Vernon T. Oi

Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and Ia Antigens

Advances in somatic cell hybridization techniques have made it possible to generate hybrid cell lines producing monospecific antibodies directed at desired antigenic determinants (1). In this paper a modification of the cell fusion procedure (2,3) was used to recover stable hybrid cell lines secreting IgG antibodies to: (a) mouse major histocompatibility complex (MHC) alloantigens (H-2K and I-A); and (b) mouse immunoglobulin (Ig) allotypes (Ig-1b, Ig-5a, and Ig-5b).

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen‐combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

Genetically engineered antibody molecules.

Publisher Summary The availability of monoclonally derived hybridoma antibodies provided an infinite supply of homogeneous reagents so that laboratories around the world could use standardized procedures. However, some limitations persisted. One approach to producing hybridoma antibodies with more desirable biological effector functions or antigen-binding specificities has been to take advantage of the high rate of somatic mutation which occurs in cultured myeloma and hybridoma cell lines. The limitation inherent in this approach is that it is possible only to switch to isotypes that lie downstream of the expressed heavy chain gene isotype in the heavy chain gene complex. An alternative approach to producing improved monoclonal antibodies is to use recombinant deoxyribonucleic acid (DNA) techniques and eukaryotic gene expression methods, the objective being to produce antibodies with improved antigen specificities and effector functions. An advantage of this approach is that producing antibodies is not a limitation as they exist in nature. Instead, design and production of antibody molecules can be done with optimized specificities and effector functions. Antigen-binding specificities can be joined to both immunoglobulin and nonimmunoglobulin sequences to provide antibody and antibody-like molecules for use in diagnosis and immunotherapy. Standard techniques of genetic engineering and gene transfection make it feasible to produce antigen-binding molecules with widely varying structures. The challenge is to define the structure that is optimal for a desired function.

Localization of murine Ig-1b and Ig-1a (IgG 2a) allotypic determinants detected with monoclonal antibodies

We have used hybridoma anti-allotype antibodies to define six allotypic determinants: four on Ig-1b molecules and two on Ig-1a molecules. By locating these determinants on fragments obtained by limited proteolysis of IgG2a, molecules and by hybridoma antibody blocking assays, five distinct allotypic sites have been defined on mouse γ2a heavy chains. These sites are located in the hinge region and the CH2 and CH3 domains. We have not been able to show genetic recombination between these sites by genetic linkage studies.

Rotational dynamics of monoclonal anti-dansyl immunoglobulins☆

The rotational motions of monoclonal mouse anti-dansyl immunoglobulins were studied by nanosecond fluorescence emission anisotropic spectroscopy using a mode-locked argon-ion laser as the pulsed excitation source. Three homogeneous antibodies of the immunoglobulin Gl (IgGl) subclass containing different V regions were prepared. The fluorescence emission maxima of these antibodies (designated as DNS1, DNS2 and DNS3) are at 515, 480 and 500 nm, respectively. Their mean rotational correlation times, 〈φ〉, are 84, 109 and 96 ns, respectively. The binding of protein A or a monoclonal anti-allotype antibody to the Fc unit of DNS1 increased 〈φ〉 to 142 and 150 ns, respectively, whereas reduction of the disulfide bond between the heavy chains decreased 〈φ〉 to 48 ns. These nanosecond measurements show that the rotational motion of the Fab arms in mouse IgGl is restricted.

Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.

Nanosecond Motions Of Genetically-Engineered Antibodies: Structural Elements Controlling Segmental Flexibility Defined By Time-Resolved Emission Anisotropy

Immunoglobulins are flexible proteins which display large-amplitude modes of motion on a nanosecond time scale. Different classes of antibodies differ markedly in their degree of segmental flexibility; a number of their essential biological functions are correlated with their nanosecond internal dynamics. These motions can be conveniently monitored by time-resolved fluorescence anisotropy measurements. An instrument built around a synch-pumped cavity-dumped dye laser and a fast time-to-digital convertor with histogramming memory has made it possible to obtain high-quality anisotropy in a few minutes on small amounts (ca. 100 pmol) of protein. Genetic engineering techniques have made it possible to construct a large number of immunoglobulins with identical binding sites for the fluorescent probe dansyllysine. These proteins differ in the heavy chain regions which are responsible for their biological effector functions and their segmental flexibility. We have analyzed a series of such constructs derived by genetic recombination between the genes coding for the mouse isotypes IgG1 and IgG2a. The results identify two regions responsible for their different degrees of segmental flexibility: the hinge region connecting the Fab and Fc portions of the antibodies, and a short stretch (residues 131-139) of sequence in the amino terminal half of the CH1 domain containing five amino acid substitutions.

Human:Mouse Antibody-Producing Transfectoma Cell Lines

A new means to generate antibodies (Ab’s) with desired antigen-specificities and biological effector functions is to create transfectoma Ab-producing cell lines. These cell lines are derived from lymphoid cell lines that have been transfected with novel immunoglobulin genes using DNA-mediated gene transfer techniques. The context is our attempts at understanding the regulation of immunoglobulin gene expression. A direct outcome of these studies has been a means of generating human Ab molecules with mouse antigen-specificities.