Bispecific Complexes Research Articles

Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer.

LyP-1, a nine-amino-acid tumor homing peptide, selectively binds to its cognate receptor, p32. Overexpression of p32 in certain tumors should allow use of LyP-1 as a targeting agent for the delivery of therapeutic or diagnostic agents. Peptide conjugates are developed for enhanced pre-targeting of MDA-MB-231 breast cancer cells with peptide-antibody bispecific complexes and targeting with multiple-drug/-fluorophore-conjugated nano-polymers. LyP-1-anti-DTPA bispecific antibody complexes (LyP-1-bsAbCx) were generated by conjugation of anti-DTPA antibody and LyP-1. LyP-1-doxorubicin (Dox), Dox-DTPA-succinyl-polylysine (Dox-DSPL), Dox-DSPL-LyP-1, DTPA-Dox-poly glutamic acid (D-Dox-PGA) or DTPA-rhodamine conjugated polylysine (DSPL-RITC) were prepared. In vitro therapeutic efficacy and targeting by immunofluorescence in MDA-MB-231 breast cancer cells were assessed with Dox-LyP-1. Immunofluorescence visualization of cancer cells was evaluated after pretargeting with LyP-1-bsAbCx and targeting with DSPL-RITC. Cytotoxicity of Dox-LyP-1 conjugates was significantly greater than free doxorubicin (p < 0.0001). For fluorescent-labeled LyP-1, internalization occurred in 30min in tumor cells. Fluorescence intensity of two-step targeted cells showed that pretargeting with LyP-1-bsAbC, followed by targeting with DSPL-RITC was greater than non-pretargeted DSPL-RITC (p < 0.05). Peptide-conjugates are effective targeting agents for MDA-MB-231 breast cancer cells in culture. LyP-1-bsAbCx and Dox-LyP-1 conjugates may allow development of novel targeted cancer therapy and diagnosis.

In vitro demonstration of enhanced prostate cancer toxicity: pretargeting with Bombesin bispecific complexes and targeting with polymer-drug-conjugates

Background: Bombesin has been used to target Bombesin receptor, a growth receptor, which is over-expressed in many cancers, including prostate cancer. Polymer-anti-neoplastic-drug-conjugates (PDC) were also developed to reduce non-specific toxicity and increase tumor toxicity utilizing the enhanced permeability and retention effect, benefitting treatment of large tumors with well-established vasculature.Purpose: If PDCs were delivered by targeted delivery to cancer cells, tumor toxicity would be enhanced and non-specific toxicity decreased.Methods: Cardiocyte toxicity was assessed in H9c2 cardiocytes with doxorubicin (Dox) or N-terminal DTPA-modified-Doxorubicin-loaded-polyglutamic acid polymers (D-Dox-PGA). Therapeutic efficacy of targeted D-Dox-PGA after pretargeting with Bombesin-conjugated anti-DTPA-antibody Bispecific Complexes (Bom-BiSpCx) was compared to that of Dox in PC3 cells. Bom-BiSpCx was generated by thioether bond between Bombesin to Anti-DTPA antibody.Results: D-Dox-PGA was demonstrated to have less cardiocyte toxicity (IC50 = 20 µg/ml) than free Dox (1.55 µg/ml, p < 0.001). However, after pre-targeting of human prostate cancer PC3 cells with Bom-BiSpCx and targeting with D-Dox-PGA, IC50 (13.2 µg/ml) was about two times less than that of Dox (28.5 µg/ml, p < 0.0001).Discussion: Targeted delivery of PDCs having lower cardiocyte toxicity enabled higher efficiency cancer cell therapy.Conclusion: This study may allow development of very efficient targeted prostate cancer pro-drug therapy.

Interleukin-15:Interleukin-15 receptor α scaffold for creation of multivalent targeted immune molecules.

Human interleukin-15 (hIL-15) and its receptor α (hIL-15Rα) are co-expressed in antigen presenting cells allowing trans-presentation of the cytokine to immune effector cells. We exploited the high-affinity interactions between hIL-15 and the extracellular hIL-15Rα sushi domain (hIL-15RαSu) to create a functional scaffold for the design of multispecific fusion protein complexes. Using single-chain T cell receptors (scTCRs) as recognition domains linked to the IL-15:IL-15Rα scaffold, we generated both bivalent and bispecific complexes. In these fusions, the scTCR domains retain the antigen-binding activity and the hIL-15 domain exhibits receptor binding and biological activity. As expected, bivalent scTCR fusions exhibited improved antigen binding due to increased avidity, whereas fusions comprising two different scTCR domains were capable of binding two cognate peptide/MHC complexes. Bispecific molecules containing scTCR and scCD8αβ domains also exhibit enhanced binding to peptide/MHC complexes, demonstrating that the IL-15:IL-15Rα scaffold displays flexibility necessary to support multi-domain interactions with a given target. Surprisingly, functional heterodimeric molecules could be formed by co-expressing the TCR α and β chains separately as fusions to the hIL-15 and hIL-15RαSu domains. Together, these properties indicate that the hIL-15 and hIL-15RαSu domains can be used as versatile, functional scaffold for generating novel targeted immune molecules.

Bispecific Anti-CD20/Anti-CD22 Antibodies with Potent Lymphoma Cytotoxicity.

BACKGROUND: Multivalent bispecific antibodies (bsMAbs) have shown improved efficacy in a number of preclinical studies. We made a pair of hexavalent bsMAbs based on epratuzumab (hLL2, anti-CD22) and veltuzumab (hA20, anti-CD20) by the Dock-and-Lock (DNL) method, and compared their properties to each other and their parental MAbs.METHODS AND RESULTS: DNL is used for the site-specific and covalent assembly of modular components, and was utilized to generate stably tethered hexavalent bispecific complexes, each composed of 4 Fab fragments conjugated to an IgG at the latter's carboxyl termini of the heavy chain. DNL-1 has 4 veltuzumab Fabs tethered to epratuzumab IgG; DNL-2 has 4 epratuzumab Fabs bound to veltuzumab IgG. DNL made each construct as a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human lymphoma cell lines demonstrated that, unlike the parental MAbs, the bsMAbs each induced translocation of both CD22 and CD20 into lipid rafts and also strong cell-cell adhesion. DNL1 or DNL2 treatment resulted in increased apoptosis vs. the parental MAbs alone or combined. DNL1 and DNL2 inhibited the growth of Ramos, Raji and Daudi Burkitt lymphoma cell lines without the requirement of crosslinking, and more potently than the combination of the parental MAbs. For Daudi cells, DNL1 and DNL2 showed similar activity, which was approximately 50-fold more potent than the combination of the parental MAbs. For Raji and Ramos, DNL1 was 8–10-fold more potent than DNL2, which in turn was 8–10-fold more potent than the combined parental MAbs. The results suggest that crosslinking of CD20 and CD22 at the cell surface is required for enhanced cytotoxicity. Veltuzumab, but neither epratuzumab nor either bsMAb, displayed CDC activity. Veltuzumab and DNL2 induced a similarly high degree of ADCC. DNL1 induced ADCC to an intermediate level between veltuzumab/DNL2 and epratuzumab. In an ex-vivo assay using fresh whole blood mixed with either Daudi or Raji, DNL1 and DNL2 each demonstrated selective killing of lymphoma cells over normal B-cells compared to veltuzumab or rituximab; the latter depleted normal B-cells with greater efficiency than the bsMAbs. PK studies in mice demonstrated that despite their large size, the bsMAbs have a significantly shorter serum half-life than the IgGs. Even without CDC activity and with a considerably shorter serum half-life, DNL2 had anti-lymphoma efficacy in the Daudi Burkitt lymphoma model in mice that was equivalent to veltuzumab. DNL1 was less potent than DNL2 in vivo, but more effective than epratuzumab and control bsMAbs comprising either epratuzumab-IgG-AD2 (22-14) or 4 veltuzumab-Fab-DDD2 groups (734-22) combined with 4 non-binding Fab-DDD2 groups or a non-binding IgG-AD2, respectively. The anti-tumor efficacy of both DNL1 and DNL2 was abolished in tumor-bearing mice in which their ADCC potential was diminished by depletion of neutrophils and NK cells. These findings suggest that ADCC is the most critical mechanism of action for lymphoma killing in these murine models. DNL1 is more potent than DNL2 in vitro yet DNL2, having the stronger ADCC activity, is more potent than DNL1 in vivo.CONCLUSIONS: These findings suggest that the DNL method can be used to make a variety of multivalent bsMAbs with potent anti-tumor activity, and having distinct properties dependent on their arrangement and composition. CD20/CD22 bsMAbs appear to have different functions than their parental MAbs, even when these were combined, and appear to be potent anti-B-cell lymphoma therapeutics.

A bispecific dsDNA×monoclonal antibody construct for clearance of anti-dsDNA IgG in systemic lupus erythematosus

High avidity anti-dsDNA IgG antibodies are believed to play an important role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) and therefore attempts have been made to reduce the concentration of these antibodies in the bloodstream of SLE patients. Previously we reported the development of an antigen based heteropolymer (AHP), a bispecific complex prepared by using the avidin–biotin system to crosslink dsDNA to a mAb specific for the human erythrocyte (E) complement receptor. Our studies indicated that this AHP could bind anti-dsDNA antibodies to E and facilitate clearance of these autoantibodies from the circulation of a monkey without E destruction. Here we report an improved covalent crosslinking procedure and purification scheme in which the AHP construct is isolated by precipitation in 50% saturated ammonium sulfate. We used a dsDNA binding dye, PicoGreen, to demonstrate specificity of binding of dsDNA to E via the AHP. The efficacy of the AHP in binding IgG anti-dsDNA antibodies to E was demonstrated in a sensitive and quantitative assay, based on the time resolved fluorescence properties of europium-labeled anti-human IgG mAbs used to probe the E. We also used this assay to screen SLE patient and normal plasmas for levels of anti-dsDNA IgG. The results of this assay correlate very well with the Farr assay, and therefore this approach may be useful in the development of informative and specific assays for a variety of autoantibodies. Treatment of SLE plasmas with E-AHP under conditions close to physiological led to substantial reductions (≥ 90%) in anti-dsDNA titers. It should be possible to test these new AHP for their ability to target and safely remove IgG anti-dsDNA antibodies from the circulation in animal models.

Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model.

We investigated the feasibility of using bispecific mAb complexes to redirect and improve the efficiency of the primate E complement receptor 1-based clearance reaction to remove a virus from the circulation. As an initial approach, we used bacteriophage phiX174 as an immunologic model for mammalian viruses. Bispecific complexes were prepared by chemically cross-linking a mAb specific for complement receptor 1 with a mAb specific for the bacteriophage phiX174. In a monkey model these complexes facilitate rapid and quantitative binding of the target bacteriophage to E in vitro and in vivo. Moreover, after in vivo binding to E, the complexes containing mAb and prototype virus are rapidly cleared from the circulation of rhesus and cynomolgus monkeys without loss of E. Our findings suggest that bispecific mAb complexes, in concert with primate E complement receptor 1, may have therapeutic utility in the treatment of diseases associated with blood-borne pathogens.

Primate erythrocyte (E) complement receptor (CR1) as an anchor site for bispecific-based therapies to clear pathogens or autoantibodies safely from the circulation.

We have prepared cross-linked, bispecific complexes [heteropolymers (HP) and antigen-based heteropolymers (AHP)] that facilitate complement-independent binding of target model pathogens or autoantibodies to primate erythrocytes (E) via complement receptors (CR1). The method is based on using monoclonal antibodies (mAb) specific for CR1 that either are cross-linked to an mAb specific for a prototype pathogen (e.g., IgE) or are cross-linked to an autoantigen (e.g., dsDNA) that is recognized by circulating pathogenic autoantibodies in the autoimmune disease systemic lupus erythematosus (SLE). The underlying assumption in this research is that complexed ligands containing IgG bound to primate E CR1 should be recognized and processed via the same mechanism by which complement-opsonized immune complexes bound to E CR1 are cleared from the circulation and phagocytosed in the liver and spleen. Our work in experimental monkey models has demonstrated that binding of substrates to primate E via this method does indeed lead to the safe and rapid clearance of the target pathogens or autoantibodies from the circulation, without any lysis or loss of the E. Although a number of questions must still be resolved, it may be possible to generalize these findings and use this CR1-based approach to develop a simple noninvasive bispecific therapy that can be used to clear pathogens or autoantibodies from the circulation.

Cyclic tetramolecular complexes of monoclonal antibodies: a new type of cross-linking reagent.

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies, when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1, yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody, stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens, and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine.