Search

Poly(styrenyl acetal trehalose) (pSAT), composed of trehalose side chains linked to a polystyrene backbone via acetals, stabilizes a variety of proteins and enzymes against fluctuations in temperature. A promising application of pSAT is conjugation of the polymer to therapeutic proteins to reduce renal clearance. To explore this possibility, the safety of the polymer was first studied. Investigation of acute toxicity of pSAT in mice showed that there were no adverse effects of the polymer at a high (10 mg/kg) concentration. The immune response (antipolymer antibody and cytokine production) in mice was also studied. No significant antipolymer IgG was detected for pSAT, and only a transient and low level of IgM was elicited. pSAT was also safe in terms of cytokine response. The polymer was then conjugated to a granulocyte colony stimulating factor (GCSF), a therapeutic protein that is approved by the Federal Drug Administration, in order to study the biodistribution of a pSAT conjugate. A site-selective, two-step synthesis approach was developed for efficient conjugate preparation for the biodistribution study resulting in 90% conjugation efficiency. The organ distribution of GCSF–pSAT was measured by positron emission tomography and compared to controls GCSF and GCSF–poly(ethylene glycol), which confirmed that the trehalose polymer conjugate improved the in vivo half-life of the protein by reducing renal clearance. These findings suggest that trehalose styrenyl polymers are promising for use in therapeutic protein–polymer conjugates for reduced renal clearance of the biomolecule.

The progress in the field of targeted α-particle therapy (TAT) has to a great extent been enhanced by developments in both recombinant DNA technology and radionuclide labeling chemistry. Advances in genomics and proteomics have promoted an increase in the identification of novel targets and molecules that can define different diseases, such as cancer. In radioimmunotherapy (RIT), the primary goal is to improve delivery to and therapeutic efficacy of the cancer cells, whilst minimizing toxicity. Different approaches have been investigated to achieve this, such as reducing the size of the carrier, pretargeting, multidosing, locoregional administration and using a cocktail of radiolabeled monoclonal antibodies for targeting multiple antigens simultaneously. Some of these approaches have been encouraging, but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available α-particle emitters. The clinical studies carried out to date have been promising, although many challenges remain in order to make TAT safe and economically feasible. In this paper a number of different targeting constructs used hitherto that may be promising carriers for TAT in the future are presented and discussed. The constructs include enzymatic cleaved antibody fragments (Fab and F(ab˙)2 fragments); genetically engineered antibody fragments (scFv monomer, dimer (i.e. diabody) and tetramer, CH2 domain deleted antibody fragments); other protein targeting constructs such as affibodies and peptides as well as liposomal delivery.

Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their half-life is controlled by their ability to interact with the protective neonatal Fc receptor (FcRn, Brambell receptor) present on endothelial cells. Here, we describe a protocol using site-specific mutagenesis of individual residues responsible for this interaction, resulting in engineered antibodies with distinct half-lives. The method is a powerful tool that enables manipulation of half-lives and is applicable to all antibodies and Fc fusion proteins for the development of agents with controlled pharmacokinetic properties. Moreover, the protocol is applicable to any situation where the structure and/or function of engineered proteins are to be studied. The protocol begins with the mutagenesis reaction at the DNA level and proceeds to describe mammalian expression and purification of recombinant proteins, radiolabeling and evaluation in vivo. The time frame for completing the procedure is about 6 months, provided that no complications are encountered.

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 ± 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 ± 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using 124I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.

An engineered antibody fragment (minibody; scFv-CH3γ1 dimer, Mr 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185HER-2 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (VL-linker-VH and VH-linker-VL) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 CH3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20–60 mg/l, demonstrated binding to the human p185HER-2 overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (KD ∼ 2–4 nM) were equivalent to that for the parental 10H8 mAb (KD ∼ 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (±1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (β-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen–antibody system.

An engineered anti‐carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti‐CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti‐CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site‐specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti‐CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C‐terminal Gly‐Gly‐Cys) existed exclusively as a disulfide‐bonded dimer. This cysteine‐modified diabody (Cys‐diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non‐covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys‐diabody could be chemically modified using a thiol‐specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys‐diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site‐specifically. This format will provide a versatile platform for targeting a variety of agents to CEA‐positive tumors.

A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the VL-VH domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a `diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcγR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-γ ΔIgM wt, scOKT3-γ ΔIgM C575S and scOKT3-γ ΔIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-γ ΔIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-γ ΔIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-α and IFN-γ) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-γ ΔIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.

Abstract: Osteosarcoma is the commonest malignant tumour of the bones. The presence of micrometastases at the time of primary diagnosis is associated with poor prognosis. Despite developments in surgery and aggressive chemotherapy, about 50% of the patients still succumb to the disease. Thus, there is a need to develop alternative treatment modalities. One such strategy is to use antibodies with improved effector functions. The two monoclonal antibodies, TP-1 and TP-3, recognize a tumour-associated antigen on human osteosarcoma cells. In the present study, we describe the cloning of the TP-1 variable genes, and the production of complete chimeric mouse/human monoclonal antibodies. Constructs containing the constant genes from human IgG1, IgG3 or a mutant IgG3 with a shortened hinge region, called m15, were expressed in the mouse myeloma cell line, NS0. The m15 mutant has been shown to be very potent in triggering complement-mediated lysis. Our goal was to investigate whether this mutant could overcome the complement protection on human osteosarcoma cells, which is generally present on all human cells. We found that the target cells expressed several membrane-bound complement inhibitors, and that masking of these inhibitors rendered the cells sensitive to lysis. The m15 mutant exhibited greater lytic activity than both IgG3 and IgG1, although it could not cause extensive killing of the target cells alone.

The variable (V) genes of TP-1 and TP-3 MAbs have been cloned and sequenced. Because of the potential use of these antibodies in the diagnosis and treatment of osteosarcoma, it is important to determine the presence and position of amino acid residues that may react with radiolabeling within the V domains. In this article, location of the tyrosine residues is determined using the knowledge of immunoglobulin structures in general. The TP-1V domains have a total of 19 tyrosines, whereas TP-3V domains have 18, with approximately half of these located within complementarity determining regions (CDRs). Thus, if equal reactivity of all tyrosines is assumed, smaller fragments of MAbs have a high probability of being radiolabeled at one of these sites with possible resultant loss of antigen binding

No relevant conflicts of interest to declare.

Abstract 723