David J. King

Binding to CD20 by Anti-B1 Antibody or F(ab')2 is sufficient for induction of apoptosis in B-cell lines

Abstract. CD20 is a B-cell-specific cell surface protein expressed on mature B lymphocytes and is a target for monoclonal antibody therapy for non-Hodgkins lymphoma (NHL). Though clear clinical efficacy has been demonstrated with several anti-CD20 antibodies, the mechanisms by which the antibodies activate CD20 and kill cells remain unclear. Proposed mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and induction of apoptosis. In this report we compared the activity of two anti-CD20 antibodies, Anti-B1 Antibody (tositumomab) and rituximab (C2B8), in a variety of cellular assays using a panel of B-cell lines. Anti-B1 Antibody showed a low level of activity in a CDC assay against complement-sensitive B-cell lines, Ramos and Daudi. We found that there is an inverse correlation between the expression of CD55 and CD59 and CDC mediated by either Anti-B1 Antibody or rituximab. Rituximab was more potent at inducing CDC when compared to Anti-B1 Antibody. Using Raji cells as target cells and human peripheral blood leukocytes as effector cells, Anti-B1 Antibody was a potent inducer of ADCC. The activities of Anti-B1 Antibody and rituximab were nearly identical in the ADCC assay. In addition, Anti-B1 Antibody showed direct induction of apoptosis in all B-cell lines tested. In general, crosslinking Anti-B1 Antibody with a goat anti-mouse Ig did not further enhance the percentage of cells undergoing apoptosis. Importantly, a F(ab)2 fragment of Anti-B1 Antibody induced apoptosis, while the Fab fragment did not, indicating that the Fc region was not required and dimerization of CD20 may be sufficient for induction of apoptosis. In contrast, rituximab, which binds to an overlapping epitope on CD20 with a three-fold lower affinity than Anti-B1 Antibody, did not efficiently induce apoptosis in the cell lines tested in the absence of crosslinking. In conclusion, these two anti-CD20 antibodies have overlapping, but distinct mechanisms of action on B-cell lines.

The Use of UCOE Vectors in Combination with a Preadapted Serum Free, Suspension Cell Line Allows for Rapid Production of Large Quantities of Protein

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.

Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy.

A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or indium-111 respectively. These molecules have been used to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG from the circulation of animals but does not lead to accumulation of these metallic radionuclides in the kidney. 90Y-labelled hA33 TFM therefore appears to be the optimal form of the antibody for radioimmunotherapy of colorectal carcinoma.

Radioimmunotherapy of colorectal carcinoma xenografts in nude mice with yttrium-90 A33 IgG and Tri-Fab (TFM).

The monoclonal antibody A33 recognises a tumour-associated antigen on human colorectal carcinoma, and has undergone preliminary evaluation in the clinic where selective localisation to hepatic metastases has been demonstrated [Welt et al. (1994) J. Clin. Oncol. 12, 1561-1571]. A33 and an A33 tri-fab fragment (TFM) were labelled with 90Y via a stable macrocyclic ligand for biodistribution and therapy studies in nude mice bearing SW1222 colon carcinoma xenografts. Biodistribution studies demonstrated tumour localisation for both A33 IgG and TFM with low bone, liver and kidney levels. Clearance of TFM from the blood was much faster than IgG and this led to lower tumour accumulation for TFM but superior tumour-blood ratios. The maximum per cent injected dose per g localised to tumour was 35.9% +/- 5.3% for A33 IgG and 12.9% +/- 4.6% for A33 TFM with tumour-blood ratios at 48 h after administration of 5.6 +/- 1.8 and 29.2 +/- 9.8 respectively. Autoradiography studies with 125I-labelled A33 IgG and TFM demonstrated a homogeneous distribution within tumour tissue which was not observed with other anti-colorectal tumour antibodies. TFM penetrated into the tumour tissue more rapidly than IgG. In therapy studies, a single dose of 90Y-A33 IgG (250 microCi per mouse) or 90Y-A33 TFM (300 microCi per mouse) led to complete regression of 2-week-old tumour xenografts with long-term tumour-free survivors. A transient drop in white blood cell count was observed with both IgG and TFM but was significantly more pronounced with IgG. The cell count fell to 8.4% of control for IgG, whereas with TFM cell counts fell to 51% of control before recovery. These results indicate that the more rapid blood clearance of 90Y-TFM confers reduced toxicity compared with 90Y-IgG although similar therapeutic effects are achieved. When the dose of 90Y-IgG was adjusted to give the same dose to tumour achieved with 300 microCi 90Y-TFM, a lesser therapeutic effect was observed. This may be owing to more rapid tumour penetration achieved with TFM. Both A33 IgG and TFM demonstrated potent anti-tumour effects against human tumour xenografts in this mouse model system. The stability of these 90Y-labelled conjugates and their effective tumour penetration are promising for the development of humanised reagents for clinical studies.

Purification of recombinant chimeric B72.3 Fab′ and F(ab′)2 using streptococcal protein G

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab fragments is described here. Chimeric mouse-human B72.3 Fab and F(ab)2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab bound weakly to the protein G-Sepharose it could be separated from F(ab)2 and eluted with a pH 7 wash whereas B72.3 F(ab)2 required elution at pH 2. Both Fab and F(ab)2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab or F(ab)2 fragments from CHO cell supernatant.

Humanization of an Anti-Mucin Antibody for Breast and Ovarian Cancer Therapy

Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.