Search

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use., (Copyright 1999 John Wiley & Sons, Ltd.)

E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human gamma4 heavy and kappa light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human gamma4 with a gamma2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.

ABL 364 is a murine monoclonal IgG3 antibody directed against the Lewis Y carbohydrate antigen (Le(y)) expressed on the surface of many epithelial cell tumors. The antibody mediates cytotoxicity via activation of human complement or human effector cells, and has been evaluated in several clinical trials including two Phase I/II trials in relapsed small cell lung cancer and metastatic breast cancer. To improve the effector functions of the antibody, increase its half-life in circulation, and avoid the human antimouse antibody response, two chimeric and several humanized antibodies were constructed for evaluation. The chimeric IgG1 is more potent than the murine IgG3 in tumor cell lysis via activation of human peripheral mononuclear cells (10-fold), but somewhat less effective in complement-dependent lysis (2-3 fold). The chimeric IgG3 is slightly less potent than the IgG1. A humanized IgG1 was constructed by combining the complementarity-determining regions of the ABL 364 antibody with human framework and constant regions. Several additional variants were subsequently constructed to improve the binding affinity and increase expression of the antibody. Two of the variants, designated I and K, differ by a single amino acid at position 75 of the heavy chain. Both variants have affinity within 2-fold of the chimeric IgG1 antibody and retain the cytolytic activities toward tumor cell lines. However, it was possible to express variant K at a significantly higher level (5- 10-fold) than variant I. Pharmacokinetics of the humanized ABL 364 antibody variant K was compared with that of the parent murine antibody in rhesus monkeys. It was shown that the terminal half-life of the humanized antibody in rhesus monkeys is 14-20 days, with a mean of 16.3 days, while that of the parent murine antibody is only 1.9 days.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.