Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-02
2001-04-17
Reeves, Julie (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S328000, C530S387300, C530S395000, C514S008100, C536S023530, C536S124000
Reexamination Certificate
active
06218149
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
Immunochemical characterization of antibodies to alpha (1→6) dextran has given insights into the size and shape of the antibody combining site and the nature of the interaction between antibodies and antigen. In this regard, it would be useful to correlate the immunochemical properties of the anti-dextran antibodies with their primary structure. In the course of these studies, cDNAs from three monoclonal anti-alpha (1→6) dextran hybridoma cell lines, 14.6b.1, 5.54.4.24.1, and 19.22.1, were cloned (1) and the nucleotide sequences of the variable regions of the heavy chain (V
H
) and of the light chain (V
L
) determined (2) (see Table I on page 22 of this application). All synthesize an identical kappa light chain with the V
kappa
-OX1 germline gene (3) rearranged to the J
kappa
2 segment and the heavy chains differ by only one or two amino acids in their complementarity-determining regions (CDRs). When compared to 14.6b.1, 5.54.4.24.1 and 19.22.1 have an identical Thr→Asn amino acid change at position 60 in the variable region of the heavy chain (V
H
); 5.54.4.24.1 has an additional change (Ser→Gly) at position 31 in complementarity-determining region 1 (CDR1). The changes in heavy chain sequence result in 5.54.4.24.1 and 19.22.1 having a (ten) 10 fold or greater reduction in their binding constant for both polymeric dextran and isomaltoheptaose (IM7) when compared to 14.6b.1 (Table I).
The Thr→Asn change in 5.54.4.24 and 19.22.1 leads to the loss of a potential N-linked glycosylation site (Asn 58-Tyr 59-Thr 60) present in 14.6b.1. One of the purposes of this study and the present invention was to determine whether this potential N-linked glycosylation site is glycosylated, and if so, whether the addition of carbohydrate to complementarity-determining region 2 (CDR2) affects the binding constant for dextran. It is difficult to demonstrate glycosylation of V
H
directly since both Immunoglobulin A (IgA) and Immunoglobulin M (IgM) isotypes are glycosylated within their C
H
1 domains and carbohydrate present in Fd could be linked to either V
H
or constant region of the heavy chain (C
H
). Fd is the product resulting from the chemical or enzymatic cleavage of the antibody and comprises the heavy chain of the variable region and the heavy chain of the constant region of the antibody. Therefore, the three V
H
regions have been transferred to the human IgG
4
constant region which is devoid of carbohydrate in its C
H
1 domain. In this invention, the presence of carbohydrate is demonstrated to be within the V
H
of 14.6b.1. Comparison of the association constants for aglycosylated, tunicamycin treated and untreated antibodies shows that the presence of carbohydrate increases the apparent association constant (aKa) of 14.6b.1 for dextran. The effect on binding is unique to the carbohydrate present in V
H
since absence of carbohydrate from C
H
2 does not change the aKa for dextran.
By introducing into an antibody a carbohydrate recognition site for the attachment of carbohydrate, purification of the antibody can be enhanced because the carbohydrate is attached to the outside of the antibody and thus, is more accessible to binding by lectin (purification).
In this invention, the carbohydrate content of an antibody may be modified by adding or deleting carbohydrate recognition sites in the constant region of the antibody. In so doing, effector functions of the antibody are modified. Carbohydrate recognition sites in the constant region can also serve as sites for labelling, e.g., radionuclides, such as
125
I.
SUMMARY OF THE INVENTION
This invention concerns a method of altering the affinity of an antibody for the antigen to which it is directed which comprises introducing into the variable region of the antibody a carbohydrate recognition site under conditions such that a carbohydrate binds to the site and thus attaches to the antibody.
This invention also concerns a method of producing an antibody which may be more readily recovered or purified which comprises introducing into the variable region of the antibody a carbohydrate recognition site under conditions such that a carbohydrate binds to the site and thus attaches to the antibody.
This invention further provides an antibody which does not occur in nature and which comprises a carbohydrate recognition site genetically engineered into a variable region of the antibody which does not naturally include a carbohydrate recognition site in such variable region.
Still further, the present invention provides a method of modifying the carbohydrate content of an antibody which comprises deleting from a constant region of the antibody a carbohydrate recognition site which naturally occurs in such constant region of such antibody.
The invention also provides a method of modifying the carbohydrate content of an antibody which comprises adding to a constant region of the antibody a carbohydrate recognition site which does not naturally occur in such constant region of such antibody.
This invention further concerns a human antibody which does not occur in nature and which is characterized by the absence of a carbohydrate recognition site in a constant region of the antibody which naturally includes such a carbohydrate recognition site in such constant region.
Further, this invention provides a human antibody which does not occur in nature and which is characterized by the presence of a carbohydrate recognition site in a constant region of the antibody which does not naturally include such a carbohydrate recognition site in such constant region.
Finally, this invention provides therapeutic agents derived from, and DNA encoding, the antibodies, of the invention as well as sensitive methods for detecting the presence of substances in a sample, methods for recovering a substance from a sample containing the substance and for purifying such substance, and diagnostic test kits.
REFERENCES:
patent: 8807089 (1988-09-01), None
Alberts et al,Molec. Biol. of Cell, 3rdEdition, Garland Publ., 136 Madison Ave, Ny, Ny, 10016, pp 344-349 and 375, 1983.*
Tachibana et al Biochem Biphys Res Commun 189(2) 625, 1992.*
Endo et al Molec Immunology 32(13):931, 1995.*
Wright et al EMBO J vol. 10(1) 2717, 1991.*
Wright et al Springer Seminars in Immunology 15(2-3) 259, 1993.*
Knight et al BioTechnology vol. 17 No. 1, Jan. 1989.*
Molecular Biology Of The Cell, Alberts et al. ed. Garland Publishing , Inc., 136 Madison Ave, NY , NY, 10016, pp. 344-349 (1983).
Labeta et al. Immunology 57: 311-317 (1986). Structure of Asymmetric Non-Preciptating Antibody .*
Zoller et al., Methods of Enzymology 154:329 (1987). Olignucleotide-Directed Mutagenesis.*
Matsuuchi et al J of Immunology 125:5 2188-2190, 1981.*
Wigzell et al 1983, Proceedings of Natl. Acad. Sci. 80: 6632-6636.*
Kabat et al 1987 J of Immunology 138:12 4472-4479.*
Morrison and Kabat 1988 J of Experimental Medicine 168: 1099-1109.*
J. Sharon et al. (1986) PNAS 83:2628-2631.
J. Sharon et al. (Apr. 1988) J. Immunol. 140(8):2666-2669.
Weiss et al. (1985) Eur. J. Immunol. 15:768-772.
Akolkar et al. (1987) The Journ of Immunol. 138(12):4472-4479.
Wilke et al. (1987) Chemical Abstracts 107(7), abst. No. 57077n.
Morrison et al (1984) Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domanis, Proc Nat'l Acad Sci USA 81, 6851-6855.
Verhoeyen et al (Mar. 25, 1988) Reshaping human antibodies: grafting an antilysozme activity, Science 239, 1534-1536.
Reichmann et al (Mar. 24, 1988) Reshaping human antibodies for therapy, Nature 332, 323-327.
Abel et al (1986) The carbohydrate content of fragments and polypeptide chains of human &ggr;G-myeloma p
Hinton Paul R.
Morrison Sherie L.
Oi Vernon T.
Cooper & Dunham LLP
Reeves Julie
The Trustees of Columbus University in the City of New York
White John P.
LandOfFree
Antibodies having modified carbohydrate content and methods... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antibodies having modified carbohydrate content and methods..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibodies having modified carbohydrate content and methods... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2467051