Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1992-03-12
1994-09-20
Elliott, George C.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4353201, 5303873, 536 2353, C12N 510, C12N 1513, C12P 2108, C07K 1528
Patent
active
053488768
DESCRIPTION:
BRIEF SUMMARY
This invention relates to modified IgG3 antibodies having human constant regions, and to a novel antibody assay system.
Human IgG consists of four subclasses which show 5% homology in the amino acid sequence of the constant domains of their heavy chains. However, the hinge regions of these molecules differ both in length and in sequence. The length of the hinge is 15 amino acids for IgG1, 12 amino acids for IgG2 and IgG4, while it is 62 amino acids for IgG3, and the sequence homology is approximately 60%. The hinge region of IgG3 is encoded by four exons separated by short introns, and consists of a NH.sub.2 -terminal 17 amino acid residue segment followed by a 15 amino acid residue segment which is consecutively repeated three times [1-3].
The subclasses show differences in effector functions, and it has been suggested that some of these could be due to differences in the flexibility of the antibody molecule, and that this in turn depends on the length of the hinge, as well as interplay between the first domain in the constant heavy chain (C.sub.H 1) and the hinge [3-7].
It has been shown, both with myeloma protein [8] and with a set of recombinant IgG molecules with identical V regions [3,9,10] that among the four IgGs, IgG3 is the most efficient in the first step of the classical complement activation pathway, namely Clq binding. Clq binding to the antibody is the first step in the complement cascade which leads to cell lysis. This effect or mechanism is of particular importance in combating microbial infection and has a role in the destruction of cancerous cells.
It is thought that the upper part of the hinge determines the flexibility of the Fab arms relative to one another, allowing rotation of the Fab arms [11]. This flexibility may facilitate efficient aggregation of antibody molecules on an antigenic surface with several epitopes, resulting in several Clq binding sites coming in close proximity and thereby increasing the affinity of Clq for antibody. The middle of the hinge contains cysteines (which participate in interchain disulphide bridges) and a high content of proline, and therefore probably adopts a relatively rigid structure, separating the Fab and Fc portions of the molecule.
In order to establish whether the efficient binding of Clq to human IgG3 is due to the structure of its particular hinge region, we have made genetically engineered chimetic IgG3 antibodies containing human constant regions, the hinges of which have been shortened and in which the amino acid sequence may have been altered.
Surprisingly, we found that where the amino acid composition of the hinge region was unmodified Clq binding was substantially independent of both the total hinge length and the length of the so-called upper hinge of IgG3. The upper hinge is defined as the stretch from the end of the C.sub.H 1 domain to the first inter-heavy chain disulphide bridge [12]. An IgG3 mutant with a hinge of 15 amino acids bound Clq just as efficiently as one with a hinge of 47 amino acids. Both these IgG3 variants bound more Clq than the wild-type, and much more than IgG1.
By modifying the amino acid sequence of the shortened hinge region however we have found that Clq binding, and thus complement mediated cytolysis can be further improved. In particular in IgG3 molecules having human constant regions, where the hinge region has been modified to approximate the sequence of a human IgG4 hinge region, improvements of up to 100 times greater complement-mediated cytolytic capacity have been obtained. Thus as little as 1/100th of the concentration of this mutant antibody is required to give 50% lysis of target cells, compared to the wild type.
A first aspect of our invention therefore provides modified IgG3 antibodies containing human constant regions which have a shorter total-hinge region compared with normal human IgG3.
The IgG3 may be modified by reducing the number of repeats of the 15 amino acid residue segment or by removing the 17 amino acid residue segment or a combination of these deletions. This produces what are re
REFERENCES:
Bindon et al., "Human Monoclonal IgG Isotypes Differ in Complement Activating Function at the Level Of C4 as Well as C1q".
Gregory et al., "The Solution Conformations of the Subclasses of Human IgG Deduced From Sedimentation and Small Angle X-Ray Scattering Studies", pp. 821-829, 1987.
Schneider et al., "Genetically Engineered Immunoglobulins Reveal Structural Features Controlling Segmental Flexibility", pp. 2509-2513, 1988.
Sandlie et al., "C1q Binding to Chimeric Monoclonal IgG3 Antibodies Consisting of Mouse Variable Regions and Human Constant Regions with Shortened Hinge Containing 15 to 47 Amino Acids", pp. 1599-1603, 1989.
Garred et al., "The IgG Subclass Pattern of Complement Activation Depends on Epitope Density and Antibody and Complement Concentration", pp. 379-382, 1989.
Tan et al., "Influence of the Hinge Region on Complement Activation, C1q Binding, and Segmental Flexibility in Chimeric Human Immunoglobulins", pp. 162-166, 1990.
Morrison et al.; Annals of the New York Academy of Sciences; vol. 507, pp. 187-198; 1988.
Bindon, et al.; Journal of Experimental Medicine; vol. 168, pp. 127-142; 1988.
Michaelsen Terje
Sandlie Inger
Dynal AS
Elliott George C.
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