Development Of Human Monoclonal Antibodies Research Articles

Intricacies in the Preparation of Patient Doses of [177Lu]Lu-Rituximab and [177Lu]Lu-Trastuzumab Using Low Specific Activity [177Lu]LuCl3: Methodological Aspects.

The development of humanized monoclonal antibodies (MAbs) with Lutetium-177 ([177Lu]Lu3+) has brought a paradigm shift in the arena of targeted therapy of various cancers. [177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab have gained prominence due to their improved therapeutic efficacy in the treatment of lymphoma and breast cancer. The clinical dose formulation of these radiolabeled MAbs, using low specific activity [177Lu]LuCl3, requires extensive optimization of the radiolabeling protocol. The present study merits the development of a single protocol which has been optimized for conjugation of Rituximab and Trastuzumab with p-NCS-benzyl-DOTA and further radiolabeling these immunoconjugates (ICs) with low specific activity [177Lu]LuCl3.Herein, we report a consistent and reproducible protocol for clinical dose formulations of [177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab (~9.25 GBq each, equivalent to ~2 patient doses) with radiochemical yield (RCY) between 84 and 86% and radiochemical purities (RCP) >99%. The in vitro stabilities of both these radioimmunoconjugates (RICs) were retained up to 120 h post-radiolabeling, upon storage with L-ascorbic acid as stabilizer (concentration: ~ 220-240 μg/37MBq) at -20 °C.The ready-to-use formulation of clinical doses[177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab has been successfully achieved by employing a single optimized protocol. While [177Lu]Lu-DOTA-Rituximab has exhibited a high degree of localization in retroperitoneal nodal mass of refractory lymphoma patient, high uptake of [177Lu]Lu-DOTA-Trastuzumab has been observed in metastatic breast carcinoma patient with multiple skeletal metastases.

Phage Display Technology as a Powerful Platform for Antibody Drug Discovery.

Antibody drugs with a high affinity and specificity are effective and safe for intractable diseases, such as cancers and autoimmune diseases. Furthermore, they have played a central role in drug discovery, currently accounting for eight of the top 20 pharmaceutical products worldwide by sales. Forty years ago, clinical trials on antibody drugs that were thought to be a magic bullet failed, partly due to the immunogenicity of monoclonal antibodies produced in mice. The recent breakthrough in antibody drugs is largely because of the contribution of phage display technology. Here, we reviewed the importance of phage display technology as a powerful platform for antibody drug discovery from various perspectives, such as the development of human monoclonal antibodies, affinity enhancement of monoclonal antibodies, and the identification of therapeutic targets for antibody drugs.

Human Monoclonal Antibodies: On the Menu of Targeted Therapeutics Against COVID-19.

Coronavirus disease 2019 (COVID-19), reminiscent of the severe acute respiratory syndrome (SARS) outbreak in 2003, has been a tragic disaster to people all over the world. As there is no specific drug for COVID-19, neutralizing antibodies are attracting more and more attention as one of the most effective means to combat the pandemic. Here, we introduced the etiological and serological characteristics of COVID-19, discussed the current stage of development of human monoclonal antibodies against SARS-CoV-2 and summarized the antigenic epitopes in the S glycoprotein, which may deepen the understanding of the profile of immune recognition and response against SARS-CoV-2 and provide insight for the design of effective vaccines and antibody-based therapies.

Live unattenuated vaccines for controlling viral diseases, including COVID‐19

Live unattenuated vaccines (LUVs) have been neglected for decades, due to widespread prejudice against their safety, even though they have successfully controlled yellow fever and adenovirus infection in humans as well as rinderpest and infectious bursal disease in animals. This review elucidated that LUVs could be highly safe with selective use of neutralizing antivirus antibodies, natural antiglycan antibodies, nonantibody antivirals, and ectopic inoculation. Also, LUVs could be of high efficacy, high development speed, and high production efficiency, with the development of humanized monoclonal antibodies and other modern technologies. They could circumvent antibody‐dependent enhancement and maternal‐derived antibody interference. With these important advantages, LUVs could be more powerful than other vaccines for controlling some viral diseases, and they warrant urgent investigation with animal experiments and clinical trials for defeating the COVID‐19 pandemic caused by the novel coronavirus SARS‐CoV‐2.

Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell–cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.

Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus.

The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens.IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis ("strep throat") to deadly necrotizing fasciitis (sometimes called "flesh-eating" disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.

PF445 INHIBITION OF ACUTE LYMPHOBLASTIC AND MYELOID LEUKEMIAS BY NOVEL CHIMERIC MOUSE‐HUMAN MONOCLONAL ANTIBODIES AGAINST ANTIGEN PRAME

Background:A number of the novel immunotherapy approaches revolutionized leukemia treatment. Among most striking ones are CAR‐T and bispecific antibodies targeting CD19 and monoclonal antibodies inhibiting immune checkpoints pathways PD‐1 and CTLA4. Nevertheless, anti‐leukemia immunotherapy is still a challenge due to the lack of suitable leukemia cell surface targets. As a matter of fact, a unique and “table breaking” target molecule CD19 is restricted only by B‐cell lineage making it rather frustrating to find a similar fulcrum in the case of myeloid and T‐cell malignancies. There are a few studies suggesting that frequently activated cancer testis antigen PRAME is not only located within the inner tumor cell compartments but also on the surface of the tumor cells including leukemia cells. We have sought to estimate the value of the tumor cell surface antigen PRAME to be a target of therapeutic monoclonal antibodies.Aims:To evaluate anti‐leukemia activity of the novel chimeric monoclonal mouse‐human antibodies.Methods:recPRAME has been used to develop a number of mouse hybridomas secreting highly specific and affine anti‐PRAME moAbs. 11 of mouse anti‐PRAME moAbs have been tested to evaluate their PRAME‐expressing anti‐leukemia/solid tumor cell lines growth inhibiting activity. Mouse hybridomas producing most efficient anti‐PRAME moAbs to inhibit tumors have been used as a basis to develop chimeric mouse‐human anti‐PRAME moAbs. Gene loci encoding VDJ domains of light and heavy chains of immunoglobulins have been sequenced and corresponding alignments studied. Proteomic MALDI analysis of chimeric mouse‐human anti‐PRAME moAbs has been performed and its result has been compared with data of RNA sequencing of immunoglobulin genes. Leukemic/solid tumor cell lines inhibiting activity of chimeric mouse‐human anti‐PRAME moAbs has been evaluated by means of MMT‐test, flow cytometry and xCELLigence dp. Among cell lines tested were K562, THP1, NOMO1, HT29, WI38, WI38 transfected by PRAME, Raji, Vero, a number of cell lines obtained from melanoma pts, and some others.Results:Distinct ability of chimeric mouse‐human anti‐PRAME moAbs to inhibit growth of PRAME expressing leukemia and solid tumor cells has been revealed. The most sensitive cell lines occured to be K562, NOMO1 and HT29. Negative for PRAME expression line of human fetal fibroblasts WI38 was resistant against anti‐PRAME chimeric moABs. Transfection of PRAME expressing gene construct into WI38 renders it to be sensitive of anti‐PRAME chimeric moABs. The more PRAME was expressed by tumor cells, the more was the observed inhibiting effect. It turned out that one of PRAME epitopes on the surface of leukemic and solid tumor cells is most susceptible as an antitumor target of monoclonal antibodies.Summary/Conclusion:We have developed novel chimeric mouse‐human monoclonal antibodies targeting PRAME antigen to efficiently inhibit growth of lymphoblastic and myeloid leukemia cells lines as well as solid tumor cell lines provided that they express PRAME gene. Our data suggest that PRAME antigen is not only presented on the surface of the PRAME expressing tumor cells but is a very useful target for antitumor drugs as it was shown in the case of PRAME‐binding monoclonal antibodies. We believe that our chimeric mouse‐human anti‐PRAME monoclonal antibodies are a valuable basis for development of humanized monoclonal antibodies and CAR‐T targeting PRAME in leukemias and other PRAME‐positive tumors. This research was supported by Ministry of Science and Higher Education of the Russian Federation (unique project identifier RFMEFI60418X0204)image

647. Characterization and Development of Human Monoclonal Antibodies to Pneumococcal Serotype 3

Background Streptococcus pneumoniae is the main bacterial cause of pneumonia in the United States and globally. Although pneumococcal conjugate vaccines are highly effective against invasive pneumococcal disease, they are less effective against pneumonia, particularly in the elderly and those with immune deficiency. Given the additional challenge of antibiotic resistance, immunotherapy holds promise for treatment of pneumococcal pneumonia. The current PCV13 vaccine is less effective against serotype (ST) 3, which carries a higher risk of mortality than other vaccine-included STs. Our group has previously identified murine monoclonal antibodies (mAb) to ST3 capsular polysaccharide (PPS3) that are protective in experimental models of sepsis and pneumonia. The aim of the present study is to isolate and develop PPS3-specific human monoclonal antibodies (humAbs) as adjunctive immunotherapy for pneumonia.MethodsWe sorted individual PPS3-specific memory B cells from PBMCs isolated on days 0 and 7 post-vaccination from pneumococcal polysaccharide (PPS)-based vaccine (Pneumovax or Prevnar13) recipients using fluorescently labeled PPS3. Immunoglobulin heavy (Igh) and light (Igl) chains were sequenced, cloned into IgG1 and κ or λ vectors, and expressed in HEK-293 cells. PPS3 specificity was confirmed using ELISA.ResultsHere, we report the first 7 PPS3-specific humAbs isolated: 5 used lambda light chains and two used kappa light chains. Six of these humAbs used variable heavy 3 (VH3) Igh gene elements. Kappa humAbs used VH3-30 or VH3-7, whilst lambda humAbs used VH3-9, VH3-72 or VH3-23. Sequence analysis revealed somatic mutations in complementary determining as well as framework regions. Initial studies show that some humAbs agglutinate ST3 in vitro. Structure-function relationship studies are ongoing to identify specific determinants of PPS3 binding and biological efficacy against ST3 in vitro and in vivo.ConclusionThe results of this study provide further understanding of the biology of PPS3 antibodies and may facilitate design of adjunctive immunotherapy to treat and prevent ST3 disease.Disclosures All authors: No reported disclosures.

The development of a synthetic scFv monoclonal antibody targeting pro-oncogenic AGR2

Event Abstract Back to Event The development of a synthetic scFv monoclonal antibody targeting pro-oncogenic AGR2 M. Aiman Mohtar1*, Teck Yew Low1, Borek Vojtesek2, Rahman Jamal1 and Ted Hupp3 1 UKM Medical Molecular Biology Institute (UMBI), Malaysia 2 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Czechia 3 Medical Research Council Institute of Genetics and Molecular Medicine (MRC), United Kingdom Background AGR2 is an endoplasmic reticulum (ER)-resident protein that belongs to the protein disulphide isomerase (PDI) family. These enzymes facilitate the rearrangement of disulphide bonds during protein maturation in the ER. AGR2 has emerged as a clinically relevant anti-tumour target since it possesses pro-oncogenic features, promotes metastasis and is overexpressed in diverse types of cancer. One unique molecular function of AGR2 is to drive certain oncogenic client proteins maturation in the ER, such as receptor EpCAM, via a specialised docking site. Methods and Results We developed synthetic single-chain variable fragment (scFv) monoclonal antibodies targeting AGR2. First, recombinant AGR2 protein was used as an antigen for screening a naïve canine phage-scFv library for specific scFvs. After four rounds of screening, nine AGR2-binding scFvs were identified; while subsequent epitope mapping highlighted the presence of four different classes of scFvs. We next selected one scFv that binds strongly to the N-terminal of AGR2 (scFv4) for further studies. We re-designed scFv4 such that the new scFv4 construct contains a C-terminal CD20 epitope tag, and ER postcodes for entry into ER as tools to manipulate the proteins inside the ER. Although all synthetic scFv scaffolds were expressed in AGR2-negative cells, the AGR2-binding scFv with an ER-postcode was selectively degraded in AGR2-positive cells. These data suggest that the expression of scFv scaffolds can be impacted in an AGR2-dependent manner and the presence of AGR2 in cells can suppress the production of a high-affinity binding protein when it is destined for the ER. Conclusion This study demonstrates the successful construction of a novel AGR2-binding scFv scaffold that can be expressed in mammalian cells. These tools were used to show the key determinants that regulate the ability of AGR2 to mediate client protein maturation in the ER and can be potentially manipulated for the development of human monoclonal antibodies for therapeutic treatment of cancer. Keywords: Secretory Pathway, Protein disulfide isomerase (PDI), Endoplasmic Reticulum, Chaperone, Anti cancer Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Mohtar M, Low T, Vojtesek B, Jamal R and Hupp T (2019). The development of a synthetic scFv monoclonal antibody targeting pro-oncogenic AGR2. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00128 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. M. Aiman Mohtar, UKM Medical Molecular Biology Institute (UMBI), Kuala Lumpur, Malaysia, m.aimanmohtar@ppukm.ukm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers M. Aiman Mohtar Teck Yew Low Borek Vojtesek Rahman Jamal Ted Hupp Google M. Aiman Mohtar Teck Yew Low Borek Vojtesek Rahman Jamal Ted Hupp Google Scholar M. Aiman Mohtar Teck Yew Low Borek Vojtesek Rahman Jamal Ted Hupp PubMed M. Aiman Mohtar Teck Yew Low Borek Vojtesek Rahman Jamal Ted Hupp Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Emerging infectious diseases: A proactive approach

Infectious diseases are now emerging or reemerging almost every year. This trend will continue because a number of factors, including the increased global population, aging, travel, urbanization, and climate change, favor the emergence, evolution, and spread of new pathogens. The approach used so far for emerging infectious diseases (EIDs) does not work from the technical point of view, and it is not sustainable. However, the advent of platform technologies offers vaccine manufacturers an opportunity to develop new vaccines faster and to reduce the investment to build manufacturing facilities, in addition to allowing for the possible streamlining of regulatory processes. The new technologies also make possible the rapid development of human monoclonal antibodies that could become a potent immediate response to an emergency. So far, several proposals to approach EIDs have been made independently by scientists, the private sector, national governments, and international organizations such as the World Health Organization (WHO). While each of them has merit, there is a need for a global governance that is capable of taking a strong leadership role and making it attractive to all partners to come to the same table and to coordinate the global approach.

PCSK9 inhibitors.

The discovery of proprotein convertase subtilisin kexin 9 (PCSK9) has considerably changed the therapeutic options in the field of lipid management. PCSK9 reduces LDL receptor recycling, leading to a decrease of low-density lipoprotein cholesterol (LDL-C) receptors on the surface of hepatocytes and a subsequent increase of circulating LDL-C levels. In observational studies, the loss-of-function mutations of PCSK9 have been associated with a reduction of LDL-C levels and cardiovascular disease (CVD) events. In contrast, humans with high levels of PCSK9 have higher level of plasma LDL-C and significantly enhanced CVD risk during their lifetime, gain-of-function mutations on PCSK9 are, for instance, causatively associated with familial hypercholesterolaemia (FH). Inhibition of PCSK9 is therefore a promising therapeutic option for the lowering of LDL-C levels. The clinical development of human monoclonal antibodies against PCSK9 has progressed, with promising results reported from phase 2 clinical studies in patients with FH or intolerant to statin with LDL-C levels not on target levels. Phase I studies demonstrated safety and efficacy. In phase II, a 60%-70% reduction in LDL-C was observed, especially when subcutaneous injections were performed regularly every two weeks. No significant side effects were observed, with the exception of injection site reactions. Three large phase III programmes with the new anti PCSK9 antibodies are currently underway in patients with acute coronary syndrome (ACS) and LDL-C inadequately controlled by standard treatments. The main objective of these studies is to evaluate the effect of PCSK9 inhibition on the occurrence of CVD events in patients with ACS.

Development of humanized monoclonal antibody by logical approach: Characterization, functional studies in vitro and immunogenicity studies in vivo in non-human primates

Monoclonal antibodies (mAbs) are widely used due to their exquisite specificity and once were hailed as the solution to cancer. However, when mouse mAbs are administered in humans, anti-antibody response (AAR) is frequently observed. Using humanized mAbs which are commonly developed by CDR-grafting method, the AAR is negligible, but loss of binding has also been consistently reported. Therefore, in an effort to produce humanized anti-C2 mAbs that retain binding properties but produce minimal AAR, humanized mAb has been developed by logical approach using IgBLAST. The purity and functionality of humanized mAb was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and cell-based assays, respectively. Although the humanized mAb developed using logical approach had reduced AAR compared to mouse anti-C2 mAb in Macaca fascicularis, however the AAR was higher, compared to humanized mAb developed using deimmunization method. Hence, for the development of functional humanized mAbs with negligible AAR, it is recommended that amphipathic mouse residues, excluding those located in the CDR or Vernier zone, be chosen for humanization. However, humanization of every amphipathic mouse residues is unnecessary because with minimal judicious amino acid substitutions, an AAR response can be minimized without jeopardizing the immunoreactivity, hence making it ideal for use in human therapeutics. Key words: Antibody humanization, cell-based assay, immunogenicity, Macaca fascicularis, site-directed overlapping-PCR mutagenesis.

Characterization of a monoclonal anti-capsid antibody that cross-reacts with three major primate lentivirus lineages

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24–27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm. Using overlapping peptides, the AG3.0 epitope was mapped in capsid to a sequence (SPRTLNA) conserved among HIV-1, HIV-2, SIVrcm, SIVsm/mac, and SIVagm related viruses. Because of its broad cross-reactivity, AG3.0 was used to develop an antigen capture assay with a lower detection limit of 100pg/ml HIV-1 Gag p24. Interestingly, AG3.0 was found to have a faster binding on/off rate for SIVagmVer and SIVmac Gag than for SIVagmSab Gag, possibly due to differences outside the SPRTLNA motif. In addition, the ribonucleic acid (RNA) coding for AG3.0 was sequenced to facilitate the development of humanized monoclonal antibodies.

Properties and Therapeutic Efficacy of Broadly Reactive Chimeric and Humanized H5-Specific Monoclonal Antibodies against H5N1 Influenza Viruses

Highly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.

Inhibitors of B7–CD28 Costimulation in Urologic Malignancies

T-cell costimulatory molecules deliver positive or negative signals to govern the ultimate fate of immune responses. These ligands and receptors that negatively costimulate T cells (including cytotoxic T-lymphocyte antigen [CTLA]-4, B7-H1, programmed death [PD]-1, B7-H3 and B7x) have received significant interest recently owing to their proposed ability to form a molecular shield for tumor cells. CTLA-4 represents the most extensively studied receptor in the costimulatory pathway and functions as a potent inhibitor of T-cell-mediated immunity. Clinical trials with anti-CTLA-4 are ongoing, although numerous objective responses have been observed in heavily pretreated patients, albeit with autoimmune side effects. In renal cell carcinoma, B7-H1, PD-1 and B7x have been observed to be expressed on tumor cells or infiltrating lymphocytes and are individually associated with adverse pathologic features and poor clinical outcome. In prostate cancer, B7-H3 and B7x immunostaining intensity correlate with disease spread, clinical cancer recurrence and cancer-specific death. External validation and prospective studies are now needed to confirm these findings, while further development of humanized monoclonal antibodies, similar to the experience with anti-CTLA-4, are underway. Herein, we review the B7-CD28 family as it applies to urologic malignancies.

B cell responses to HIV and the development of human monoclonal antibodies.

In this review B cell responses in HIV-infected individuals are summarized together with the techniques used to date to produce human monoclonals to HIV and the properties of these antibodies. Profound disturbances in B cell responses are apparent both in vivo and in vitro. While there is evidence in vivo of marked polyclonal B cell activation, primary and secondary antibody responses are impaired. Similarly these cells exhibit spontaneous immunoglobulin secretion upon in vitro culture but do not readily respond to B cell mitogens and recall antigens including HIV. Furthermore, certain of these defects can be reproduced in normal B cells in vitro by incubation with HIV or HIV coded peptides. Individuals infected with HIV develop antibodies to HIV structural proteins (e.g. p17, p24, gp41 and gp120) and regulatory proteins (e.g. vif, nef, RT). Autoantibodies against a number of immunologically important molecules are also frequently observed. The anti-HIV antibodies are predominantly of the IgG1 isotype and exhibit a variety of effects on the virus in vitro. To date, using conventional immortalization strategies, an appreciable number of human monoclonals to HIV have been developed. These have been specific for gp41, gp120 and gag with antibodies of the former specificity predominating. The majority of these antibodies have been of the IgG1 isotype. Only a small number of the antibodies neutralize virus in vitro and most of these react with gp120. The neutralizing antibodies recognize conformational and carbohydrate epitopes or epitopes in amino acid positions 306-322. The predominant epitopes recognized by the anti-gp41 antibodies were in amino acid positions 579-620 and 644-662. A high percentage (congruent to 25%) of these antibodies enhance viral growth in vitro. The problems relating to the production of human monoclonals to HIV are discussed together with strategies that could be used in the future.

Targeting Nerve Growth Factor in Pain

Chronic pain presents a huge economic and social burden, with existing treatments largely unable to satisfy medical needs. Recently, studies have shown that nerve growth factor (NGF) is a major mediator of inflammatory and neuropathic pain, providing a new therapeutic target. Although originally discovered as a trophic factor for sympathetic and sensory neurons during development, it now appears that in adults, levels of NGF are elevated in many acute and chronic pain conditions. Furthermore, preclinical animal models of inflammatory and neuropathic pain also show increased NGF levels, while the sequestration of NGF alleviates the associated hyperalgesia. The molecular mechanisms involved are being elucidated. This review briefly examines pain signaling pathways and describes currently available analgesics. It then investigates the approaches taken in targeting NGF-mediated pain. Current options being explored include the development of humanized monoclonal antibodies to NGF or its tyrosine kinase receptor TrkA (also known as neurotrophic tyrosine kinase receptor, type 1 [NTRK1]), and the sequestration of NGF using TrkA domain 5 (TrkAd5), a soluble receptor protein that binds NGF with picomolar affinity. Administration of either antibodies or TrkAd5 has been shown to be effective in a number of preclinical models of pain, including cystitis, osteoarthritis, UV irradiation (sunburn), and skeletal bone pain due to fracture or cancer. Other possible future therapies examined in this review include small-molecule TrkA antagonists, which target either the extracellular NGF binding domain of TrkA or its intracellular tyrosine kinase domain.

Accelerated purification process development of monoclonal antibodies for shortening time to clinic: Design and case study of chromatography processes

For accelerating the purification process development of human monoclonal antibodies (hmAbs) for pharmaceutical drugs, we designed a standardized method for setting the conditions of the purification process, which could be applied to hmAbs for the early phase of pharmaceutical development. The process includes three sequential chromatography steps: Protein A affinity chromatography (AFC), anion-exchange chromatography (AIEC) and cation-exchange chromatography (CIEC), and also includes a low pH virus inactivation step after the AFC step. We predicted the elution pH in the AFC and elution salt concentration in the CIEC from the amino acid sequences of hmAbs, as described in our previous paper. The mobile phase pH in AIEC and the pH for virus inactivation were also predicted based on the amino acid sequence of hmAb. As a case study, six hmAbs (two of IgG 1, two of IgG 2 and two of IgG 4) were purified with the standardized method. The recovery, purity and clearance of impurities (DNA, host cell proteins (HCP), and Protein A) were examined. All the six hmAbs were purified with high recovery and high clearance of the impurities. Factors affecting the impurities level in the purified products are also discussed.

Development of human monoclonal antibodies against diseases caused by emerging and biodefense-related viruses

Polyclonal antibodies have a century-old history of being effective against some viruses; recently, monoclonal antibodies (mAbs) have also shown success. The humanized mAb Synagis® (palivizumab), which is still the only mAb against a viral disease approved by the US FDA, has been widely used as a prophylactic measure against respiratory syncytial virus infections in neonates and immunocompromised individuals. The first fully human mAbs against two other paramyxoviruses, Hendra and Nipah virus, which can cause high (up to 75%) mortality, were recently developed; one of them, m101, showed exceptional potency against infectious virus. In an amazing pace of research, several potent human mAbs targeting the severe acute respiratory syndrome coronavirus S glycoprotein that can affect infections in animal models have been developed months after the virus was identified in 2003. A potent humanized mAb with therapeutic potential was recently developed against the West Nile virus. The progress in developing neutralizing human mAbs against Ebola, Crimean–Congo hemorrhagic fever, vaccinia and other emerging and biodefense-related viruses is slow. A major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies, is the viruses’ heterogeneity and mutability. A related problem is the low binding affinity of crossreactive antibodies able to neutralize a variety of primary isolates. Combinations of mAbs or mAbs with other drugs, and/or the identification of potent new mAbs and their derivatives that target highly conserved viral structures, which are critical for virus entry into cells, are some of the possible solutions to these problems, and will continue to be a major focus of antiviral research.

Control of Eosinophil Toxicity in the Lung

The inappropriate accumulation of eosinophils and the subsequent release of their potent pro-inflammatory mediator arsenal are thought to be important contributors to the pathogenesis of asthma and other allergic diseases. It is also becoming apparent that eosinophils may play a role in the orchestration of immune responses in the asthmatic lung. There is therefore much interest in the development of strategies to limit or prevent eosinophil-induced toxicity. The mechanisms by which eosinophils accumulate in the peribronchial tissues of the lung are complex and include enhanced differentiation and release from the bone marrow, selective adhesion and transendothelial migration, directed movement in response to specific chemotactic mediators and finally prolonged survival as a consequence of delayed apoptosis. Thus it can be appreciated that there are many points at which the toxicity of eosinophils can be limited or even prevented. Important areas for potential advances in glucocorticoid (GC) development include efforts to dissociate their anti-inflammatory effects from unwanted side effects. Other areas include the development of humanized monoclonal antibodies against IL-4, IL-13 and IL-5 together with the inhibition of adhesion pathways and/or chemokines responsible for eosinophil accumulation in the asthmatic lung. Several avenues of research are currently underway in an attempt to define mechanisms by which pro-inflammatory cells such as eosinophils can be safely removed from the asthmatic lung through apoptosis induction and their subsequent ingestion by phagocytes. This review will discuss both the potential and shortcomings of these diverse approaches to limit eosinophil toxicity in the asthmatic lung.