Grafting of “Abbreviated” Complementarity-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody

Abstract

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the “abbreviated” CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients’ sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.

Antibodies VRC01 and 10E8 Neutralize HIV-1 with High Breadth and Potency Even with Ig-Framework Regions Substantially Reverted to Germline

“…In the case of 10E8-0fH/4fL, post-hoc analysis of the 10E8-MPER peptide structure indicated that additional reversions of mutations that do not directly interact with the antigen or CDR regions may be possible (e.g., S2Y, Figure 5). Further, the reversion analysis here was focused solely on framework residues; however, it should also be possible to selectively revert CDR mutations as not all mutations within the CDR regions will be important for antigen recognition or overall antibody structure (33, 34). This process could allow for the generation of partially reverted antibody variants with even lower degrees of mutation and with similar, if not better, neutralization activity as their mature bNAb counterparts.…”

Overcoming the Blood–Brain Barrier: The Role of Nanomaterials in Treating Neurological Diseases

“…[280,281] (Inherent immunogenicity also previously posed a challenge to therapeutic development, however, recent advances-including the development of fully humanized antibodies-have rendered this less of a problem nowadays. [282][283][284] ) Single-domain antibodies times lighter in molecular weight compared to regular IgG antibodies (150-160 kDa). [285] Despite their smaller makeup, sdAbs have proven capable of having similar specificity as normal antibodies, and in some cases have demonstrated greater robustness.…”

Minimizing the Immunogenicity of Antibodies for Clinical Application

“…The murine anti-CEA MAb COL-1 was humanized through 'abbreviated' CDR grafting, referred to as HuCOL-1 ABR . Compared to the CDR-grafted humanized COL-1 (Hu-COL-1), HuCOL-1 ABR shows only a minimal decrease in its antigen-binding affi nity while showing a signifi cant decrease in its reactivity to the sera of patients previously administered murine COL-1 in clinical trials [12] .…”

“…In L-CDR1, the COL-1 sequence differed from the human template X93639 at positions 27, 27d, 28, 29, 30, 33 and 34, of which 27 and 33 are designated as non-SDR positions ( table 2 ). HuCOL-1 ABR , previously developed by grafting 'abbreviated' CDRs, contains a human substitution at position 27 (K27Q), validating this residue as a non-SDR [12] . A variant containing a human substitution at position 33 (M33L) in addition to the one at 27 was generated.…”

“…The most common approach in choosing the human template is to select the human antibody with the highest sequence homology to the target murine antibody [10][11][12] . It has been suggested that limiting the choice of human templates to germline sequences precludes the potential immunogenicity of residues that are generated as a result of somatic hypermutations [13][14][15] .…”