Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies
As of 2006 there were 18 monoclonal antibodies approved for therapeutic use in US, and 14 of them were molecules of the IgG isotype. Among these, 50% are humanized IgG1 anti-bodies. The antibody molecule is formed by two identical heavy chains (~450 residues each) and two identical light chains (~220 residues each).
The chains fold into domains, of about 110 residues, that have a characteristic beta-sheet architecture known as the "immunoglobulin fold." There are two immunoglobulin domains in the light chain, VL and CL, and four immunoglobulin domains in the heavy chain, VH, CH1, CH2, and CH3 ("V" and "C" stand for "variable" and "constant," respectively). Digestion of human IgG1 antibody by papain results in two types of fragments of similar size: the Fc fragment (composed of two CH2 and two CH3 domains) and the Fab fragment (composed of CH1, CL, VH, and VL).
Mouse-derived antibodies are often subject to immune recognition when administered to humans and their effector functions governed by species-specific Fc fragments are diminished. This has been overcome by "humanization" of murine antibodies, which involves the transfer of complementarity determining regions (CDRs) from the murine antibody along with some adjacent framework regions into the variable domains of the heavy and light chains of a human antibody.To identify the key residues to be transferred with CDRs, computer models are used to generate the 3-D structure of the variable domains that can incorporate the CDRs, while preserving the binding specificity and affinity for the desired target.There is a limited number of existing human variable domain frameworks (coded in the germline), and the selection of the framework is done based on the sequence homology with the murine antibody.
Temperature-induced unfolding of monoclonal antibodies measured by differential scanning calorimetry (DSC) has become an indispensable tool used for monitoring production consistency, clone selection, protein structure characterization, and may potentially have a value for formulation development. Consequently, correct interpretation of the observed transitions is essential. Temperature or pH-induced unfolding of intact antibodies has been studied, however, mostly with antibodies of non-human origin, such as murine or rabbit immunoglobulins. Temperature-induced unfolding of human IgG1 and its Fab and Fc fragments was studied with the myeloma IgG1 Van by DSC in the pH range 3.5-5.5 and compared to the thermograms obtained from other human isotypes (IgG2, IgG3, and IgG4).
The unfolding of the human myeloma IgG1 at pH 5.5 presents two transitions with the melting temperatures (Tm) around 70°C and 82°C, and with the amplitude of the first peak much larger than that of the second peak. Studies on the Fab and Fc fragments of the same antibody reported a melting temperature of 70°C for the Fab fragment and melting temperatures of 66°C and 82°C for the Fc fragment. Based on these results one may infer that the first unfolding event in the intact human IgG1 antibody is associated with the melting of the CH2 domain in the Fc fragment and the melting of the Fab fragment, while the second transition represents mainly the unfolding of the CH3 domain. Analogous studies using murine antibodies and their corresponding Fc and Fab fragments presented a similar trend in the unfolding events, with the unfolding of the Fab fragment occurring at lower temperatures than that of the Fc fragment.
Using three different humanized IgG1 antibodies we show here that different melting profiles can be observed depending on the stability of the Fab fragment. We also demonstrate that the stability of the Fab fragment is significantly affected by the sequence of the variable domains. Therefore, one cannot assign a priori the first transition observed by DSC of the intact antibody to the unfolding of the Fab fragment; rather it is the experimental enthalpy of unfolding, as determined by the peak area in the DSC thermogram, which may be used as the indication for which transition represents the Fab fragment unfolding. This approach relies on the assumption that the Fab fragment unfolds in a cooperative manner, that is, only one transition is observed in the thermogram of the Fab fragment. If the coupling among the domains in the Fab is disrupted by CDR grafting and humanization process, the thermogram of the Fab fragment may present multiple transitions, and the interpretation of the DSC profile for an intact antibody will become more complex.
As will be shown below, multiple transitions observed in Fab unfolding should be carefully examined because may represent artifacts of papain digestion. One has to emphasize that the amplitude criterion can be used for DSC measurements only, and cannot be extended to other methods which track the temperature-induced unfolding by a spectroscopic signal (like circular dichroism or fluorescence).