Humanized anti-tag-72 monoclonal antibodies using human...

Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...

Reexamination Certificate

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C424S001490, C424S001530, C424S138100, C424S156100, C424S178100, C435S007230, C530S387300, C530S388850, C530S391300, C530S391500, C530S391700, C530S866000, C530S867000

Reexamination Certificate

active

06495137

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to the fields of immunology and genetic engineering.
BACKGROUND OF THE INVENTION
The following information is provided for the purpose of making known information believed by the applicants to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the following information constitutes prior art against the present invention.
Antibodies are specific immunoglobulin (Ig) polypeptides produced by the vertebrate immune system in response to challenges by foreign proteins, glyco-proteins, cells, or other antigenic foreign substances. The binding specificity of such polypeptides to a particular antigen is highly refined, with each antibody being almost exclusively directed to the particular antigen which elicited it.
Two major methods of generating vertebrate antibodies are presently utilized: generation in situ by the mammalian B lymphocytes and generation in cell culture by B cell hybrids. Antibodies are generated in situ as a result of the differentiation of immature B lymphocytes into plasma cells (see Gough (1981),
Trends in Biochem Sci,
6:203). Even when only a single antigen is introduced into the immune system of a particular mammal, a uniform population of antibodies does not result, i.e., the response is polyclonal. The limited but inherent heterogeneity of polyclonal antibodies is overcome by the use of hybridoma technology to create “monoclonal” antibodies in cell cultures by B cell hybridomas (see Kohler and Milstein (1975),
Nature,
256:495-497). In this process, a mammal is injected with an antigen, and its relatively short-lived, or mortal, splenocytes or lymphocytes are fused with an immortal tumor cell line. The fusion produces hybrid cells or “hybridomas” which are both immortal and capable of producing the genetically-coded antibody of the B cell.
In many applications, the use of monoclonal antibodies produced in non-human animals is severely restricted where the monoclonal antibodies are to be used in humans. Repeated injections in humans of a “foreign” antibody, such as a mouse antibody, may lead to harmful hypersensitivity reactions, i.e., an anti-idiotypic, or anti-mouse antibody (HAMA), response (see Shawler et al. (1985),
Journal of Immunology,
135:1530-1535; and Sear et al.,
J. Biol. Resp. Modifiers,
3:138-150).
Various attempts have already been made to manufacture human-derived monoclonal antibodies by using human hybridomas (see Olsson et al. (1980),
Proc. Natl. Acad. Sci. USA,
77:5429; and Roder et al. (1986),
Methods in Enzymology,
121:140-167). Unfortunately, yields of monoclonal antibodies from human hybridoma cell lines are relatively low compared to mouse hybridomas. In addition, human cell lines expressing immunoglobulins are relatively unstable compared to mouse cell lines, and the antibody producing capability of these human cell lines is transient. Thus, while human immunoglobulins are highly desirable, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with required antigenic specificities can be easily obtained.
Thus, antibodies of nonhuman origin have been genetically engineered to create chimeric or humanized antibodies. Such genetic engineering results in antibodies with a reduced risk of a HAMA response compared to that expected after injection of a human patient with a mouse antibody. In a chimeric antibody, non-human regions of immunoglobulin constant sequences are replaced by corresponding human ones (see U.S. Pat. No. 4,816,567 to Cabilly et al., Genentech); in a humanized antibody, complementarity determining regions (CDRs) are grafted onto human framework regions (FR) (see European Patent Office Application (EPO) 0 239 400 to Winter). Some researchers have produced Fv antibodies (see U.S. Pat. No. 4,642,334 to Moore, DNAX) and single chain Fv (SCFV) antibodies (see U.S. Pat. No. 4,946,778 to Ladner, Genex).
The above patent publications only show the production of antibody fragments in which some portion of the variable domains is coded for by nonhuman V gene regions. Humanized antibodies to date still retain various portions of light and heavy chain variable regions of nonhuman origin: the chimeric, Fv and single chain Fv antibodies retain the entire variable region of nonhuman origin and CDR-grafted antibodies retain CDR of nonhuman origin.
Such nonhuman-derived regions are expected to elicit an immunogenic reaction when administered into a human patient (see Brüggemann et al. (1989),
J. Exp. Med.,
170:2153-2157; and Lo Buglio (1991), Sixth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, Calif.). Thus, it is most desirable to obtain a human variable region which is capable of binding to a selected antigen.
One known human carcinoma tumor antigen is tumor-associated glycoprotein-72 (TAG-72), as defined by monoclonal antibody B72.3 (see Thor et al. (1986)
Cancer Res.,
46:3118-3124; and Johnson, et al. (1986),
Cancer Res.,
46:850-857). TAG-72 is associated with the surface of certain tumor cells of human origin, specifically the LS174T tumor cell line (American Type Culture Collection (ATCC) No. CL 188), which is a variant of the LS180 (ATCC No. CL 187) colon adeno-carcinoma line.
Numerous murine monoclonal antibodies have been developed which have binding specificity for TAG-72. Exemplary murine monoclonal antibodies include the “CC” (colon cancer) monoclonal antibodies, which are a library of murine monoclonal antibodies developed using TAG-72 purified on an immunoaffinity column with an immobilized anti-TAG-72 antibody, B72.3 (ATCC HB-8108) (see EP 394277, to Schlom et al., National Cancer Institute). Certain CC antibodies were deposited with the ATCC: CC49 (ATCC No. HB 9459); CC83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458); CC92 (ATCC No. HB 9454); CC30 (ATCC NO. HB 9457); CC11 (ATCC No. 9455) and CC15 (ATCC No. HB 9460). Various antibodies of the CC series have been chimerized (see, for example, EPO 0 365 997 to Mezes et al., The Dow Chemical Company).
It is thus of great interest to develop antibodies against TAG-72 containing a light and/or heavy chain variable region(s) derived from human antibodies. However, the prior art simply does not teach recombinant and immunologic techniques capable of routinely producing an anti-TAG-72 antibody in which the light chain and/or the heavy chain variable regions have specificity and affinity for TAG-72 and which are derived from human sequences so as to elicit expectedly low or no HAMA response. It is known that the function of an immunoglobulin molecule is dependent on its three dimensional structure, which in turn is dependent on its primary amino acid sequence. A change of a few or even one amino acid can drastically affect the binding function of the antibody, i.e., the resultant antibodies are generally presumed to be a non-specific immunoglobulin (NSI), i.e., lacking in antibody character, (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al., Genentech).
Surprisingly, the present invention is capable of meeting many of these above mentioned needs and provides a method for supplying the desired antibodies. For example, in one aspect, the present invention provides a cell capable of expressing a composite antibody having binding specificity for TAG-72, said cell being transformed with (a) a DNA sequence encoding at least a portion of a light chain variable region (V
L
) effectively homologous to the human Subgroup IV germline gene (Hum4 V
L
); and a DNA sequence segment encoding at least a portion of a heavy chain variable region (V
H
) capable of combining with the V
L
into a three dimensional structure having the ability to bind to TAG-72. As is customary in the art, the term “Hum4 V
L
” which is itself derived from the designation of a protein—i.e. the variable domain of a light chain belonging to Subgroup IV of the class of human &kgr; light chains—can indicate this protein and/or the gene(s) or DNA sequence(s) which encode it.
In one aspect, the

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