Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-08-01
2004-12-21
Helms, Larry R. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S350000, C424S130100
Reexamination Certificate
active
06833441
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of immunology. Specifically, the invention relates to the generation of chimeric heteromultimers such as non-single-chain antigen-binding units using unique heterodimerization sequences. This invention also relates to the generation of single-chain antigen-binding units stabilized by the subject heterodimerization sequences. The compositions and methods embodied in the present invention are particularly useful for identifying antigen-binding units that are of major diagnostic and/or therapeutic potential.
BACKGROUND OF THE INVENTION
Antibodies or immunoglobulins are molecules that recognize and bind to specific cognate antigens. Because of their exclusive specificities, antibodies, particularly monoclonal antibodies, have been widely used in the diagnosis and treatment of a variety of human diseases.
The basic immunoglobulin (Ig) in vertebrate systems is composed of two identical light (“L”) chain polypeptides (approximately 23 kDa), and two identical heavy (“H”) chain polypeptides (approximately 53 to 70 kDa). The four chains are joined by disulfide bonds in a “Y” configuration. At the base of the Y, the two H chains are bound by covalent disulfide linkages. The L and H chains are organized in a series of domains. The L chain has two domains, corresponding to the C region (“CL”) and the other to the V region (“VL”). The H chain has four domains, one corresponding to the V region (“VH”) and three domains (CH1, CH2 and CH3) in the C region. The antibody contains two arms (each arm being a Fab fragment), each of which has a VL and a VH region associated with each other. It is this pair of V regions (VL and VH) that differ, from one antibody to another (due to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen-binding site. More specifically, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDR's are the most variable part of the variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation and selection.
Research in recent years has demonstrated that the function of a binding antigen can be performed by fragments of a whole antibody. Exemplary antigen binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the dAb fragment (Ward, E. S. et al,
Nature
341, 544-546 (1989) which consists of a VH domain; (iv) isolated CDR regions; and (v) F(ab′)
2
fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (vi) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is the smallest functional unit required for high affinity binding of antigen.
One major challenge in the antibody field has been to reconstitute a vast diverse repertoire of immunoglobulins that mimics the immunoglobulin pool in the human immune system. Such a repertoire generally has a complexity ranging from 10
8
to 10
13
distinct immunoglobulins. The generation of such a repertoire would greatly facilitate the identification and production of immunoglobulins capable of interacting specifically with therapeutic targets. However, the design and production of such a repertoire has traditionally been hampered by the lack of a stabilizing means for assembly of the minimal functional unit, namely the Fv fragment. It is a well-known problem in the art that the VH and VL regions, when expressed alone, have very low interaction energy (Glockshuber et al (1990)
Biochemistry
29(6):1362-1367). The two components dissociate at low protein concentrations and are too unstable for many applications at physiological body temperature. It is also a long-recognized technical obstacle that large proteins, such as whole antibodies (albeit extremely stable), do not express at an appreciable level in the host cell, thus rendering the construction of a highly diverse antibody repertoire very difficult.
More recently, three approaches have been developed to generate stable VL and VH complexes. However, each of these techniques bears a number of intrinsic limitations; and none of them circumvents the aforementioned technical hurdles completely. The first approach uses a peptide linker to connect the VL and VH as a single-chain (“scFv”) (Huston et al (1988)
Proc. Natl. Acad. Sci. U.S.A
85:5879-5883). While the resulting scFv exhibits substantial antigen-binding activity, not all antibodies can be made as single chains and still retain high binding affinity (Huston et al (1988)
Proc. Natl. Acad. Sci. U.S.A.
85:5879-5883; Stemmer et al (1993)
Biotechniques
14(2): 256-265). In part, this is due to the interference of linker sequences with the antigen binding sites. The second approach involves inserting a pair of cysteine residues in the VL and VH regions to generate a disulfide-bond stabilized Fv (“dsFv”) (Brinkmann et al (1993)
Proc. Natl. Acad. Sci. U.S.A.
90(16): 7538-7542). The incorporated disulfide linkage, however, is unstable under reducing conditions in many host cells. For instance, in cytosol of
E. Coli
, the inter-molecular disulfide bond is often insufficient to stabilize the VL and VH complex. Moreover, this method typically requires 3-dimensional structural information of the V regions to ensure that the cysteine pair is inserted in a proper place without disruption the binding activity. Because the 3-dimensional information of a vast majority of the existing antibodies is unknown, this approach has little practical utility, and is particularly unsuited for antibody library construction, especially for constructing antibody repertoires derived from B cells. The third approach for stabilizing the VL and VH regions utilizes the disulfide bonds native to the CH1 and CL domains. This method proceeds with grafting a disulfide-bond linked CH1 and CL domains to the C-termini of the VL and VH regions in order to reconstitute a Fab fragment. While the resulting Fab fragment is generally more stable and often exhibits higher binding affinity than scFv, Fab is not optimal for high level expression and antibody repertoire construction due to its large size.
Certain dimerization sequences that form coiled-coil structures have also been employed to assemble multivalent antibodies. Specifically, U.S. Pat. No. 5,932,448 describes a bispecific F(ab′)
2
heterodimer linked by the Fos and Jun leucine zippers. The Fos and Jun leucine zippers are well-characterized sequences known to preferentially form heterodimers. However, they still exhibit significant propensity to form homodimers under physiological buffer conditions and/or at physiological body temperature (O'Shea et al. (1992)
Cell
68: 699-708; Vidal et al. (1996)
Proc. Natl. Acad. Sci. U.S.A.
). In fact, the Jun/Jun homodimer is so stable that formation of Fos/Jun heterodimer in vitro requires dissociation of the Jun/Jun homodimer by first heating or reduction with 2-mercaptoethanylamine (see U.S. Pat. No. 5,910,573 column 7 lines 35-37; U.S. Pat. No. 5,932,448, column 16 lines 15-30). When tested in vivo, both Fos and Jun yield detectable amounts of homodimers (see, e.g. column 15, lines 41-43 of U.S. Pat. No. 5,932,448; and Vidal et al. (1996)
Proc. Natl. Acad. Sci. U.S.A
). While the existence of some homodimerization propensity may not be of substantial concern for the production of a single antibody species, such propensity presents a serious problem for antibody repertoire construction, where high efficiency of heterodimerization between VL and VH regions is required.
Aside from Fos and Jun leucine zippers, U.S. Pat. No. 5,824,483 by Houston et al. describes the construction of a combinatorial library of coiled-coil dimerization peptides. Houston et al. proposes that the library is useful for identifying a polypeptide that is capabl
Li Shengfeng
Liu Shengjiang
Luo Peizhi
Wang Caili
Wang Xinwei
Abmaxis, Inc.
Helms Larry R.
Wong Karen K.
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