A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain.

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Harvesting Tissue from Immunized Mice, Rats, and Hamsters.

Immunologically active organs such as the spleen and lymph nodes are rich sources of antibodies; they are also major sites in the body where antibody-producing B cells accumulate. The lymph nodes, in particular, can be targeted when deciding where to inject an animal with antigen. Once an immunized animal has developed a sufficient serum antibody titer, these organs can be harvested for hybridoma fusion and monoclonal antibody production.

Micro-environmentally restricted hybridoma cell growth within polysaccharide hydrogel microbeads.

The mechanism of micro-environmentally restricted hybridoma cell growth caused by action of local mechanical compression stress generated within various polysaccharide hydrogel matrixes is estimated by comparing the growth of hybridoma cells within (1) 1.5% Ca-alginate microbeads from Bugarski et al. [in: Fundamentals of Animal Cells Immobilization and Microencapsulation, M.F.A. Goosen, ed., CRC Press, Boca Raton, FL, 1993, p. 267] and (2) 1.3% alginate-agarose microbeads from Shen et al. [Animal Cell Technology: Basic & Applied Aspects, H. Murakami ed., Kluwer Academic Publishers, The Netherlands, 1992, p. 173].Consideration of restricted cell growth dynamics based on developed kinetic model and kinetic 3D Monte Carlo simulation include: (1) changes the fraction of active proliferating cells in the exponential phase and (2) changes of non-proliferating cell concentration in the plateau phase.Higher value of the specific decrease of active fraction of proliferating cells κ is obtained for 1.3% alginate-agarose compared to 1.5% alginate microbeads. It corresponds to higher compression stress generated within hydrogel matrix during cell growth obtained for 1.3% alginate-agarose microbeads.

Inhibition of cell division in mouse B-cell hybridomas: an overlooked property of 2-mercaptoethanol and its impact on in vitro antibody production.

Thiol 2-mercaptoethanol (2-ME) has been reported to enhance growth in lymphocytes by various investigators. Some have used 50 µM for growing hybridomas in vitro. Concentrations of 50 and 5 µM in 5% FBS supplemented D-MEM were tested to determine their effects on the growth of 5 monoclonal antibody secreting mouse B cell hybridomas and the myeloma Sp2/O-Ag14. Viability after 24 and 48 h exposure was determined by Trypan blue exclusion. Analysis by one-way ANOVA confirmed that 50 µM 2-ME has a significant negative impact (p<0.05) on hybridoma as well as on myeloma growth, whereas no significant difference (p>0.05) between the control and the 5 µM treatment group was observed after 48 h. Also, no significant difference (p>0.05) in the mortality rates between the control and the treatment groups was found. When combined with the observed protracted doubling time in the 50 µM treatment group, these results indicate that the impact of 2-ME is due to inhibition of cell division. The degree of inhibition was observed to vary between the different hybridomas as well as the myeloma. Although the impact of 2-ME on mitosis has been demonstrated in organisms such as the ciliated protozoan Tetrahymena pyriformis, the yeast Saccharomycess cerevisiae, and the egg of the echinoid the sand dollar Dendraster excentricus, this work demonstrates for the first time that 2-ME impedes the growth of mouse B cell hybridomas. We conclude that adding 2-ME to mouse B cell hybridoma growth media may not be beneficial.

Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development.

The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation.

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 µs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.

Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation.

Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A(b) complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses.

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy.

During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of approximately 14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.

Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes.

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein-Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0-G1 state and this is translated in lower numbers of dead cells.

Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection.

The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 +/- 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.

Effect of alpha-ketoglutarate on monoclonal antibody production of hybridoma cell lines in serum-free and serum-containing medium.

Process development and optimization for increase population growth and protein productivity in mammalian cell culture have been studied for many years. In this study, the behavior of hybridoma cells was investigated using six-well micro-titer plate systems with a working volume of 4 ml. Mouse hybridoma cell lines D2 and 2C83G2 were seeded in serum-free and serum-containing media and cultured for 8 days. alpha-Ketoglutarate is an integral component of the tricarboxylic acid (TCA) cycle and is produced from glutamine via glutamate. To study its effect on cell growth, metabolism, and monoclonal antibody (mAb) production, 2 mM alpha-ketoglutarate (pH 7.2) was added in both media at the beginning of the cultivation and in another set after 72 h. High cell density was observed in D2 cell culturing in serum-free medium, while 2C83G2 cell line showed high cell density in serum-containing medium. However, both cell lines cultured in serum-free medium gave viability above 70% when grown for 8 days. The supplement of 2 mM alpha-ketoglutarate supported cell growth and mAb production of both hybridoma cell lines in serum-free and serum-containing medium. The addition of alpha-ketoglutarate at the beginning of the batch cultivation gave better result in cell growth and mAb production as compared to alpha-ketoglutarate supplementation after 72 h. However, addition after 72 h was better than no addition at all. This indicates that alpha-ketoglutarate have a positive effect on production and release of antibody.

T84-intestinal epithelial exosomes bear MHC class II/peptide complexes potentiating antigen presentation by dendritic cells.

BACKGROUND & AIMS: Intestinal epithelial cells release antigen-presenting vesicles (exosomes) bearing major histocompatibility complex class II/peptide complexes stimulating specific immune responses in vivo. To characterize further the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, bind immune target cells, and induce T-cell activation was analyzed in vitro. METHODS: The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide (3)H-HSA 64-76 and to activate a HLA-DR4-restricted T-cell hybridoma was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of fluorescein isothiocyanate-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. RESULTS: T84-derived exosomes, enriched in CD9, CD81, CD82, and A33 antigen, were capable of binding specifically human serum albumin (HSA) 64-76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T-cell hybridoma directly but induced a productive T-cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (x10(-3)). CONCLUSIONS: Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to major histocompatibility complex class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces.

Characterization of a monoclonal antibody specific for human peripheral myelin protein 22 and its use in immunohistochemical studies of the fetal and adult nervous system.

We produced a mouse monoclonal antibody using cDNA and peptide immunization against the putative second extra-cellular domain of human peripheral myelin protein 22 (PMP22). It reacted specifically with human PMP22 and not with other human myelin proteins and did not react with bovine, rat, or mouse PMP22. The antibody stained the compact myelin of human peripheral nerve motor and sensory axons and did not stain central nervous system tissue. PMP22 reactivity was detected in the spinal roots of the human fetus at 19-20 weeks of gestation. The staining pattern of the PMP22 antibody resembled that of a monoclonal antibody directed against the myelin protein zero.

Polyreactive myelin oligodendrocyte glycoprotein antibodies: Implications for systemic autoimmunity in progressive experimental autoimmune encephalomyelitis.

Two myelin oligodendrocyte glycoprotein (MOG92-106) monoclonal antibodies (mAbs) were produced from an A.SW mouse with progressive experimental autoimmune encephalomyelitis. Polyreactivity/specificity of the mAbs was demonstrated by ELISA. Functionality and a potential role in pathogenesis of systemic autoimmunity were demonstrated in vitro in a lymphocytotoxicity assay and in vivo upon injection into naïve mice. Injection of MOG mAb producing hybridomas into naïve mice resulted in immunoglobulin deposition in kidneys and liver. This model will be useful in determining whether transitional forms between CNS (organ)-specific and systemic autoimmune diseases exist, and whether progressive multiple sclerosis has features of a systemic autoimmune disease.

Statistical methods in media optimization for batch and fed-batch animal cell culture.

Hybridoma 130-8F producing anti-F monoclonal antibodies (MAb) were grown in batch and fed-batch mode with glutamine as the limiting substrate. The initial concentration of glucose varied between 10 and 25 mM but was not growth limiting. Monoclonal antibody production was identified as being partially growth associated. Employing the cumulative cell hour concept, external metabolic flux estimates were calculated during the exponential growth phase for MAb, glucose, amino acids, ammonia and lactate. Through nutritional profiling using principal component analysis (PCA) followed by partial least squares regression (PLS), key metabolites were identified and grouped for significant positive, significant negative, low level, and negligible correlation to MAb production, cellular growth, glucose consumption, and ammonia and lactate production. Significant relationships peculiar to Hybridoma 130-8F were identified, such as demand for two normally non-essential amino acids (asparagine and aspartic acid), and the positive correlation between MAb and ammonia production.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells.

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semi-quantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs.

Monoclonal antibodies to distinct regions of human myelin proteolipid protein simultaneously recognize central nervous system myelin and neurons of many vertebrate species.

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50-69, 100-123, 139-151, 178-191, 200-219, 264-276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti-100-123 mAb did not recognize DM-20, the PLP isoform that lacks residues 116-150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116-150; the anti-100-123 mAb did not stain their myelin. In addition to myelin, the anti-178-191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti-human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti-PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti-PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions.

Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA.

BACKGROUND: The amplification of variable regions of immunoglobulins has become a major challenge in the cloning of antibody genes, whether from hybridoma cell lines or splenic B cells. Using conventional protocols, the heavy-chain variable region genes often are not amplified successfully from the hybridoma cell lines. RESULTS: A novel method was developed to design the degenerated primer of immunoglobulin cDNA and to amplify cDNA ends rapidly. Polymerase chain reaction protocols were performed to recognize the VH gene from the hybridoma cell line. The most highly conserved region in the middle of the VH regions of the Ig cDNA was identified, and a degenerated 5'primer was designed, using our algorithms. The VH gene was amplified by both the 3'RACE and 5'RACE. The VH sequence of CSA cells was 399 bp. CONCLUSION: The new protocol rescued the amplifications of the VH gene that had failed under conventional protocols. In addition, there was a notable increase in amplification specificity. Moreover, the algorithm improved the primer design efficiency and was shown to be useful both for building VH and VL gene libraries and for the cloning of unknown genes in gene families.

Estimating the growth kinetics of experimental tumors from as few as two determinations of tumor size: implications for clinical oncology.

Clinical information on tumor growth is often limited to a few determinations of the size of the tumor burden taken at variable time. As a consequence, fitting of growth equations to clinical data is hampered by the small number of available data. On the other hand, characterising the tumor growth kinetics in terms of clinically relevant parameters, such as the doubling time of the tumors, is increasingly required to optimize and personalise treatments. A computational method is presented which can estimate the growth kinetics of tumors from as few as two determinations of its size taken at two successive time points, provided the size at which tumor growth saturates is known. The method is studied by using experimental data obtained in vitro with multicell tumor spheroids and in vivo with tumors grown in mice, and its outputs are compared to those obtained by fitting of experimental data with the Gompertz growth equation. Under certain assumptions and limitations the method provides comparable estimates of the doubling time of tumors with respect to the classical nonlinear fitting approach. The method is then tested against simulated tumor growth trajectories spanning the range of tumor sizes observed in the clinics. The simulations show that a relative classification of tumors on the basis of their growth kinetics can be obtained even if the size at which tumor growth saturates is not known. This result opens the possibility to classify patients bearing fast or slow growing tumors and, hence, to adapt therapeutic regimens under a more rationale basis.