Selecting the Antigen.

The classical method for generating polyclonal or monoclonal antibodies relies on the in vivo humoral response of animals. Here we describe the factors that antigens can have that might influence the strength and quality of an antibody response. This introduction is divided into three sections: (1) an overview of immunogenicity, (2) choosing the best form for the immunogen, and (3) methods for modifying antigens to make them more immunogenic.

Making Weak Antigens Strong: Preparing Immune Complexes for Injection.

If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.

Making Weak Antigens Strong: Coupling Antigens to Red Blood Cells.

Coupling antigens to red blood cells (RBCs) can increase the immunogenicity of weak antigens. Their size slows dispersal; that, and their particulate nature, also make them good targets for phagocytosis. If the source of the cells is different from the animal to be injected, they can also provide good targets for MHC class II-T-cell receptor binding. The choice of coupling method will depend on the antigen, but because of the complexity of proteins found on the surface of the RBCs, almost all chemical groups are available for coupling. Commonly used coupling methods include tannic acid, chromic chloride, and glutaraldehyde.

Preparing mFc- and hFc-Fusion Proteins from Mammalian Cells.

Fc-fusion proteins are composed of an immunoglobulin Fc domain that is directly linked to the antigen of interest. Typically, these vectors will contain an amino-terminal signal sequence that permits trafficking to the cell surface and secretion into the media and a carboxy-terminal Fc receptor that enables purification on Protein A-Sepharose. Fc-fusion proteins have several applications in protein microarrays, oncological therapies, and vaccine and antibody development. Presence of the Fc domain significantly increases the plasma life of the fusion partner, which prolongs therapeutic activity. Furthermore, the Fc domain enhances the solubility and stability of the partner molecule. Because Fc-fusion proteins are secreted into the culture medium, purification by affinity chromatography is relatively easy and cost-effective. Immunizing a murine host with mFc-fusion protein generates an antigen-specific immune response because the Fc domain is recognized as "self" by the host.

Preparing Live Cells for Immunization.

Generating monoclonal antibodies against cell-surface (i.e., membrane) proteins can be challenging because membrane and membrane-associated proteins often lose their native conformation during the purification process. This also makes fusion screening very difficult. One widely used technique to overcome this issue is to overexpress the target protein in HEK 293T cells and then immunize the host with these cells. The advantage of immunizing with native cells is that the target protein is expressed and presented to the immune system in a correctly folded form with all of its secondary posttranslational structure in place. This is essential for conformational or discontinuous epitopes, and for transmembrane proteins that weave in and out of the cell membrane multiple times. Transient or stable transfectants can be used for immunization and for screening using fluorescence-activated cell sorting, western blot, or immunoprecipitation. Although transfectants often have higher expression levels than do native cells, care should be taken to ensure that the transfectant expresses a functionally active version of the target protein, as otherwise minor folding issues or modifications in structure can result in antibodies that recognize the transfected, but not the native, protein. Care must also be taken when using cells as immunogens because numerous antigenic proteins coimmunize with the target protein. Screening hybridomas using the same cells and counterscreening them on untransfected cells will enable the selection of specific hybridomas.

Preparing GST-, His-, or MBP-Fusion Proteins from Bacteria.

An excellent source of antigens is to overexpress cloned genes in bacteria. A wide variety of coding regions can be expressed in bacteria either on their own or as fusion proteins. Indeed, it is often convenient to use vectors that express the antigen fused to an affinity tag, such as glutathione-S-transferase (GST), maltose-binding protein (MBP), or poly-histidine (e.g., 6×His). Each of these tags reliably binds to a specific affinity column enabling the antigen to be conveniently eluted under appropriate conditions. It should be noted that GST adds ∼25 kDa and MBP adds 40 kDa to the expressed protein. Because bacteria will reliably express proteins that are <80 kDa, the mass of the affinity tag should be included when designing your recombinant protein construct.

Sarkosyl Preparation of Antigens from Bacterial Inclusion Bodies.

In many cases, solubility and proper folding of fusion proteins expressed in bacteria pose a major challenge in protein purification and crystallization. This is especially true when the fusion proteins are of eukaryotic origin. They form aggregates or become packaged into inclusion bodies, which makes protein purification extremely difficult. Sarkosyl is widely used to extract misfolded proteins from inclusion bodies in soluble form.

Detecting Protein Antigens in Sodium Dodecyl Sulfate-Polyacrylamide Gels.

In some cases, a native protein can be isolated in its pure form from cell lysates or tissue preparation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antigens purified this way often induce good antibody responses. After electrophoresis, the band of protein of interest must be located in the gel. A variety of identification methods can be used, all of which are designed to avoid excessive fixation of the protein in the gel matrix. The choice of method depends partly on the abundance of the polypeptide. Three methods are commonly used: (1) staining side strips cut from the edge of the gel, (2) light staining of the gel itself, and (3) locating the band by radioactive labeling of the antigen. Staining strips of the gel cut from its sides avoids the need to fix the gel. When isolating abundant proteins that are well separated from other bands, staining side strips is a useful method. If the protein is not abundant or is located close to a contaminating band making a clean excision difficult, use one of the other staining methods. If the protein is reasonably abundant, then a light staining of the proteins in the gel with Coomassie Blue G will permit localization without fixing. Alternatively, the bands in the gel can be visualized by immersing the gel in sodium acetate or copper chloride. If the protein is radiolabeled with 125I, 32P, or 35S, then use an autoradiogram as a template to excise the band of interest.

Preparing Protein Antigens from Sodium Dodecyl Sulfate-Polyacrylamide Gels for Immunization.

Some native proteins can be isolated in pure form from cell lysates or tissue preparation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Antigens purified this way often induce good antibody responses. There are many different ways to process a gel fragment containing the protein of interest for injection. Such samples can be processed into small pieces and then injected, either by fragmenting the gel by passing it repeatedly through a syringe, or drying the entire gel slice and grinding it into a powder. Injections using the whole gel fragment should only be used with larger animals such as rabbits. For mice or other small animals, electroelution or electrophoretic transfer of the protein should be used to prepare the protein for injection. In the latter method, the protein is transferred to a suitable membrane, such as nitrocellulose or PVDF. The location of the desired protein is identified by staining, and the protein band is then cut from the surrounding membrane, minced, and dissolved in a small amount of dimethyl sulfoxide before subcutaneous injection into the animal.

Transfected Dendritic Cell Immunizations.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). An efficient way of generating antibodies is to introduce antigens of interest into DCs and then inject them into the host. This will result in initiation of an antigen-specific immune response mediated by T-cell immunity. Apoptosis of DCs expressing transgenic proteins results in enhanced immunity through cross-presentation by endogenous DCs in vivo. The major advantage of this technology is prolonged presentation of antigens that are synthesized endogenously and their presentation by both modified DCs as well as endogenous DCs to the immune system of the host.

Preparing Antigens Using a Baculovirus Expression System.

Baculovirus-produced recombinant proteins circumvent many of the issues that limit bacterial protein production. Baculoviruses contain double-stranded, circular, supercoiled DNA in a rod-shaped capsid. The viral life cycle includes three major phases: (1) early (or virus synthesis) phase (0.5-6 h after infection), which includes viral attachment, uncoating early viral gene expression, and shutting off host gene expression; (2) late phase (6-12 h after infection), which includes viral replication and formation of budded virus containing host plasma membrane and glycoprotein (gp)64, which is required for virus entry; and (3) very late phase (24-96 h after infection), which includes formation of polyhedral inclusion bodies and cell lysis. Large proteins can thus be produced readily in abundant quantities. Furthermore, posttranslational modifications of the protein similar to those of mammals (such as phosphorylation, disulfide-bond formation, and even glycosylation) will occur; indeed, because typical baculovirus constructs produce secreted proteins, most products will be glycosylated. Baculovirus expression systems are safe, because baculoviruses are nonpathogenic to mammals and plants, and insect cells can be grown easily in suspension culture, which makes protein production easy to scale up. Counterbalancing this approach, however, is the need to perform a two-step process: first cloning the gene into the recombination donor vector and then generating the viral construct. At that point, the virus can be used as a mixture or plaque-purified. Insect cells are then infected with the virus, and the proteins are produced.

Making Weak Antigens Strong: Cross-Linking Peptides to KLH with Maleimide.

Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. KLH-hapten conjugates are commonly used in antibody generation in a variety of hosts such as mice, rats, and rabbits. Because KLH is harvested from mollusks, it is physiologically distant from mammalian species and less likely to produce antibodies that cross-react mammalian antigens. Maleimide activation of carrier proteins makes it possible to conjugate sulfhydryl-containing haptens, and hence this chemistry is widely used for conjugating KLH with haptens.

Making Weak Antigens Strong: Modifying Antigens by Dinitrophenol or Arsynyl Coupling.

Many compounds on their own do not have all of the properties needed to induce a strong antibody response. However, small changes in the structure of an antigen can often greatly alter the immunogenicity of a compound. Common methods for doing so include the addition of small modifying groups such as dinitrophenol or arsenate to the molecules. These techniques either alter regions of the immunogen to provide better sites for T-cell binding or expose new epitopes for B-cell binding. The techniques are rapid and easy, and have been used extensively as a general procedure to increase the chances of raising antisera, particularly against well-conserved antigens.

Making Weak Antigens Strong: Modifying Protein Antigens by Denaturation.

Many molecules can be made more immunogenic by denaturation. This treatment will change the structure of many compounds, particularly proteins, and expose new epitopes. In addition, heating will often cause protein antigens to aggregate, and, because aggregated antigens are often more immunogenic, this can increase the antibody response. Either heating alone or heating with sodium dodecyl sulfate (SDS) is the usual treatment. Injecting denatured antigens will be more likely to produce an antibody response against epitopes that are not found on the native antigen. If antibodies against totally denatured proteins are desired, the SDS-heat treatment is normally best. These treatments will generate antibodies that should be particularly good for immunoblots, for screening bacterial expression libraries, and for immunoprecipitation of proteins synthesized during in vitro translations.

Developing a Phosphospecific IHC Assay as a Predictive Biomarker for Topoisomerase I Inhibitors.

Phosphorylation is the most extensively studied posttranslational modification of proteins. There are approximately 500 kinases known in the human genome. The kinase-activated pathways regulate almost every aspect of cell function and a deregulated kinase cascade leads to impaired cellular function. Impaired regulation of several kinase cascades, including the epidermal growth factor receptor (EGFR) pathway, leading to tumor pathogenesis, is well documented. Thus, a phosphospecific test with prognostic or predictive value was expected in oncology. However, no phosphospecific IHC test is used in oncology clinics. Human topoisomerase I (topoI) inhibitors, camptothecin and its analogues (CPT), are used extensively to treat various solid tumors. Depending on tumor type, the response rate is only 13-32%. We have demonstrated that the deregulated kinase cascade is at the core of CPT resistance. DNA-PKcs, a kinase central to the DNA-double-strand break (DSB) response pathway, phosphorylates topoI at serine 10 (topoI-pS10), and cells with higher basal levels of topoI-pS10 degrade topoI rapidly and are resistant to this class of drug. The higher basal level of topoI phosphorylation is due to continual activation of DNA-PKcs, and one potential mechanism of this pathway activation is failure of upstream effector phosphatases such as phosphatase and tensin homolog (PTEN). Based on this understanding, we have developed an IHC-based test (P-topoIDx) that can stratify the responder and non-responder patient population.

Cross-reactivity of the BRAF VE1 antibody with epitopes in axonemal dyneins leads to staining of cilia.

Antibodies that recognize neo-epitopes in tumor cells are valuable tools in the evaluation of tissue biopsy or resection specimens. The VE1 antibody that recognizes the V600E-mutant BRAF protein is one such example. We have recently shown that the vast majority of papillary craniopharyngiomas-tumors that arise in the sellar or suprasellar regions of the brain-harbor BRAF V600E mutations. The VE1 antibody can be effective in discriminating papillary craniopharyngioma from adamantinomatous craniopharyngioma, which harbors mutations in CTNNB1 and not BRAF. While further characterizing the use of the VE1 antibody in the differential diagnosis of suprasellar lesions, we found that the VE1 antibody stains the epithelial cells lining Rathke's cleft cysts with very strong staining of the cilia of these cells. We used targeted sequencing to show that Rathke's cleft cysts do not harbor the BRAF V600E mutation. Moreover, we found that the VE1 antibody reacts strongly with cilia in various structures-the bronchial airways, the fallopian tubes, the nasopharynx, and the epididymis-as well as with the flagella of sperm. In addition, VE1 reacts strongly with the cilia of the ependymal lining of the brain and with the cilia-containing microlumens of ependymoma tumors. There is significant sequence homology between the synthetic peptide (amino acid 596-606 of BRAF V600E: GLATEKSRWSG) that was used to generate the VE1 antibody and regions of multiple axonemal dynein heavy chain proteins (eg, DNAH2, DNAH7, and DNAH12). These proteins are major components of the axonemes of cilia and flagella where they drive the sliding of microtubules. In ELISA assays, we show that the VE1 antibody recognizes epitopes from these proteins. A familiarity with the cross-reactivity of the VE1 antibody with epitopes of proteins in cilia is of value when evaluating tissues stained with this important clinical antibody.

No evidence for interference of h&e staining in DNA testing: usefulness of DNA extraction from H&E-stained archival tissue sections.

Although histochemical staining has been believed to inhibit the DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of H&E staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained, and 10 H&E-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among the 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis, in DNA amplification by real-time polymerase chain reaction (PCR), in microsatellite PCR fragment analyses, or in a PCR-pyrosequencing assay. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on more than 1,300 colorectal cancers using DNA extracted from H&E-stained tissue sections. Our data provide no evidence for an interfering effect of H&E staining on DNA testing, suggesting that DNA from H&E-stained sections can be effectively used for routine DNA testing.

Functional interaction of plasmacytoid dendritic cells with multiple myeloma cells: a therapeutic target.

Multiple myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile, and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting toll-like receptors with CpG oligodeoxynucleotides both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM.

Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma.

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138(+) MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib-induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-kappaB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma.

Extensive gene promoter methylation in colorectal carcinoma has been termed the CpG island methylator phenotype (CIMP). Previous studies on CIMP used primarily methylation-specific polymerase chain reaction (PCR), which, unfortunately, may detect low levels of methylation that has little or no biological significance. Utilizing quantitative real-time PCR (MethyLight), we measured DNA methylation in a panel of 5 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 459 colorectal carcinomas obtained from 2 large prospective cohort studies. CIMP was defined as tumors that showed methylation in >or=4/5 promoters. CIMP was significantly associated with the presence of mucinous or signet ring cell morphology, marked Crohn's-like lymphoid reaction, tumor infiltrating lymphocytes, marked peritumoral lymphocytic reaction, tumor necrosis, tumor cell sheeting, and poor differentiation. All these features have previously been associated with microsatellite instability (MSI). Therefore, we divided the 459 colorectal carcinomas into 6 subtypes, namely, MSI-high (MSI-H)/CIMP, MSI-H/non-CIMP, MSI-low (MSI-L)/CIMP, MSI-L/non-CIMP, microsatellite stable/CIMP, and micro satellite sstable/non-CIMP. Compared with MSI-H/non-CIMP, MSI-H/CIMP was associated with marked tumor infiltrating lymphocytes, tumor necrosis, sheeting, and poor differentiation (all P