Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-10
2004-12-21
Wehbé, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S325000, C536S023100, C800S013000, C800S018000
Reexamination Certificate
active
06833268
ABSTRACT:
BACKGROUND OF THE INVENTION
A quarter century after the discovery of monoclonal antibodies (mAbs) [G. Kohler and C. Milstein,
Nature
256:495-497 (1975)], their therapeutic utility is finally being realized. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. Many monoclonal antibodies are in late stage clinical trials to treat a broad range of disease indications. As a result, mAbs represent one of the largest classes of drugs currently in development.
The utility of mAbs stems from their specific recognition of a complex target followed by high affinity binding to that target. Because different C
H
isotypes have different effector functions, it is desirable to tailor the mAb isotype to the desired effector function. For, example, a mAb bearing a constant region with effector functions, e.g., human IgG
1
, can be used to direct complement dependent cytotoxicity or antibody-dependent cytotoxicity to a target cell. Alternatively, a mAb with a constant region essentially lacking effector function, e.g., human IgG
2
or IgG
4
, can be used to block signal transduction, either by binding to and neutralizing a ligand, or by blocking a receptor binding site.
Many therapeutic applications for monoclonal antibodies require repeated administrations, especially for chronic diseases such as autoimmunity or cancer. Because mice are convenient for immunization and recognize most human antigens as foreign, mAbs against human targets with therapeutic potential have typically been of murine origin. However, murine mAbs have inherent disadvantages as human therapeutics. They require more frequent dosing to maintain a therapeutic level of mAb because of a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine immunoglobulin creates the likelihood that the human immune system will recognize the mouse protein as foreign, generating a human anti-mouse antibody (HAMA) response. At best, a HAMA response will result in a rapid clearance of the murine antibody upon repeated administration, rendering the therapeutic useless. More likely is that a HAMA response can cause a severe allergic reaction. This possibility of reduced efficacy and safety has lead to the development of a number of technologies for reducing the immunogenicity of murine mAbs.
In order to reduce the immunogenicity of antibodies generated in mice, various attempts have been made to replace murine protein sequences with human protein sequences in a process now known as humanization. The first humanization attempts utilized molecular biology techniques to construct recombinant antibodies. For example, the complementarity determining regions (CDR) from a mouse antibody specific for a hapten were grafted onto a human antibody framework, effecting a CDR replacement. The new antibody retained the binding specificity conveyed by the CDR sequences. [See P. T. Jones et al.
Nature
321: 522-525 (1986)]. The next level of humanization involved combining an entire mouse VH region (HuVnp) with a human constant region such as &ggr;1. [S. L. Morrison et al.,
Proc. Natl. Acad. Sci.,
81, pp. 6851-6855 (1984)]. Such chimeric antibodies, which still contain greater than 30% xenogeneic sequences, are sometimes only marginally less immunogenic than totally xenogeneic antibodies. [M. Bruggemann et al.,
J. Exp. Med.,
170, pp. 2153-2157 (1989)].
Subsequently, attempts were carried out to introduce human immunoglobulin genes into the mouse, thus creating transgenic mice capable of responding to antigens with antibodies having human sequences. [See Bruggemann et al.
Proc. Nat'l. Acad. Sci.
USA 86:6709-6713 (1989)]. These attempts were thought to be limited by the amount of DNA which could be stably maintained by available cloning vehicles. As a result, many investigators concentrated on producing mini-loci containing limited numbers of V region genes and having altered spatial distances between genes as compared to the natural or germline configuration. [See U.S. Pat. No. 5,569,825 to Lonberg et al., (1996)]. These studies indicated that producing human sequence antibodies in mice is possible, but serious obstacles remained regarding obtaining sufficient diversity of binding specificities and effector functions (isotypes) from these transgenic animals to meet the growing demand for antibody therapeutics.
In order to provide additional diversity, work has been conducted to add large germline fragments of the human Ig locus into transgenic mammals. For example, a majority of the human V, D, and J region genes arranged with the same spacing found in the unrearranged germline of the human genome and the human C
&mgr;
and C
&dgr;
constant regions was introduced into mice using yeast artificial chromosome (YAC) cloning vectors. [See PCT patent application WO 94/02602 to Kucherlapati et al.]. A 22 kb DNA fragment comprising sequences encoding a human gamma-2 constant region and the upstream sequences required for class-switch recombination was latter appended to the foregoing transgene. In addition, a portion of a human kappa locus comprising V
k
, J
k
and C
k
region genes, also arranged with substantially the same spacing found in the unrearranged germline of the human genome, was introduced into mice using YACS. Gene targeting was used to inactivate the murine IgH & kappa light chain immunoglobulin gene loci and such knockout strains were bred with the above transgenic strains to generate a line of mice having the human V, D, J, C
&mgr;
, C
&dgr;
and C&ggr;2 constant regions as well as the human V
k
, J
k
and C
k
region genes all on an inactivated murine immunoglobulin background. [See PCT patent application WO 94/02602 to Kucherlapati et al.; see also Mendez et al.,
Nature Genetics
15:146-156 (1997)].
Yeast artificial chromosomes as cloning vectors in combination with gene targeting of endogenous loci and breeding of transgenic strains provided one solution to the problem of antibody diversity. Several advantages were obtained by this approach. One advantage was that YACs can be used to transfer hundreds of kilobases of DNA into a host cell. Therefore, use of YAC cloning vehicles allows inclusion of substantial portions of the entire human Ig Heavy and light chain regions into a transgenic animal thus approaching the level of potential diversity available in the human. Another advantage of this approach is that the large number of V genes has been shown to restore full B cell development in mice deficient in murin immunoglobulin production. This ensures tht thers reconstituted mice are provided with the requisite cells for mounting a robust human antibody response to any given immunogen. [See PCT patent application WO 94/02602 to Kucherlapati et al.; L. Green and A. Jakobovits,
J. Exp. Med.
188:483-495 (1998)]. A further advantage is that sequences can be deleted or inserted onot the YAC by utilizing high frequency homologous recombination in yeast. This provides for facile engineering of the YAC transgenes.
As mentioned above, there are several strategies that exist for the generation of mammals that produce human antibodies. In particular, there is the “minilocus” approach that is typified by work of GenPharm International, Inc. and the Medical Research Council, YAC introduction of large and substantially germline fragments of the Ig loci that is typified by work of Abgenix, Inc. (formerly Cell Genesys), and introduction of entire or substantially entire loci through the use microcell fusion as typified by work of Kirin Beer Kabushiki Kaisha. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V
H
genes, one or more D
H
genes, one or more J
H
genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This
Davis C. Geoffrey
Green Larry L.
Ivanov Vladimir E.
Abgenix, Inc.
Fish & Neave
Wehbe Anne M.
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