Alessandro Sidoli

The three-step pretargeting approach reduces the human anti-mouse antibody response in patients submitted to radioimmunoscintigraphy and radioimmunotherapy

The scant clinical application of radiolabelled monoclonal antibodies (MoAbs) in the diagnosis and therapy of tumours has been attributed to their slow pharmacokinetics, poor target accumulation and human anti-mouse antibody (HAMA) production [1]. Tumour to background ratlos have been improved by the advent of pretargeting strategies, in which MoAbs and radiolabel are administered separately. In particular, the avidin-biotin system has been validated in hundreds of patients with a variety of tumours [2, 3]. In immunoscintigraphic studies (RIS), the injection of 2 mg of murine biotinylated MoAbs (lst step) is followed, 36 h later, by 5 mg of avidin and 5 mg of streptavidin (2nd step), in order to clear circulating biotinylated MoAbs and at the same time to target the tumour cells, allowing adequate localization of the subsequently (24 h later) administered radiolabelled biotin (3rd step). Recently, the system has been applied in therapy trials (RIT) with ten-fold increased amounts of MoAbs and avidin-streptavidin in order to deliver higher doses of radioactivity to the tumour [4]. We evaluated the HAMA, the human anti-avidin antibody (HAVA) and the human anti-streptavidin antibody (HASA) responses in patient sera obtained 1 month after RIS (group A) and RIS plus RIT (group B) with an ELISA test in 96-well microtitre plates coated respectively with a mouse MoAb, avidin and streptavidin. After incubation with the serum samples, specific human antibodies were revealed with rabbit anti-human immunoglobulins IgA, IgG and IgM, conjugated to horseradish peroxidase. In order to standardize the assays, a positive serum was selected and used in all the tests as a reference curve, assigning an arbitrary value, The results are reported in Table 1 and permit the following conclusions:

Human recombinant antibody fragments specific for a rye-grass pollen allergen: Characterization and potential applications

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Recombinant allergen Lol p II: Expression, purification and characterization

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

LOL pII Allergen

Grass pollen allergens are a major cause of allergy affecting about 75% of allergic patients (1). The perennial rye grass Lolium perenne is the most important species that produces allergenic pollen world-wide, due to its large use for forage and turf. Earlier studies showed that this pollen contains at least 17 allergens ranging in size from 12 to 89 kDa (2). They have been grouped on the basis of molecular size and electrophoretic mobility, and it has been recognised that most of these proteins exist in multiple isoforms which may differ in allergenicity. Recent work to isolate, clone and sequence cDNAs for the various allergens has somehow simplified the analysis of these proteins and it is expected to produce new concepts for diagnosis and therapy of grass pollen allergies (3). It has been recognised that Lol pI and Lol pV are the major IgE binding allergens, they have similar size (27–35 Kda) and no significant amino acid sequence homology (4—7). They both exist in multiple forms (6,7), the origin and allergenicity of which have not been clarified. Lol pI is localised in the cytoplasm and it is exported on the surface of the pollen (8), while LolpV is addressed to amyloplasts (9), a localisation that seems to facilitate the diffusion of the allergen in the air and to trigger attacks of asthma (10).