Immunotherapeutic mechanisms of anti-CD20 monoclonal antibodies

The anti-CD20, B-cell-specific mAb rituximab (RTX) has been approved for treatment of non-Hodgkin's B cell lymphoma and rheumatoid arthritis. Under conditions of high B cell burden, exhaustion of the body's effector mechanisms, for example, NK-cell-mediated killing, may lead to substantial decreases in the immunotherapeutic efficacy of this mAb. Moreover, RTX treatment of patients with chronic lymphocytic leukemia and high levels of circulating B cells can lead to removal of CD20 from the cells, thus allowing them to persist and resist clearance. RTX therapy for several autoimmune diseases has proven to be effective, but in numerous instances there has been little correlation between reductions in disease activity and changes in titers of pathogenic autoantibodies. This paradox may be explained by a separate mechanism: Binding of RTX to B cells generates immune complexes that act as decoys to attract monoycte/macrophages and thus reduce their inflammatory activity in certain autoantibody-mediated diseases. Several second-generation anti-CD20 mAbs with enhanced cytotoxic action have been developed and are being tested in the clinic for treatment of cancer and autoimmune diseases. The application of these mAbs, potentially in combination with immune effector modifying drugs, may successfully address the shortcomings of current anti-CD20 immunotherapy.     

Immunotherapeutic potential of monoclonal antibodies againstPseudomonas aeruginosa protein F

European Journal of Clinical Microbiology & Infectious Diseases - To unambiguously demonstrate the immunotherapeutic potential of outer membrane porin protein F fromPseudomonas aeruginosa, a...     

Raised bispecific anti-p-azophenylphosphorylcholine antibodies reactive with nitroiodohydroxyphenylacetyl

Rabbit antisera raised against the p-azophenylphosphorylcholine group (APPC) were found to contain bispecific antibodies which bound the 3-nitro-5-iodo-4-hydroxyphenylacetyl group (NIP) as well as the APPC group. Binding of the NIP group occurred only with restricted components of the total anti-APPC antibodies, as shown by isoelectric focusing patterns visualized with radiolabeled protein conjugates of APPC and NIP. The bispecific antibody isolated on NIP immunoadsorbent bound NIP acid with an association constant of 5 × 10³M⁻¹ which was two orders of magnitude lower than the association constant of 4 × 10⁵M⁻¹ for p-nitrophenylphosphorylcholine (NPPC). The binding by bispecific antibody of either structure could be inhibited by both haptens, as shown by inhibition of hemolysis of NIP- and APPC-coupled red cells. When stimulated by NIP-KLH, a rabbit previously immunized with APPC-KLH gave rise to an increased amount of NIP binding anti-APPC antibody with the same isoelectric focusing properly as that exhibited by the bispecific antibody originally produced by APPC-KLH immunization. Antibody-forming cells from this rabbit produced antibody that could bind either haptenic structure, as shown by the fact that lymph node cells from the rabbit contained plaque forming cells (PFC) which could lyse either NIP- or APPC-coupled red cells. Both PFC responses could be inhibited by either NIP acid or NPPC. Furthermore, complete lysis was seen in plaques formed with a mixed target cell population containing both NIP-and APPC-coupled red cells. These responses showed that the same antibody-forming cells were stimulated by either haptenic structure and that the resultant antibodies were bispecific.     

Monoclonal and bispecific antibodies as novel therapeutics

Gene amplification, over-expression, and mutation of growth factors, or the receptors themselves, causes increased signaling through receptor kinases, which has been implicated in many human cancers and is associated with poor prognosis. Tumor growth has been shown to be decreased by interrupting this process of extensive growth factor-mediated signaling by directly targeting either the surface receptor or the ligand and thereby preventing cell survival and promoting apoptosis. Monoclonal antibodies have long been eyed as a potential new class of therapeutics targeting cancer and other diseases. Antibody-based therapy initially entered clinical practice when trastuzumab/Herceptin became the first clinically approved drug against an oncogene product as a well-established blocking reagent for tumors with hyperactivity of epidermal growth factor signaling pathways. In the first part of this review we explain basic terms related to the development of antibody-based drugs, give a brief historic perspective of the field, and also touch on topics such as the “humanization of antibodie” or creation of hybrid antibodies. The second part of the review gives an overview of the clinical usage of bispecific antibodies and antibodies “armed” with cytotoxic agents or enzymes. Further within this section, cancer-specific, site-specific, or signaling pathway-specific therapies are discussed in detail. Among other antibody-based therapeutic products, we discuss: Avastin (bevacizumab), CG76030, Theragyn (pemtumomab), daclizumab (Zenapax), TriAb, MDX-210, Herceptin (trastuzumab), panitumumab (ABX-EGF), mastuzimab (EMD-72000), Erbitux (certuximab, IMC225), Panorex (edrecolomab), STI571, CeaVac, Campath (alemtuizumab), Mylotarg (gemtuzumab, ozogamicin), and many others. The end of the review deliberates upon potential problems associated with cancer immunotherapy.     

Antigen targeting to dendritic cells with bispecific antibodies

We have developed a universal DC targeting vehicle that could be a convenient method to deliver any type of antigen to DC. P125, a quadroma (hybrid-hybridoma) secreting bispecific monoclonal antibodies (bsmAb), with one paratope specific for mouse DC DEC-205 and another paratope specific for biotin, was developed by PEG-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter. The bsmAb were purified using a biotin-Agarose column and the bsmAb activity was demonstrated using ELISA method employing mouse bone marrow DC and biotinylated BSA. Both confocal microscopy and ELISA studies have shown enhanced binding and internalization of biotinylated and FITC-labelled M13 to DC cell in the presence of bsmAb. In vivo studies in mice with biotinylated OVA has shown that in the presence of bsmAb and anti-CD40 mAb, both humoral and cell-mediated responses can be augmented. In addition, only a low concentration of antigen (500 fold less) is required using bsmAb to achieve a similar immune response in mice that were immunized using complete Freund's adjuvant. In the absence of traditional adjuvants, bsmAb targeting of biotinylated antigens to DC could be an alternative, convenient method to deliver antigens to DC. Moreover, this method could be an alternative method to ex vivo stimulation of DC to overcome DC defects and for treatment of cancer.     

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.     

Antigen binding properties of highly purified bispecific antibodies.

A panel of three bispecific monoclonal antibodies (bsMAbs) binding to follitropin (FSH) and to beta-galactosidase have been prepared by fusion of hybridoma cell lines resistant to oubain and neomycin. One of these bispecific antibodies contains heavy chains of the same IgG subclass, and two are composed of heavy chains of different IgG subclasses. We have investigated methods for the purification of bispecific antibodies from hybrid hybridoma supernatant fluids grown in serum-free medium. Following ammonium sulfate precipitation, bispecific antibodies can be purified in a single step by mixed mode ion-exchange HPLC on Bakerbond Abx columns. In one case, three species were resolved by ion-exchange HPLC and functional analysis showed that two peaks contained parental antibodies, and the third contained the bispecific. Ion-exchange HPLC purification of serum-free preparations from two other hybrid hybridomas resolved seven protein-containing peaks, only one of which was active in a bispecific ELISA. The equilibrium affinity constants for each of the parental antibodies for both FSH and beta-galactosidase were determined and found to be similar to those of the purified bsMAbs. Further, the association of FSH to one binding site on a bispecific antibody was shown to have no effect on the equilibrium binding constant for beta-galactosidase binding to the other site. Our results suggest that bsMAbs can be readily purified from hybrid hybridomas by a simple and rapid method, and the binding of antigen to one binding site on a bsMAb is independent of antigen binding to the second site.     

Adoptive immunotherapy with bispecific antibodies: targeting through macrophages.

We report on two applications of bispecific antibodies to enhance the antitumoral function of human macrophages: (1) use of rhuIFN gamma (recombinant human IFN gamma) encapsulated in human red blood cells coated with anti-Fc gamma RI/anti-RhD+ bispecific antibodies to target and to activate human macrophages; encapsulated rhuIFN gamma was more potent than free IFN gamma in activating mature macrophages in vitro, demonstrating the efficacy of this delivery system to initiate in situ activation of macrophages and also to maintain a high antitumoral efficacy of macrophages with less side effects than after systemic injection of IFN gamma; (2) targeting of activated macrophages to tumours by bispecific antibodies directed against macrophage Fc gamma RI and against human adenocarcinoma antigen; differentiated human macrophages became cytotoxic for human adenocarcinoma in vitro and in vivo (tumours implanted in nude mice) when activated by rhuIFN gamma; this effect was increased in the presence of bispecific antibodies. These two approaches were aimed at increasing the efficacy of cellular immunotherapies using activated macrophages as effector cells (macrophage-activated killer, or MAK), an adoptive therapy which we have developed. Bispecific antibodies could increase specific homing and activation of cytotoxic MAK effectors at tumour sites.     

Use of bispecific hybrid antibodies for the development of a homogeneous enzyme immunoassay

Hybrid bispecific monoclonal antibodies reacting with carcinoembryonal antigen (CEA) and with the E. coli enzyme beta-galactosidase (GZ) were produced by fusion of hybridomas or chemical linkage of half-antibodies. Since the original anti-GZ antibody used in these experiments was capable of protecting GZ from thermal denaturation, it was possible, by hybridizing it with two different non-competitive anti-CEA antibodies, to design a homogeneous enzyme immunoassay for quantitation of CEA. In fact, a mathematical analysis of the reaction indicates that, under appropriate concentrations of the reactants, circular complexes can be formed which contain the two hybrid antibodies, the GZ enzyme and the CEA antigen. The stability of these complexes can be expected to be substantially greater than that of the more labile CEA-free GZ-antibody complexes, prompting a significant increase in the amount of enzyme molecules which are bound to antibody and are consequently protected from thermal denaturation. These expectations were supported by experimental results: under appropriate conditions, heat-resistant enzyme activity was indeed proportional to concentration of CEA in the range up to 75 ng/ml. As predicted by theory, however, in the presence of excess CEA - in fact at CEA concentrations which are higher than those of possible clinical relevance - circular complexes tended to open up, leading to a marked prozone effect.     

The assembly of single domain antibodies into bispecific decavalent molecules

Bispecific antibodies present unique opportunities in terms of new applications for engineered antibodies. However, designing ideal bispecific antibodies remains a challenge. Here we describe a novel bispecific antibody model in which five single domain antibodies (sdAbs) are fused via a linker sequence to the N-terminus of the verotoxin B (VTB) subunit, a pentamerization domain, and five sdAbs are fused via a linker sequence to the VTB C-terminus. Fifteen such decavalent bispecific molecules, termed decabodies, were constructed and characterized for the purpose of identifying an optimal decabody design. One of the fifteen molecules existed in a non-aggregated decavalent form. In conjunction with the isolation of sdAbs with the desired specificities from non-immune phage display libraries, the decabody strategy provides a means of generating high avidity bispecific antibody reagents, with good physical properties, relatively quickly.     

Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin

We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semipreparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60-360 mM phosphate buffer gradient was found to give better separation than elution with a 60-180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.     

Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies

Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.     

A simple metabolic system for selection of hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies

A metabolic selective system has been proposed for the selection of hybrid hybridomas (tetradomas) based on the introduction in one of the parental cell lines of two traits simultaneously--a recessive one (resistance to 8-azaguanine) and a dominant one (multidrug resistance). Tetradomas were selected in the presence of two selective agents: aminopterin and actinomycin D. Using this approach we produced tetradomas secreting bispecific MAbs to horseradish peroxidase and human alpha-fetoprotein.     

Specific and effective targeting cancer immunotherapy with a combination of three bispecific antibodies

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we previously constructed two kinds of bispecific antibodies (bsAbs), anti-MUC1 x anti-CD3 (M x 3) and anti-MUC1 x anti-CD28 (M x 28), which activate T cells and form bridges between them and MUC1-expressing tumor cells. In our previous studies [Cancer Res. 56 (1996) 4205] specific targeting therapy (STT) consisting of i.v. administration of lymphokine activated killer cells with a T cell phenotype (T-LAK) sensitized with two kinds of bsAbs to human BDC-grafted severe combined immunodeficient (SCID) mice demonstrated remarkable inhibition of tumor growth. However, complete cures could not be obtained. In order to improve antitumor efficacy, we have paid attention to anti-CD2 monoclonal antibodies (mAbs), thought to play an important roles in signal transduction in T cell activation or control of T cell receptor (TCR)-driven activation. Therefore, we developed another bsAb, anti-MUC1 x anti-CD2 (M x 2), in order to examine if this would show synergism with the two previously described bsAbs. The combination of the three bsAbs (M x 3, M x 28 and M x 2 bsAbs) showed highest cytotoxicity against MUC1-expressing BDC cells when given simultaneously with peripheral blood mononuclear cells (PBMCs) or T-LAK cells in vitro. When 2 x 10(7) T-LAK cells sensitized with different combinations of bsAbs were administered four times i.v. to BDC-grafted SCID mice, the best therapeutic result was obtained with a combination of all three bsAbs. These results indicate usefulness of combination of three bsAbs for targeting cancer immunotherapy.     

Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies

Studies with gene-modified mice have recently reinforced the importance of Fc receptor-mediated effector mechanisms for the therapeutic efficacy of rituxan and herceptin - two clinically approved antibodies for the treatment of tumor patients. We investigated Fc receptor-dependent tumor cell killing by mononuclear and granulocytic effector cells - comparing human IgG1 antibodies against CD20 or HER-2/neu with their respective FcgammaRI (CD64)-, FcgammaRIII (CD16)-, or FcalphaRI (CD89)-directed bispecific derivatives. With blood from healthy donors as effector source, human IgG1 and FcgammaRIII (CD16)-directed bispecific antibodies proved most effective in recruiting mononuclear effector cells, whereas tumor cell killing by granulocytes was most potently triggered by FcalphaRI-directed bispecific constructs. Granulocyte-mediated tumor cell lysis was significantly enhanced when blood from G-CSF- or GM-CSF-treated patients was investigated. Interestingly, however, both myeloid growth factors improved effector cell recruitment by different mechanisms, which were furthermore dependent on the tumor target antigen, and on the selected cytotoxic Fc receptor.     

Anti-human CD138 monoclonal antibodies and their bispecific formats: generation and characterization

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a co-receptor for growth factors and chemokines and is a molecular marker associated with the epithelial–mesenchymal transition during development and carcinogenesis. In this study, we generated two specific mouse anti-human CD138 monoclonal antibodies (mAbs, clone ID: 480CT5.4.3, 587CT7.3.6.5) using hybridoma technology and identified their immunological characteristics. After hybridoma sequencing, the single-chain variable fragments (ScFvs) cloned from two hybridoma cells were combined with anti-CD3 OKT-3 ScFv to generate two recombinant bispecific antibodies (h-STL002, m-STL002) against CD138 and CD3 molecules, respectively. The bispecific antibodies were able to specifically target CD138?+?multiple myeloma (MM) cells and CD3?+?T cells, and showed the potent cytotoxicity against MM RPMI-8226 cell line through T cell activation. However, these bispecific antibodies without T cells did not cause toxic side effect on MM cells. Overall, the two hybridoma clones and their bispecific formats have great potential to promote diagnosis and immunotherapy of plasma cell malignancy.     

Potential of Immunotherapeutic Treatment for Recurrent Pregnancy Loss

The problem of recurrent pregnancy loss frustrates couples and physicians alike. One major reason for these frustrations is that the precise aetiology of recurrent pregnancy loss is rarely determined during the routine clinical evaluation. In at least 50% of couples, no obvious aetiology is ascertained, and this fact has led many investigators to suggest an immunological cause for the disorder. Various immunotherapeutic regimens have been developed, including paternal leucocyte immunisation and intravenous immunoglobulin. Thus far, properly randomised and blinded trials have shown only a modest benefit from immunotherapy for recurrent pregnancy loss. One major problem is that there are no reliable and reproducible tests to identify any subsets of women with recurrent pregnancy loss who might potentially benefit from immunotherapy. Until there is a better understanding of the basic pathophysiology of recurrent pregnancy loss, all immunotherapies for the disorder will be empirical. Therefore, new immunotherapeutic regimens should be tested in randomised prospective trials prior to general acceptance by the medical community.     

Immunotherapeutic strategies in autoimmune uveitis

Autoimmune uveitis is an organ-specific disorder characterized by irreversible lesions to the eye that predominantly affect people in their most productive years and is among the leading causes of visual deficit and blindness. Currently available therapies are effective in the treatment of a wide spectrum of uveitis, but are often associated with severe side effects. Here, we review ongoing research with promising immunomodulatory therapeutic strategies, describing their specific features, interactions and the responses triggered by the targeted immune molecules that aim to minimize clinical complications and the likelihood of disease relapse. We first review the main features of the disease, diagnostic tools, and traditional forms of therapy, as well as the animal models predominantly used to understand the pathogenesis and test the novel intervention approaches aiming to control the acute immune and inflammatory responses and to dampen chronic responses. Both exploratory research and clinical trials have targeted either the blockade of effector pathways or of their companion co-stimulatory molecules. Examples of targets are T cell receptors (CD3), their co-stimulatory receptors (CD28, CTLA-4) and corresponding ligands (B7-1 and B7-2, also known as CD80 and CD86), and cytokines like IL-2 and their receptors. Here, we summarize the available evidence on effectiveness of these treatments in human and experimental uveitis and highlight a novel CD28 antagonist monovalent Fab′ antibody, FR104, which has shown preclinical efficacy suppressing effector T cells while enhancing regulatory T cell function and immune tolerance in a humanized graft-versus-host disease (GVHD) mice model and is currently being tested in a mouse autoimmune uveitis model with encouraging results.