Improved tumor targeting with chemically cross-linked recombinant antibody fragments.
The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.
King
D J
DJ
Celltech Ltd., Berkshire, United Kingdom.
Turner
A
A
Farnsworth
A P
AP
Adair
J R
JR
Owens
R J
RJ
Pedley
R B
RB
Baldock
D
D
Proudfoot
K A
KA
Lawson
A D
AD
Beeley
N R
NR
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism / Q000627:therapeutic use*; D007128:Immunoglobulin Fragments / Q000378:metabolism / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000532:radiotherapy*; D016499:Radioimmunotherapy; D011994:Recombinant Proteins / Q000378:metabolism / Q000627:therapeutic use; D014183:Transplantation, Heterologous; D015021:Yttrium Radioisotopes / Q000627:therapeutic use
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D015021:Yttrium Radioisotopes; C055966:immunoglobulin Fv