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1.  The Therapeutic Value of Monoclonal Antibodies Directed Against Immunogenic Tumor Glycoproteins 
Journal of Cancer  2010;1:209-222.
Monoclonal antibodies developed against immunogenic proteins (Tumor Specific Antigens/TSA's) that are expressed in human cancers, display a unique behavioral pattern. They appear to serve in a dual role. This includes the early recognition of these immunogenic membrane proteins that can serve as diagnostic markers, and the targeting of such markers for the destruction of the tumor, primarily thru ADCC.
The monoclonals (mAbs) that we have developed against specific immunogenic tumor membrane proteins have been studied in detail. These tumor proteins, when first defined, were referred to as tumor associated antigens. With the ability of the mAbs to demonstrate therapeutic antitumor activity in those patients with relatively advanced malignancies, the term tumor specific was introduced. Monoclonals that we were able to develop from tumor specific proteins derived from colon and pancreas cancer were found capable of targeting those tumors to induce apoptosis. We were also able to define immunogenic membrane proteins from lung (squamous and adenoCa) as well as prostate neoplasms. Monoclonals developed from these tumor antigens are in the initial phases of investigation with regard to their specificity and antitumor activity.
Mabs capable of targeting the malignancies noted above were produced following immunization of BALBc mice with the Tumor Specific Antigens. The hybridomas that were screened and found to express the antibodies of interest appeared for the most part as IgG2a's. It became apparent after a short period of time that stability of the Fab CDR loops as well as the therapeutic efficacy of the hybridoma mAbs could be lost. Stability was achieved by chimerization and or humanization. The resulting mAbs were found to switch their isotypes to an IgG1 subsequent to chimerization and or humanization, when expressed in CHO cells. The monoclonals, so produced, were not only more efficient in controlling tumor growth but minimized the development of a HAMA response.
Because of 1) the specificity of this group of monoclonal antibodies in targeting well defined immunogenic proteins that were expressed on the tumor cell membrane,2) their lack of cross reactivity to normal tissue, 3) relatively low toxicity when delivered intravenously, 4) rapid targeting of tumor cell populations (4-6 hrs in vitro) and their 5) ability to destroy xenograft transplants (in vivo) within days of delivery, these mAbs were felt to be ideal for possible use in the treatment of patients with recurrent and or metastatic tumors.
Initial clinical studies have been planned for following the filing of an IND. It is required by FDA that the potential effects of tumor control and toxicity be defined using the naked antibodies produced under GMP conditions, In those situations where patients with recurrent malignancies are to be studied we have come to realize that a number of factors can influence the response to monoclonal therapy. This includes the amount of shed antigen in the serum at the time of treatment that could initiate immune complex formation as well as the shedding of inhibitory material into the serum possibly effecting an immune response. As such we plan to eventually employ the therapeutic mAbs in combination with chemotherapy as a means of enhancing the immunogenicity of the tumor system being treated and to possibly weaken the malignant growth for easier destruction by the mAb. We will also look at the combination of mAbs with immunostimulants such as GMCSF and IL-2 (fusion proteins) and eventual conjugation of the mAbs with alpha and possibly B-emitters to help in targeting bystander cells. The present paper reviews the potential therapeutic value of such mAbs in the treatment of recurrent malignancies, especially those having failed chemotherapy in established clinical trials.
PMCID: PMC2974238  PMID: 21060731
Monoclonal antibodies; hybridomas; chimeric antibodies; ADCC; apoptosis; tumor specific antigens (TSA).
2.  Trial Watch 
Oncoimmunology  2014;3:e27048.
In 1997, for the first time in history, a monoclonal antibody (mAb), i.e., the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug Administration for use in cancer patients. Since then, the panel of mAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has not stopped to expand, nowadays encompassing a stunning amount of 15 distinct molecules. This therapeutic armamentarium includes mAbs that target tumor-associated antigens, as well as molecules that interfere with tumor-stroma interactions or exert direct immunostimulatory effects. These three classes of mAbs exert antineoplastic activity via distinct mechanisms, which may or may not involve immune effectors other than the mAbs themselves. In previous issues of OncoImmunology, we provided a brief scientific background to the use of mAbs, all types confounded, in cancer therapy, and discussed the results of recent clinical trials investigating the safety and efficacy of this approach. Here, we focus on mAbs that primarily target malignant cells or their interactions with stromal components, as opposed to mAbs that mediate antineoplastic effects by activating the immune system. In particular, we discuss relevant clinical findings that have been published during the last 13 months as well as clinical trials that have been launched in the same period to investigate the therapeutic profile of hitherto investigational tumor-targeting mAbs.
doi:10.4161/onci.27048
PMCID: PMC3937194  PMID: 24605265
bevacizumab; brentuximab vedotin; cetuximab; nimotuzumab; trastuzumab; tumor-associated antigen
3.  Therapeutic monoclonal antibodies for multiple myeloma: an update and future perspectives 
Multiple myeloma (MM) still remains incurable in most of the patients. Despite of treatments with high-dose chemotherapy, stem cell transplantation and other novel therapies, most patients will become refractory to the therapies and relapse. Thus, it is urgent to develop new approaches for MM treatment. Currently, antibody-targeted therapy has been extensively utilized in hematological malignancies, including MM. Several novel monoclonal antibodies (mAbs) against MM have been generated and developed over the past several years. These mAbs aim to target not only tumor cells alone but also tumor microenvironment, including interaction of tumor-bone marrow stromal cells and the components of bone marrow milieu, such as cytokines or chemokines that support myeloma cell growth and survival. These include mAbs specific for CD38, CS1, CD40, CD74, CD70, HM1.24, interleukin-6 and β2-microglobulin (β2M). We have shown that anti-β2M mAbs may be a potential antitumor agent for MM therapy due to their remarkable efficacy to induce myeloma cell apoptosis in tumor cell lines and primary myeloma cells from patients in vitro and in established myeloma mouse models. In this article, we will review advances in the development and mechanisms of MM-targeted mAbs and especially, anti-β2M mAbs. We will also discuss the potential application of the mAbs as therapeutic agents to treat MM.
PMCID: PMC3207269  PMID: 22065141
Multiple myeloma; monoclonal antibodies; anti-β2M mAbs; therapy
4.  Therapeutic monoclonal antibodies for multiple myeloma: an update and future perspectives 
Multiple myeloma (MM) still remains incurable in most of the patients. Despite of treatments with high-dose chemotherapy, stem cell transplantation and other novel therapies, most patients will become refractory to the therapies and relapse. Thus, it is urgent to develop new approaches for MM treatment. Currently, antibody-targeted therapy has been extensively utilized in hematological malignancies, including MM. Several novel monoclonal antibodies (mAbs) against MM have been generated and developed over the past several years. These mAbs aim to target not only tumor cells alone but also tumor microenvironment, including interaction of tumor-bone marrow stromal cells and the components of bone marrow milieu, such as cytokines or chemokines that support myeloma cell growth and survival. These include mAbs specific for CD38, CS1, CD40, CD74, CD70, HM1.24, interleukin-6 and β2-microglobulin (β2M). We have shown that anti-β2M mAbs may be a potential antitumor agent for MM therapy due to their remarkable efficacy to induce myeloma cell apoptosis in tumor cell lines and primary myeloma cells from patients in vitro and in established myeloma mouse models. In this article, we will review advances in the development and mechanisms of MM-targeted mAbs and especially, anti-β2M mAbs. We will also discuss the potential application of the mAbs as therapeutic agents to treat MM.
PMCID: PMC3207269  PMID: 22065141
Multiple myeloma; monoclonal antibodies; anti-β2M mAbs; therapy
5.  Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue 
Diagnostic Pathology  2014;9:30.
Background
A T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 is a multifunctional molecule associated with various proteins such as adenosine deaminase, caveolin-1, CXCR4, collagen, and fibronectin, while playing an important role in the regulation of inflammatory responses and tumor biology. We have focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and have developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which is currently being evaluated in a phase I clinical trial for patients with CD26-expressing tumors, including malignant mesothelioma. Since detection of tumor CD26 expression is required for determining potential eligibility for YS110 therapy, the development of anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in the formalin-fixed paraffin-embedded tissues is critical.
Methods
To develop novel anti-CD26 mAbs capable of binding to the denatured CD26, we immunized mice with CD26 protein denatured in urea buffer. After the fusion of splenocytes and myeloma cells, the mAbs were screened for specific reactivity with human CD26 by flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry. The binding competitiveness of novel anti-CD26 mAbs with the humanized anti-CD26 mAb YS110 was also examined.
Results
We have succeeded in developing novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in formalin-fixed tissue sections with reliable clarity and intensity. Importantly, some of these mAbs exhibit no cross-reactivity with the humanized anti-CD26 mAb.
Conclusions
These novel mAbs are potentially useful as companion diagnostic agents to analyze CD26 expression in the clinical setting while advancing future CD26-related research.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5987140221097729
doi:10.1186/1746-1596-9-30
PMCID: PMC3944398  PMID: 24502396
CD26/dipeptidyl peptidase 4; Immunohistochemical staining; Companion diagnostic drug; Malignant mesothelioma; T cell costimulation
6.  Immunotherapy of malignant disease with tumor antigen (TA)-specific monoclonal antibodies: does its therapeutic efficacy require cooperation with TA-specific CTL? 
A few tumor antigen (TA)-specific monoclonal antibodies (mAb) have been approved by FDA for the treatment of several major malignant diseases and are commercially available. Once in the clinic, mAb have an average success rate of ~30% and are well tolerated. These results have changed the face of cancer therapy, bringing us closer to more specific and more effective biologic therapy of cancer. The challenge facing tumor immunologists at present is represented by the identification of the mechanism(s) underlying patients’ differential clinical response to mAb-based immunotherapy. This information is expected to lead to the development of criteria to select patients to be treated with mAb-based immunotherapy. In the past in vitro and in vivo evidence has shown that TA-specific mAb can mediate their therapeutic effect by inducing tumor cell apoptosis, inhibiting the targeted antigen function, blocking tumor cell signaling and/or mediating complement-or cell-dependent lysis of tumor cells. More recent evidence suggests that TA-specific mAb can induce TA-specific cytotoxic T cell responses by enhancing TA uptake by dendritic cells (DC) and cross-priming of T cells. In this manuscript, we briefly summarize the TA-specific mAb that have received FDA approval. Next we review the potential mechanisms underlying the therapeutic efficacy of TA-specific mAb with emphasis on the induction of TA-specific cellular immune responses and their potential to contribute to the clinical efficacy of TA-specific mAb-based immunotherapy. Lastly, we discuss the potential negative impact of immune escape mechanisms on the clinical efficacy of TA-specific mAb-based immunotherapy.
doi:10.1158/1078-0432.CCR-09-2345
PMCID: PMC2802861  PMID: 20028761
ADCC; antibody; cancer; complement; cytotoxic T lymphocyte; HLA antigen; Immunoglobin Fc receptor; immune escape; immunotherapy; monoclonal antibody; NK cell
7.  Immunology and Immunotherapy of Neuroblastoma 
Seminars in cancer biology  2011;21(4):229-237.
Purpose
This review demonstrates the importance of immunobiology and immunotherapy research for understanding and treating neuroblastoma.
Principal results
The first suggestions of immune system-neuroblastoma interactions came from in vitro experiments showing that lymphocytes from patients were cytotoxic for their own tumor cells and from evaluations of tumors from patients that showed infiltrations of immune system cells. With the development of monoclonal antibody (mAb) technology, a number of mAbs were generated against neuroblastoma cells lines and were used to define tumor associated antigens. Disialoganglioside (GD2) is one such antigen that is highly expressed by virtually all neuroblastoma cells and so is a useful target for both identification and treatment of tumor cells with mAbs. Preclinical research using in vitro and transplantable tumor models of neuroblastoma has demonstrated that cytotoxic T lymphocytes (CTLs) can specifically recognize and kill tumor cells as a result of vaccination or of genetic engineering that endows them with chimeric antigen receptors. However, CTL based clinical trials have not progressed beyond pilot and phase I studies. In contrast, anti-GD2 mAbs have been extensively studied and modified in pre-clinical experiments and have progressed from phase I through phase III clinical trials. Thus, the one proven beneficial immunotherapy for patients with high-risk neuroblastoma uses a chimeric anti-GD2 mAb combined with IL-2 and GM-CSF to treat patients after they have received intensive cyto-reductive chemotherapy, irradiation, and surgery. Ongoing pre-clinical and clinical research emphasizes vaccine, adoptive cell therapy, and mAb strategies. Recently it was shown that the neuroblastoma microenvironment is immunosuppressive and tumor growth promoting, and strategies to overcome this are being developed to enhance anti-tumor immunotherapy.
Conclusions
Our understanding of the immunobiology of neuroblastoma has increased immensely over the past 40 years, and clinical translation has shown that mAb based immunotherapy can contribute to improving treatment for high-risk patients. Continued immunobiology and pre-clinical therapeutic research will be translated into even more effective immunotherapeutic strategies that will be integrated with new cytotoxic drug and irradiation therapies to improve survival and quality of life for patients with high-risk neuroblastoma.
doi:10.1016/j.semcancer.2011.09.012
PMCID: PMC3658311  PMID: 21971567
neuroblastoma; immunology; immunotherapy; microenvironment; anti-GD2
8.  Combinatorial efficacy of anti-CS1 monoclonal antibody elotuzumab (HuLuc63) and bortezomib against multiple myeloma 
Molecular cancer therapeutics  2009;8(9):2616-2624.
Monoclonal antibody (mAb) therapy for multiple myeloma, a malignancy of plasma cells, has not been clinically efficacious in part due to a lack of appropriate targets. We recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, is currently in a Phase I clinical trial in relapsed/refractory myeloma. In this report we investigated whether the activity of elotuzumab could be enhanced by bortezomib, a reversible proteasome inhibitor with significant activity in myeloma. We first showed that elotuzumab could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model. We next showed that CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), both for OPM2 myeloma cells using natural killer (NK) or peripheral blood mononuclear cells (PBMC) from healthy donors and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo anti-tumor activity. These findings provide the rationale for a clinical trial combining elotuzumab and bortezomib, which will test the hypothesis that combining both drugs would result in enhanced immune lysis of myeloma by elotuzumab and direct targeting of myeloma by bortezomib.
doi:10.1158/1535-7163.MCT-09-0483
PMCID: PMC2748787  PMID: 19723891
multiple myeloma; CS1; bortezomib; antibody therapy; natural killer cells
9.  Targeting glioblastoma with NK cells and mAb against NG2/CSPG4 prolongs animal survival 
Oncotarget  2013;4(9):1527-1546.
Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated NK cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/ microglia (TAM) ex vivo. Taken together, these findings indicate that NK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.
PMCID: PMC3824525  PMID: 24127551
Microglia; NK cells; glioblastoma; immunotherapy; NG2/CSPG4
10.  A monoclonal antibody specific for a common determinant of the human T cell receptor gamma/delta directly activates CD3+WT31- lymphocytes to express their functional program(s) 
In an attempt to select mAbs specific for human TCR-gamma/delta, a polyclonal CD3+ 4-8-WT31- (TCR-gamma/delta+) cell line (MV1) was used for mice immunization. An mAb, termed BB3, reacted with MV1 cells but not with a large panel of CD3+ WT31+ (TCR-alpha/beta+) cell populations or clones. In addition, BB3 mAb reacted with the majority of CD3+ WT31- clones derived from six different donors. Double-color fluorescence experiments and FACS analysis showed that BB3+ cells were restricted to the CD3+ fraction of peripheral blood lymphocytes; in addition, in several donors the percentages (0.5-8% of total PBL) of BB3+ cells paralleled those of CD3+ WT31- cells. Surface molecules recognized by BB3 were susceptible to antibody-induced modulation; in addition, cell treatment with either BB3 or anti-CD3 mAb caused the simultaneous downregulation of the two molecules. That BB3 molecules are physically linked to CD3 antigen was further supported by immunoprecipitation experiments. Thus, under conditions that preserve the TCR-CD3 association, both BB3 and anti-CD3 mAb precipitated from 125I-labeled MV1 cells the same set of molecules. These consisted in the 18-28-kD CD3 molecules and in three bands of approximately 44, 42, and 38 kD under reducing conditions. When cell lysis was performed in 1% NP-40, the molecules immunoprecipitated by BB3 mAb were represented by an 80- kD band under nonreducing conditions, which resolved, under reducing conditions, in the three 44-, 42-, and 38-kD bands. Similar disulphide- linked forms of the TCR molecules were revealed in all of the other eight CD3+ WT31- BB3+ clones analyzed. Analysis of TCR molecules by electrophoresis (NEPHGE) showed that BB3 or anti-CD3 precipitated a 44- kD molecule displaying a basic PI (approximately 7.5) and two more acidic proteins (PI approximately 6) with a mol mass of 42 and 38 kD. Studies aimed to define whether stimuli directly acting on TCR- gamma/delta could induce CD3+ WT31- cell activation revealed that (a) In the presence of PMA, soluble BB3 mAb induced IL-2 production by MV1 cell line and by three other CD3+ WT31- BB3+ clones analyzed. (b) BB3 mAb-producing hybridoma used as triggering target, was efficiently lysed by CD3+ WT31- BB3+ effector cells (but not by CD3+ WT31+ BB3- conventional CTL). (c) Soluble BB3 mAb induced CD3+ WT31- BB3+ effector cells to lyse the Fc receptor-positive P815 target cells. (d) BB3-TCR- gamma/delta interaction on CD3+ WT31- BB3+ cells induced a rapid increase of [Ca2+]i levels, similar to that observed in response to anti-CD3 mAbs.
PMCID: PMC2188975  PMID: 2456364
11.  Combinatorial PD-1 Blockade and CD137 Activation Has Therapeutic Efficacy in Murine Cancer Models and Synergizes with Cisplatin 
PLoS ONE  2013;8(12):e84927.
There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 106 ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8+ T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8+ T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8+ T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical ‘translation’.
doi:10.1371/journal.pone.0084927
PMCID: PMC3868659  PMID: 24367702
12.  Combination Immune Therapies to Enhance Anti-Tumor Responses by NK Cells 
Natural killer (NK) cells are critical innate immune lymphocytes capable of destroying virally infected or cancerous cells through targeted cytotoxicity and further assisting in the immune response by releasing inflammatory cytokines. NK cells are thought to contribute to the process of tumor killing by certain therapeutic monoclonal antibodies (mAb) by directing antibody-dependent cellular cytotoxicity (ADCC) through FcγRIIIA (CD16). Numerous therapeutic mAb have been developed that target distinct cancer-specific cell markers and may direct NK cell-mediated ADCC. Recent therapeutic approaches have combined some of these cancer-specific mAb with additional strategies to optimize NK cell cytotoxicity. These include agonistic mAb targeting NK cell activating receptors and mAbs blocking NK cell inhibitory receptors to enhance NK cell functions. Furthermore, several drugs that can potentiate NK cell cytotoxicity through other mechanisms are being used in combination with therapeutic mAb. In this review, we examine the mechanisms employed by several promising agents used in combination therapies that enhance natural or Ab-dependent cytotoxicity of cancer cells by NK cells, with a focus on treatments for leukemia and multiple myeloma.
doi:10.3389/fimmu.2013.00481
PMCID: PMC3870292  PMID: 24391651
NK cells; immunotherapy of cancer; antibodies; monoclonal; ADCC; multiple myeloma
13.  Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma. 
British Journal of Cancer  1996;74(5):735-744.
The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma.
Images
PMCID: PMC2074712  PMID: 8795576
14.  Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition 
The Journal of Experimental Medicine  1990;172(6):1589-1598.
In previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3-CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183- NK cells. To this end, mice were immunized with the PE29 (CD3-CD16+GL183-) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) triggered the PE29 clone, but not a GL183+ clone used as a control. EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3-CD16+CD56+ cell populations (only less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183-; EB6+GL183+; EB6-GL183+; EB6-GL183-). Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROV-I, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA). Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6+GL183- or EB6+GL183+ clones). In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules. Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the EB6 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions. We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2188758  PMID: 2147946
15.  Anti-CD38 Antibody Therapy: Windows of Opportunity Yielded by the Functional Characteristics of the Target Molecule 
Molecular Medicine  2013;19(1):99-108.
In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule’s regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.
doi:10.2119/molmed.2013.00009
PMCID: PMC3667209  PMID: 23615966
16.  Trial Watch 
Oncoimmunology  2014;3:e27297.
Immunostimulatory monoclonal antibodies (mAbs) exert antineoplastic effects by eliciting a novel or reinstating a pre-existing antitumor immune response. Most often, immunostimulatory mAbs activate T lymphocytes or natural killer (NK) cells by inhibiting immunosuppressive receptors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1 (PDCD1, best known as PD-1), or by engaging co-stimulatory receptors, like CD40, tumor necrosis factor receptor superfamily, member 4 (TNFRSF4, best known as OX40) or TNFRSF18 (best known as GITR). The CTLA4-targeting mAb ipilimumab has been approved by the US Food and Drug Administration for use in patients with unresectable or metastatic melanoma in 2011. The therapeutic profile of ipilimumab other CTLA4-blocking mAbs, such as tremelimumab, is currently being assessed in subjects affected by a large panel of solid neoplasms. In the last few years, promising clinical results have also been obtained with nivolumab, a PD-1-targeting mAb formerly known as BMS-936558. Accordingly, the safety and efficacy of nivolumab and other PD-1-blocking molecules are being actively investigated. Finally, various clinical trials are underway to test the therapeutic potential of OX40- and GITR-activating mAbs. Here, we summarize recent findings on the therapeutic profile of immunostimulatory mAbs and discuss clinical trials that have been launched in the last 14 months to assess the therapeutic profile of these immunotherapeutic agents.
doi:10.4161/onci.27297
PMCID: PMC3961485  PMID: 24701370
CD137; checkpoint blockade; immunogenic chemotherapy; immunosuppression; lirilumab; IPH2101; PD-L1
17.  Increasing the Clinical Efficacy of NK and Antibody-Mediated Cancer Immunotherapy: Potential Predictors of Successful Clinical Outcome Based on Observations in High-Risk Neuroblastoma 
Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD2), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD2 mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD2 mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role.
doi:10.3389/fphar.2012.00091
PMCID: PMC3353262  PMID: 22623917
ADCC; KIR; FcR; neuroblastoma; immunocytokine; mAb; IL2
18.  Invited Article: Human natural autoantibodies in the treatment of neurologic disease 
Neurology  2009;72(14):1269-1276.
Naturally occurring autoantibodies are molecules that are part of the normal immunoglobulin repertoire. This review focuses on three distinct groups of human monoclonal antibodies (mAb). These are human natural autoantibodies that, when injected into an animal model of human disease, stimulate remyelination in CNS demyelinating diseases, protect neurons and extend neuronal processes in CNS axonal disorders, and activate immune dendritic cells to produce cytotoxic T cells to clear metastatic tumors. Natural autoantibodies react to self antigens and are of relatively low affinity. They are derived from germline immunoglobulin genes and are usually polyreactive. Our experiments demonstrated CNS entry by autoradiography of labeled mAb and by MRI. Remyelinating mAb rHIgM22 clusters beta-integrin and mouse mAb O4 recognizes sulfatide. Neuronal outgrowth mAbs sHIgM42 and sHIgM12 appear to target carbohydrates on the surface of neurons. The mAb sHIgM12 (B7-DC-Xab) also is promising as therapeutic against metastatic tumors. It functions by binding and cross-linking the antigen B7-DC on dendritic cells, inducing tumor-specific cytotoxic T cells. All these mAbs activate a transient increase in intracellular calcium, signal via NFκb, and prevent apoptosis. The mAbs engage downstream signaling events that induce the primary function of the cell (that is, remyelination for oligodendrocytes, axonal preservation and neurite extension for neurons, or antigen presentation for dendritic cells). Natural human auto mAbs are a potentially important therapeutic technique in combating a wide spectrum of disease processes.
GLOSSARY
= immunoglobulin;
= monoclonal antibodies;
= multiple sclerosis.
doi:10.1212/01.wnl.0000345662.05861.e4
PMCID: PMC2677483  PMID: 19349608
19.  Improving effector functions of antibodies for cancer treatment: Enhancing ADCC and CDC 
As platforms for therapeutic agents, monoclonal antibodies (MAbs) have already been approved, and several MAbs have demonstrated clinical effectiveness in a variety of malignancies. However, several issues have also been emerging in antibody therapy, such as high cost and insufficient drug action. Recently, to improve MAb activity in humans, effector functions have been subjects of focus, especially antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Extensive efforts have been made to enhance these effector functions of MAbs, and successful approaches have been reported by us and others, wherein the binding activity of MAbs to FcγRIIIa or C1q is increased by introducing amino acid mutations into heavy chain constant regions or through glyco-modification of Fc-linked oligosaccharides. In addition, one of the next approaches to optimizing therapeutic antibodies would be to combine multiple enhancing modifications into a single antibody platform to overcome the diverse mechanisms of clinical resistance of tumor cells. For this aim, we have recently developed a successful combination composed of ADCC-enhancing modification by the fucose depletion from Fc-linked oligosaccharides and CDC-enhancing modification by IgG1 and IgG3 isotype shuffling in heavy chains, which could be of great value for the development of third-generation antibody therapeutics.
PMCID: PMC2769226  PMID: 19920917
ADCC; CDC; effector functions; Fc oligosaccharides; IgG isotypes; nonfucosylated IgG
20.  Potential therapeutics specific to c-MET/RON receptor tyrosine kinases for molecular targeting in cancer therapy 
Acta Pharmacologica Sinica  2010;31(9):1181-1188.
Products of proto-oncogenes c-MET and RON belong to a subfamily of receptor tyrosine kinases that contribute significantly to tumorigenic progression. In primary tumors, altered c-MET/RON expression transduces signals regulating invasive growth that is characterized by cell migration and matrix invasion. These pathogenic features provide the basis for targeting c-MET/RON in cancer therapy. In the last decade, various approaches have been investigated to suppress c-MET/RON-transduced oncogenesis. Among the therapeutics developed, monoclonal antibodies (mAbs) and small-molecule inhibitors (SMIs) have emerged as promising candidates. The mechanism of these therapeutic candidates is the disruption of tumor dependency on c-MET/RON signals for survival. The mAbs specific to hepatocyte growth factor (AMG102) and c-MET (MetMAb) are both humanized and able to block c-MET signaling, leading to inhibition of tumor cell proliferation in vitro and inhibition of tumor growth in xenograft models. The mAb AMG102 neutralizes hepatocyte growth factor and enhances the cytotoxicity of various chemotherapeutics to tumors in vivo. AMG102 is currently in phase II clinical trials for patients with advanced solid tumors. IMC-41A40 and Zt/f2 are RON-specific mAbs that down-regulate RON expression and inhibit ligand-induced phosphorylation. Both mAbs inhibit tumor growth in mice mediated by colon and pancreatic cancer cells. SMIs specific to c-MET (ARQ107 and PF-02341066) are in various phases of clinical trials. Therapeutic efficacy has also been observed with dual inhibitors such as Compound I, which is specific to c-MET/RON. However, a potential issue is the emergence of acquired resistance to these inhibitors. Clearly, development of c-MET/RON therapeutics provides opportunities and challenges for combating cancer in the future.
doi:10.1038/aps.2010.106
PMCID: PMC4002297  PMID: 20694025
receptor tyrosine kinase; therapeutic antibody; tyrosine kinase inhibitor; targeted cancer therapy; cellular mechanism; acquired resistance
21.  A Novel Monoclonal Antibody to Human Laminin α5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521 
PLoS ONE  2013;8(1):e53648.
Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
doi:10.1371/journal.pone.0053648
PMCID: PMC3538678  PMID: 23308268
22.  Engineering and Characterization of a Mouse/Human Chimeric Anti-Phencyclidine Monoclonal Antibody 
Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human κ and IgG2 constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5’s PCP-binding variable domains and human κ and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a KD of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a “more human” version of murine mAb6B5, retains mAb6B5’s unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse – introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.
doi:10.1016/j.intimp.2007.09.020
PMCID: PMC2238695  PMID: 18068094
Phencyclidine; Arylcyclohexylamines; Substance abuse; Therapeutic antibody; Chimeric antibody
23.  Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia 
Molecular Cancer  2011;10:42.
Background
The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells.
Results
The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5.
Conclusions
Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.
doi:10.1186/1476-4598-10-42
PMCID: PMC3103468  PMID: 21504579
24.  Prophylactic and Therapeutic Efficacy of Human Monoclonal Antibodies against H5N1 Influenza 
PLoS Medicine  2007;4(5):e178.
Background
New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.
Methods and Findings
Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).
Conclusions
These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.
Cameron Simmons and colleagues provide proof of concept that human monoclonal antibodies with neutralizing activity can be rapidly generated from peripheral blood of convalescent patients and are effective in preventing and treating H5N1 infection in a mouse model.
Editors' Summary
Background.
Every year, millions of people catch influenza, a viral disease of the nose, throat, and airways. Although most recover, influenza outbreaks (epidemics) kill about half a million people annually. Epidemics occur because small but frequent changes in the viral proteins (antigens) to which the immune system responds mean that an immune response produced one year provides only partial protection against influenza the next year. Human flu viruses also occasionally appear that contain major antigenic changes. People have little or no immunity to such viruses (which often originate in animals or birds), so these viruses can start deadly pandemics—global epidemics. The Spanish flu pandemic in 1918/9, Asian flu in 1957, and Hong Kong flu in 1968 all killed millions. Experts believe that another pandemic is overdue and may be triggered by the avian H5N1 influenza virus—the name indicates that this bird virus carries type 5 hemagglutinin and type 1 neuraminidase, the two major flu antigens. H5N1, which rapidly kills infected birds, is now present in flocks around the world and, since 1997, it has caused 258 cases of human flu and 153 deaths. People have caught H5N1 through close contact with infected birds but, luckily, H5N1 rarely passes between people.
Why Was This Study Done?
H5N1 might acquire the ability to move between people and start a human influenza pandemic at any time. Some of the H5N1 viruses are resistant to the antiviral drugs used to treat flu and there will inevitably be a lag of some months between the emergence of a human pandemic H5N1 strain and the bulk production of a vaccine effective against it. Thus, new preventative and therapeutic strategies are needed to combat human infections with H5N1. One possibility is passive immunotherapy—treating people with antibodies (proteins that recognize antigens) that can stop H5N1 from infecting cells (so-called neutralizing antibodies). In this study, the researchers have generated neutralizing human monoclonal antibodies (laboratory-produced preparations that contain one type of human antibody) and tested their ability to halt viral growth in mice infected with H5N1.
What Did the Researchers Do and Find?
Patients who have survived infection with H5N1 make neutralizing antibodies, so the researchers isolated and immortalized the immune cells making these antibodies from the patients' blood. They grew up each cell separately and purified the antibody that the cells made. These monoclonal antibodies were then tested for their ability to neutralize H5N1 and other flu viruses in the laboratory. The researchers identified several that neutralized the H5N1 strain with which the patients were originally infected and chose two for further study. In the test tube, the four antibodies neutralized closely related H5N1 viruses and an H5N1 virus from a different lineage (clade) that has also caused human disease, in addition to the original H5N1 virus, although with different efficacies. In mice, the antibodies provided protection from infection with the original virus when given a day before or one to three days after infection. Three antibodies also partly protected the mice against H5N1 from a different clade. Finally, the researchers showed that the antibodies protected mice by limiting viral replication, by lessening the deleterious effects of the virus in the lungs, and by stopping viral spread out of the lungs.
What Do These Findings Mean?
These results indicate that passive immunotherapy with human monoclonal antibodies could help to combat avian H5N1 if (or when) it starts a human pandemic. Passive immunotherapy is already used to prevent infections with several other viruses. In addition, a crude form of the approach—early treatment of patients with plasma (the liquid portion of blood) from convalescent patients—halved the death rate during the Spanish flu pandemic. Large amounts of pure monoclonal antibodies can be relatively easily made for clinical use, and this study indicates that some monoclonal antibodies neutralize H5N1 viruses from different clades. The researchers sound a note of caution, however: Before passive immunotherapy can help to halt an H5N1 pandemic, they warn, the monoclonal antibodies will have to be tested to see whether they can neutralize not only all the currently circulating H5N1 viruses but also any emerging pandemic versions, which might be antigenically distinct.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040178.
US Centers for Disease Control and Prevention information about influenza for patients and professionals including key facts about avian influenza
US National Institute of Allergy and Infectious Disease feature on seasonal, avian, and pandemic flu
World Health Organization factsheet on influenza and information on avian influenza, including latest figures for confirmed human cases
UK Health Protection Agency information on seasonal, avian, and pandemic influenza
Wikipedia pages on passive immunity and monoclonal antibodies (note: Wikipedia is an online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040178
PMCID: PMC1880850  PMID: 17535101
25.  Therapeutic Efficacy of Antibodies Lacking FcγR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies 
PLoS Pathogens  2013;9(2):e1003157.
Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.
Author Summary
The four dengue virus serotypes (DENV1-4) cause the most prevalent mosquito-transmitted viral disease globally, infecting 50–100 million people annually in tropical and sub-tropical regions worldwide, yet no vaccine or therapy has been licensed to prevent or treat dengue. The greatest risk factor for severe dengue disease is a previous infection with a different serotype, which is thought to be due in part to a phenomenon known as antibody-dependent enhancement (ADE) whereby anti-DENV antibodies from a prior infection augment DENV infection of target Fcg receptor (FcgR)-expressing cells. We previously developed a mouse model that demonstrates antibody-enhanced lethal DENV disease and showed that genetically-modified antibodies incapable of interacting with the FcgR eliminate ADE in vitro and in vivo. In this study, we studied a larger panel of modified MAbs that recognize different regions of the DENV envelope protein. While all modified MAbs acted therapeutically to prevent a lethal, virus-only DENV infection, only certain MAbs effectively protected mice following an antibody-enhanced lethal infection. We determined that therapeutically effective MAbs following an ADE infection worked by competing for binding of enhancing antibodies on the DENV virion. Based on this, we designed an in vitro suppression-of-enhancement assay that predicted the ability of modified MAbs to act therapeutically in vivo.
doi:10.1371/journal.ppat.1003157
PMCID: PMC3573116  PMID: 23459315

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