation. Notably, a simple nucleotide-based convolutional neural network modeling SHM outperformed state-of-the-art protein language models, including one trained exclusively on antibody sequences. Furthermore, incorporating estimates of selection based on a custom deep mutational scanning experiment brought only modest improvement in predictive power. To support further research, we introduce EPAM (Evaluating Predictions of Affinity Maturation), a benchmarking framework to integrate evolutionary principles with advances in language modeling, offering a road map for understanding antibody evolution and improving predictive models.

Allosteric Coupling in Full-Length Lyn Kinase Revealed by Molecular Dynamics and Network Analysis

2025-06-18

Mina Rabipour , Floyd Hassenrück , Elena Pallaske +5 more | International Journal of Molecular Sciences

Lyn is a multifunctional Src-family kinase (SFK) that regulates immune signaling and has been implicated in diverse types of cancer. Unlike other SFKs, its full-length structure and regulatory dynamics remain … Lyn is a multifunctional Src-family kinase (SFK) that regulates immune signaling and has been implicated in diverse types of cancer. Unlike other SFKs, its full-length structure and regulatory dynamics remain poorly characterized. In this study, we present the first long-timescale molecular dynamics analysis of full-length Lyn, including the SH3, SH2, and SH1 domains, across wildtype, ligand-bound, and cancer-associated mutant states. Using principal component analysis, dynamic cross-correlation matrices, and network-based methods, we show that ATP binding stabilizes the kinase core and promotes interdomain coordination, while the ATP-competitive inhibitor dasatinib and specific mutations (e.g., E290K, I364N) induce conformational decoupling and weaken long-range communication. We identify integration modules and develop an interface-weighted scoring scheme to rank dynamically central residues. This analysis reveals 44 allosteric hubs spanning SH3, SH2, SH1, and interdomain regions. Finally, a random forest classifier trained on 16 MD-derived features highlights key interdomain descriptors, distinguishing functional states with an AUC of 0.98. Our results offer a dynamic and network-level framework for understanding Lyn regulation and identify potential regulatory hotspots for structure-based drug design. More broadly, our approach demonstrates the value of integrating full-length MD simulations with network and machine learning techniques to probe allosteric control in multidomain kinases.

N-glycosylation patterns of plasma immunoglobulin G in anti-synthetase syndrome disease

2025-06-18

Jing Zhao , Yanhong Li , Yunfei Ling +7 more | Frontiers in Immunology

Introduction Anti-synthetase syndrome (ASS) is a subtype of idiopathic inflammatory myopathy (IIM) characterized by characteristic rash, myositis, and interstitial lung disease (ILD). The etiology of ASS is unknown, and patients … Introduction Anti-synthetase syndrome (ASS) is a subtype of idiopathic inflammatory myopathy (IIM) characterized by characteristic rash, myositis, and interstitial lung disease (ILD). The etiology of ASS is unknown, and patients have a poor quality of life and are prone to pulmonary infection. Recent studies have elucidated the potential role of abnormal glycosylation of immunoglobulin G (IgG) in the pathogenesis of autoimmune diseases. However, the pattern of patient-specific IgG N-glycosylation in ASS has not been fully elucidated. Methods the GlycoQuant method was used to quantify the intact N-glycopeptides of IgG from 30 ASS patients and 30 healthy controls (HCs). Results and Discussion Thirteen differentially expressed intact N-glycopeptides were identified (p&lt;0.05). Notably, we observed increased fucosylation (p&lt;0.0001) and decreased N-acetylneuraminic acid (p&lt;0.05) in ASS patients. In addition, specific glycosylation patterns correlated with lung function parameters. Our study revealed the IgG glycosylation profile in ASS patients and provided a valuable reference for further investigation of its potential diagnostic and prognostic applications.

Crystal structures reveal how the multispecific antibody G2 achieves binding to different peptides.

2025-06-17

Yuya Hanazono , Saaya Yabuno , Takahiro Hayashi +4 more | PubMed

Monoclonal antibody G2, obtained via immunization with the chicken prion protein (ChPrP), has unique antigen-binding specificity. G2 specifically binds to the ChPrP-derived peptide (Pep18mer) as expected, but also to three … Monoclonal antibody G2, obtained via immunization with the chicken prion protein (ChPrP), has unique antigen-binding specificity. G2 specifically binds to the ChPrP-derived peptide (Pep18mer) as expected, but also to three other peptides. In this study, we determined the crystal structures of G2 single-chain Fv antibodies covalently linked to Pep18mer and another peptide, PepH4P6. Both bound peptides formed similar U-shaped structures that stuck into the G2 antigen-binding pocket. Their three-dimensional structures were stabilized by interactions within the peptides, and the structure of the bound Pep18mer was similar to that of the corresponding ChPrP region. G2 acquired the binding ability to both Pep18mer and PepH4P6 via deletion of the 95th residue of the light chain during affinity maturation, consistent with our structural analysis.

Reliable Quantification of Conjugated Peptides from T-DM1 In Vivo by Two-Step Enrichment and Internal Standards from Derivatized Analytes

2025-06-17

Meiling Qi , Ying Xiong , Meiling Chen +7 more | Analytical Chemistry

Antibody-drug conjugates (ADCs) are the conjugation of antibody with cytotoxic payloads. Conjugated peptides that originate from ADCs are commonly used for pharmacokinetics (PK) study. However, a reliable and sensitive method … Antibody-drug conjugates (ADCs) are the conjugation of antibody with cytotoxic payloads. Conjugated peptides that originate from ADCs are commonly used for pharmacokinetics (PK) study. However, a reliable and sensitive method for the quantitative analysis of conjugated peptides in vivo remains elusive, especially for randomly conjugated ADCs. In this study, a strategy that was based on a liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify conjugated peptides from trastuzumab emtansine (T-DM1) in vivo. To correct variations introduced during sample processing and LC-MS detection, a dimethyl labeling strategy was developed to generate one-to-one structural analogues for each conjugated peptide from T-DM1, serving as internal standards (ISs). For sample preparation, protein A/G bead enrichment from the protein level and high-pH reverse-phase fractionation from the peptide level were utilized to enrich conjugated peptides from plasma, leading to the detection of more conjugated peptides. The established method was then validated and applied to quantify conjugated peptides in plasma from rats administered with T-DM1, resulting in the detection of 17 distinct conjugated peptides. Notably, differences in stability across various conjugation sites were observed for the first time, leading to different PK profiles depending on the analytes used. This method is applicable to ADCs and peptide-drug conjugates, considering that they have complex structures and the stable isotope-labeled internal standard (SIL-IS) of conjugated peptide is unavailable.

Editorial: Next generation therapeutic modality to cure autoimmune diseases

2025-06-17

G.T Chen , Ce Wang , Qi Wan +2 more | Frontiers in Immunology

Identification of immunogenic commensal antigens using phage display

2025-06-17

Sheenam Verma , Samantha Kimmel , Oliver J. Harrison | Nature Protocols

Production of monoclonal antibodies against carcinoembryonic antigen-related cell adhesion molecule 6 and detection of their binding affinities

2025-06-17

Yun‐Hsin Wang , Yau‐Hung Chen | Tzu Chi Medical Journal

A BSTRACT Objectives: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycophosphatidylinositol-anchored member of the immunoglobulin superfamily, often overexpressed in various malignancies. Targeting CEACAM6 by suppressing its expression can … A BSTRACT Objectives: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycophosphatidylinositol-anchored member of the immunoglobulin superfamily, often overexpressed in various malignancies. Targeting CEACAM6 by suppressing its expression can potentially reverse these effects, making it a promising therapeutic target. In this study, we generated five monoclonal antibodies (CEAS1, CEAS2, CEAS3, CEAS4, and CEAS5; CEAS1-S5) against the recombinant CEACAM6 protein. Materials and Methods: Through enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay, we demonstrated that each antibody specifically binds to CEACAM6 without interfering with the binding of others. SPR analysis further revealed the rate of association (K a ), dissociation (K d ), and equilibrium dissociation constants (K D ) for each antibody. Results: The K D values ranged from 5.089 × 10 −11 to 1.213 × 10 −13 M, with CEAS5 exhibiting the highest binding affinity. In addition, CEAS5, unlike CEAS1-S4, could bind to both CEACAM5 and CEACAM6, indicating its bivalent nature. Conclusion: These findings highlight the strong antigen-binding capabilities of CEAS1-S5, warranting further investigation.

The spectrum of nuclear patterns with stained metaphase chromosome plate: morphology nuances, immunological associations, and clinical relevance

2025-06-17

Wilson de Melo Cruvinel , Gonçalo Alexandre Martins Ferreira , Laís Laura de Souza +4 more | Clinical Chemistry and Laboratory Medicine (CCLM)

Abstract The indirect immunofluorescence assay (IFA) on HEp-2 cells is the prevailing method used to screen for autoantibodies in the investigation of systemic autoimmune diseases (SAID). When positive, the titer … Abstract The indirect immunofluorescence assay (IFA) on HEp-2 cells is the prevailing method used to screen for autoantibodies in the investigation of systemic autoimmune diseases (SAID). When positive, the titer provides a semi-quantitative assessment of the autoantibody serum concentration whereas the immunofluorescence pattern indicates the possible autoantibody specificities. The Brazilian Consensus on ANA Patterns (BCA) and the International Consensus on ANA Patterns (ICAP) provide recommendations for the harmonization on the pattern nomenclature and test reporting. Nuclear patterns are among the most frequent in the clinical laboratory and some of them are highly relevant in the diagnosis of SAID. Nuclear patterns with stained metaphase plate (MP) indicate autoantibodies against chromatin components or against chromatin-bound antigens. These include the nuclear homogeneous (AC-1), nuclear dense fine speckled (AC-2), Topo 1-like (AC-29), and nuclear fine speckled with stained MP (AC-30) patterns. The Brazilian consensus has also classified the quasi -homogeneous nuclear pattern (QH). The correct identification of these patterns is important because each one is associated with different autoantibody specificities and clinical scenarios. However, the recognition of the nuances in texture of the staining pattern and other specific features that characterize each of them may be challenging for the analyst at the microscope. This review focuses on the morphological characteristics, immunological identities, and clinical relevance of nuclear patterns with stained MP. The aim is to assist laboratory analysts and clinicians in identifying and interpreting these patterns, thus optimizing the use of the HEp-2 IFA test in the investigation of patients under suspicion of SAID.

Surface Plasmon Resonance (SPR)-Based Workflow for High-Throughput Discovery of CD28-Targeted Small Molecules

2025-06-17

Laura Calvo‐Barreiro , Hossam Nada , S. Upadhyay +1 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract CD28 is a critical costimulatory receptor involved in T cell activation and immune regulation, making it a compelling target for immunomodulatory therapies. Despite its therapeutic relevance, small molecule CD28 … Abstract CD28 is a critical costimulatory receptor involved in T cell activation and immune regulation, making it a compelling target for immunomodulatory therapies. Despite its therapeutic relevance, small molecule CD28 inhibitors remain largely underexplored. To address this gap, we developed a high-throughput screening (HTS) workflow using surface plasmon resonance (SPR) to identify novel CD28-targeted small molecules. To our knowledge, this work represents the first SPR-based HTS platform applied to the discovery of small molecules targeting a stimulatory immune checkpoint receptor. A chemical library composed of diverse 1,056 small molecules was screened using a 384-well format. Compounds were evaluated based on level of occupancy (LO), binding response, and dissociation kinetics, resulting in 12 primary hits (1.14% hit rate). Follow-up dose–response SPR screening confirmed micromolar-range affinities for three compounds. Molecular docking and 100 ns molecular dynamics (MD) simulations of the top hit, DDS5 , revealed a stable complex with CD28, maintained by hydrogen bonding and a persistent interaction with Phe93. Functional validation using a competitive ELISA confirmed that DDS5 inhibited the CD28–CD80 interaction. These results demonstrate that our SPR-based HTS platform is a robust and efficient strategy for discovering CD28-targeted small molecules. The integration of computational evaluation and orthogonal validation further underscores the potential of DDS5 as an early-stage immunomodulatory agent.

Inference of multi-enhancer interactions in T lymphocytes using Hi-Cociety

2025-06-17

Sora Yoon , Golnaz Vahedi | bioRxiv (Cold Spring Harbor Laboratory)

Three-dimensional (3D) enhancer communities are key regulators of gene expression, shaping cell fate decisions and contributing to disease pathogenesis. Assays such as H3K27ac HiChIP have been used to map enhancer-enhancer … Three-dimensional (3D) enhancer communities are key regulators of gene expression, shaping cell fate decisions and contributing to disease pathogenesis. Assays such as H3K27ac HiChIP have been used to map enhancer-enhancer interactions and define enhancer communities; however, their reliance on antibody-based enrichment restricts scalability and cross-cell-type applicability. In contrast, Hi-C provides an unbiased, genomewide view of chromatin architecture but lacks direct annotation of regulatory elements, limiting its utility for enhancer-focused analyses. To bridge this gap, we introduce Hi-Cociety - a graph-based computational framework and accompanying R package that infers 3D enhancer communities directly from Hi-C data, without relying on histone modification or chromatin accessibility measurements. Hi-Cociety constructs a network of significant interactions and applies clustering algorithms to define chromatin interaction modules. Applying Hi-Cociety to Hi-C measurements in T lymphocytes, we identified highly connected modules enriched for active transcription, chromatin accessibility, and histone acetylation. Notably, modules identified in T cells pinpoint critical genes central to T cell biology. Hi-Cociety also detects cell-type-specific differences in chromatin organization, highlighting dynamic regulatory rewiring across T cell states. Our findings underscore the importance of network properties - connectivity, transitivity, and centrality - in shaping gene regulation through 3D genome organization. Hi-Cociety provides a scalable and versatile tool for mapping enhancer communities at scale, advancing our understanding of immune cell identity and the regulatory logic encoded in 3D chromatin structure.

Improving Recombinant Antibody Production Using FcBAR: An In Situ Approach to Detect and Amplify Protein-Protein Interactions

2025-06-17

Mina Wu , Frances Rocamora , Mojtaba Samoudi +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell … Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the in situ detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production in situ, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.

Divergent effects of a Treg selective IL-2 mutein on Influenza-specific T cell responses

2025-06-16

Joseph R. Albe , Anita Chaudhary , Asheema Khanna +5 more | bioRxiv (Cold Spring Harbor Laboratory)

Enhancing regulatory T cell (Treg) function offers a compelling therapeutic strategy for autoimmune disease. Engineered IL-2 muteins selectively expand functional Tregs with minimal impact on other immune cells, but their … Enhancing regulatory T cell (Treg) function offers a compelling therapeutic strategy for autoimmune disease. Engineered IL-2 muteins selectively expand functional Tregs with minimal impact on other immune cells, but their potential to compromise antiviral immunity remains largely unexplored. Here, we used a murine model of Influenza A virus (Flu) infection to determine how IL-2 mutein shapes T cell responses to respiratory virus infection. IL-2 mutein administration prior to infection suppressed Flu-specific (Flu-sp) CD8 T cell responses and altered their localization and phenotype within the lungs, without affecting bystander CD8 T cells. This suppression correlated with reduced antigen presentation molecule expression on conventional dendritic cells (cDCs) early after infection but did not impact Flu-sp CD8 T cell priming. In contrast, administering IL-2 mutein during infection exacerbated disease and drove CD25-dependent expansion of Flu-sp CD8 T cells. Despite these opposing effects on effector responses, Fc.Mut24-treated mice generated robust antibody responses and protective T cell memory which were maintained for at least 170 days. These findings reveal that Fc.Mut24 has temporally distinct effects on antiviral immunity, dampening early effector responses when given before infection, but enhancing effector expansion and disease severity when delivered during infection. Our results provide critical context for the therapeutic application of IL-2 muteins and highlight the importance of treatment timing in balancing immune modulation with protective immunity.

Improving the solubility of single domain antibodies using <scp>VH</scp>‐like hallmark residues

2025-06-16

Y. Uto , Makoto Nakakido , Takanori Yokoo +7 more | Protein Science

Abstract Single domain antibodies (sdAbs) can be generated from variable regions of heavy‐chain antibodies, which lack light chain and CH1 region. They have attracted attention due to their small size … Abstract Single domain antibodies (sdAbs) can be generated from variable regions of heavy‐chain antibodies, which lack light chain and CH1 region. They have attracted attention due to their small size and molecular characteristics. Hydrophilic hallmark amino acids at framework region 2 (FR2) are key residues involved in the solubility of sdAbs. Nevertheless, previous studies reported that several sdAbs with human VH‐like hydrophobic hallmark residues were soluble in a monomeric state and suggested that solubility also depends on the amino acid sequences in the complementarity‐determining region. In this study, we obtained two sdAbs (sdAb A and B) with VH‐like hallmark residues and low solubility from an alpaca immune library. We introduced VHH‐like mutations (V37Y, G44E, L45R, W47L) into the hallmark residues in FR2 of both sdAb A and B. We were able to prepare sdAb A as a monomer without an additive in the buffer, but sdAb B was polydispersed when arginine was not added to the buffer. We also predicted the hydrophobicity of the sdAb B surface by spatial aggregation propensity calculations and identified W99 as the residue responsible for its low solubility. Subsequently, we obtained the sdAb B mutant as a monomer by introducing the W99A mutation. We characterized the engineered sdAbs using structural, physicochemical, and biophysical analyses and found that the solubility‐improved sdAbs retained their functionality. Our findings can be applied to improving the solubility of sdAbs even in the absence of structural information.

Development of a novel system for the expression and production of recombinant monoclonal antibody, tocilizumab, using Pichia pastoris

2025-06-16

Prabir Kumar Das , Ansuman Sahoo , Veeranki Venkata Dasu | International Journal of Biological Macromolecules

A high-affinity split-HaloTag for live-cell protein labeling

2025-06-15

Yin‐Hsi Lin , Julian Kompa , De‐en Sun +7 more | bioRxiv (Cold Spring Harbor Laboratory)

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent … We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHaloΔ3 and in fixed cells through incubation with cpHaloΔ3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.

Computational Discovery of RSV Pre-F Inhibitors via Reinforcement Learning-Driven ab Initio Design from Natural Fragment Libraries

2025-06-14

Shengpeng Wang , Yongzhu Zhao , Jia‐Wei Li +6 more | Computational Biology and Chemistry

Rational Generation of Monoclonal Antibodies and Intrabodies Selective for Pathogenic TDP-43

2025-06-14

Beibei Zhao , Sarah Louadi , Juliane Coutts +7 more | bioRxiv (Cold Spring Harbor Laboratory)

TAR DNA–binding protein 43 (TDP–43), encoded by the TARDBP gene, is a ribonucleoprotein associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Under … TAR DNA–binding protein 43 (TDP–43), encoded by the TARDBP gene, is a ribonucleoprotein associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Under physiological conditions, TDP–43 is predominantly localized in the nucleus, where it participates in a variety of cellular functions related to RNA splicing, transport, and stability, as well as miRNA biogenesis. In disease, it is characteristically mislocalized to the cytoplasm where it forms aggregates, which contribute to neurotoxicity and prion–like cell–to–cell propagation of pathogenic TDP–43. Targeting of misfolded aggregates of TDP–43 represents an attractive therapeutic strategy. However, development of effective immunotherapeutic agents remains a challenge, as they require stringent selectivity for misfolded TDP–43 in order to maintain the essential functions of physiologically native TDP–43. To address this issue, monoclonal antibodies (mAbs) and intrabodies were generated against an epitope in the N–terminal domain of TDP–43 that is only exposed when the protein is misfolded, but not in its properly folded form. We show that mouse and rabbit mAbs against this epitope displayed high binding affinities by surface plasmon resonance analysis and selectively reacted with pathological TDP–43 in post–mortem tissues from ALS, FTD, and AD patients. In a cell line system, human embryonic kidney (HEK) 293T cells, mAbs and corresponding intrabodies specifically reacted with cytoplasmic aggregates of transfected misfolded TDP-43 lacking the nuclear localization signal, TDP-43 ΔNLS . Functionally, mAbs inhibited cell-to-cell transmission of misfolded TDP–43 and the seeding activity of misfolded TDP–43 from FTLD brain homogenates by novel RT–QuIC assay. Intrabodies promoted the degradation of intracellular aggregates of TDP–43 in HEK293T cells and in induced pluripotent stem cell–-–derived motor neurons (iPSC–MNs) from ALS patients. The results provide proof–of–concept evidence that supports selective targeting of misfolded toxic aggregates of TDP[ndash]43 as a potentially safe and effective avenue to treat neurodegenerative diseases associated with TDP–43 proteinopathy.

Advances in targeted therapy for triple-negative breast cancer: a review of key antigens and recent advances

2025-06-14

Bahman Akbari , Md Mehedi Hasan , Shahidul M. Islam | Journal of drug targeting

The most aggressive subtype of breast cancer is triple-negative breast cancer (TNBC), which affects about 10-15% of all breast cancer cases. TNBC is associated with a poor prognosis and drug … The most aggressive subtype of breast cancer is triple-negative breast cancer (TNBC), which affects about 10-15% of all breast cancer cases. TNBC is associated with a poor prognosis and drug resistance due to the lack of oestrogen, progesterone, and HER2 receptors. Developing targeted immunotherapy for TNBC was challenged by identifying TNBC-specific antigens that can be suitable targets for antibody and nanobody-based therapies. Evidence from cancer- targeted therapy demonstrates that treatment outcomes are more successful when the target antigen is either overexpressed in tumour tissue or exhibits tumour specificity. Several antigens have been overexpressed in TNBC, including programmed cell death protein 1 (PD-1), programmed death-ligand 1(PD-L1), mesothelin (MSLN), trophoblast cell-surface antigen 2 (Trop-2), tumour endothelial marker 8 (TEM8), etc. There have been investigations targeting these antigens with antibodies, nanobodies, small molecules, peptides, and miniproteins for targeted treatment of TNBC. Antibodies known as Aembrolizumab, Atezolizumab, and Sacituzumab Govitecan-hziy have been approved by the FDA, and many are under investigation. The present review discusses the antigens with high expression in TNBC, their role in cancer development and progression, and the targeted therapies like antibodies, recombinant proteins, and antibody-drug conjugates (ADC).