High affinity humanized anit-cea monoclonal antibodies

Abstract

Novel humanized monoclonal antibodies, fragments or derivatives thereof which specifically bind carcinoembryonic antigen (CEA) are provided as well as methods for their manufacture. These humanized antibodies are useful in the treatment of cancers which express CEA as well as for diagnostic purposes, e.g., for in vivo imaging of tumors or cancer cells which express CEA.

Description

[0001] The present invention relates to humanized monoclonal antibodies and fragments or derivatives thereof which specifically bind carcinoembryonic antigen (CEA), which is an antigen expressed by various human carcinomas including breast, lung, and gastrointestinal carcinomas such as stomach and colon cancers. More specifically, the present invention relates to humanized monoclonal antibodies and humanized antibody fragments and derivatives thereof which are derived from murine monoclonal antibody COL-1, a high affinity anti-CEA antibody. The present invention further relates to methods for producing such humanized monoclonal antibodies specific to CEA, pharmaceutical and diagnostic compositions containing such humanized monoclonal antibodies, and methods of use thereof for the treatment or diagnosis of cancer.[0002] The identification of antigens expressed by tumor cells and the preparation of monoclonal antibodies which specifically bind such antigens is well known in the art. A...

AI Technical Summary

Benefits of technology

[0061] As discussed above, humanized antibodies afford potential advantages over murine and also chimeric antibodies, e.g., reduced immunogenicity in humans. This is advantageous because it should reduce and potentially eliminate the eliciting of a HAMA response when such humanized antibodies are administered in vivo, e.g., either for treatment of cancer or for diagnosis of cancer as by tumor imaging. Also, such antibodies may exhibit improved plasma clearance, pharmacokinetic, and tumor targeting properties.

[0067] Alternatively, the amino acid sequences of the FRs of the antibody to be humanized (e.g., COL-1) will be compared to those of known human FRs, and the human FRs to be used for CDR-grafting will be selected based on their comprising sequences highly similar to those of the parent antibody, e.g., a murine antibody which binds CEA. Numerous human FRs have been isolated and their sequences reported in the literature. This enhances the likelihood that the resultant CDR-grafted "humanized" antibody, which contains the CDRs of the parent (e.g., murine) antibody grafted onto the selected human FRs, will substantially retain the antigen binding structure and thus retain the binding affinity of the parent antibody. As a result of such studies, the FRs of REI and NEWM antibodies have been identified as having amino acid sequences which are likely to allow the CDRs of COL-1 To retain a significant degree of antigen binding affinity. As noted, the selected human framework regions will preferably be those that are expected to be suitable for in vivo administration, i.e., not immunogenic. Based on their amino acid sequences, REI and NEWM human framework regions are expected to be substantially non-immunogenic.

[0076] Human constant domain sequences are well known in the art, and have been reported in the literature. Preferred human constant light chain sequences (C.sub.L) include the kappa and lambda constant light sequences. Preferred human constant heavy chain sequences include human gamma 1, human gamma 2, human gamma 3, human gamma 4, and mutated versions thereof which provide for altered effect or function, e.g., enhanced in vivo half-life, reduced Fc receptor binding, and the like.

[0081] The phrase "substantially homologous" is used in regard to the similarity of a subject amino acid sequence (of an oligo- or poly-peptide or protein) to a related, reference amino acid sequence. This phrase is defined as at least about 75% "correspondence"--i.e. the state of identical amino acid residues being situated in parallel--between the subject and reference sequences when those sequences are in "alignment," i.e. when a minimal number of "null" bases have been inserted in the subject and / or reference sequences so as to maximize the number of existing bases in correspondence between the sequences. "Null" bases are not part of the subject and reference sequences; also, the minimal number of "null" bases inserted in the subject sequence may differ from the minimal number inserted in the reference sequence. In this definition, a reference sequence is considered "related" to a subject sequence where both amino acid sequences make up proteins or portions of proteins which are either .alpha.CEA antibodies or antibody fragments with .alpha.CEA binding affinity. Each of the proteins comprising these .alpha.CEA antibodies or antibody fragments may independently be antibodies or antibody fragments or bi- or multi-functional proteins, e.g., such as fusion proteins, bi- and multi-specific antibodies, single chain antibodies, and the like.

[0104] As noted above, another preferred application of the subject humanized antibodies or fragments thereof is in the Radioimmunoguided System.RTM. (RIGS.RTM.). This technique involves the intravenous administration of a radiolabeled monoclonal antibody or its fragment prior to surgery. After allowing for tumor uptake and blood clearance of radioactivity, the patient is taken to the operating room where surgical exploration is effected with the aid of a hand-held gamma activity probe, e.g., the Neoprobe.RTM. 1000. This helps the surgeon identify the tumor metastases and lessen the complications of excision.

Problems solved by technology

However, while murine antibodies, such as COL-1 and other anti-CEA murine antibodies, have applicability as therapeutic agents in humans, they are disadvantageous in some respects.

This may result in a neutralizing antibody response--a human anti-murine antibody (HAMA) response--which is particularly problematic if the antibodies are desired to be administered repeatedly, e.g., for treatment of a chronic or recurrent disease condition.

This is a significant drawback, as some cancer treatments are effected over a prolonged time period, e.g., over several years or longer.

However, while such chimerized monoclonal antibodies typically exhibit lesser immunogenicity, they are still potentially immunogenic in humans because they contain murine variable sequences which may elicit antibody responses.

Thus, there is the possibility that these chimeric antibodies may elicit an anti-idiotypic response if administered to patients.

However, CDR-grafting by itself may not yield the desired result.

Given these effects of changes in amino acid residues, although humanized antibodies are desirable because of their potential low immunogenicity in humans, their production is unpredictable.

For example, sequence modification of antibodies may result in substantial or even total loss of antigen binding affinity, or loss of binding specificity.

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