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
1996-06-03
2001-07-24
Nolan, Patrick J. (Department: 1644)
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...
C435S007100, C435S975000
Reexamination Certificate
active
06265551
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to human T cell activation antigens.
BACKGROUND OF THE INVENTION
Human CD26, a Type II membrane glycoprotein with intrinsic dipeptidylpeptidase IV (DPPIV) activity and ability to bind Adenosine Deaminase Type I (ADA-1), is expressed on epithelial cells constitutively, but on T lymphocytes its expression is regulated.
Initially identified as a 105 kDa T cell activation antigen defined by the monoclonal antibody Ta1 (
1
), the CD26 antigen was subsequently shown to delineate the T cell subset responding to recall antigens (
2
,
3
). Although the antigen is expressed in the liver, kidney and intestine (
4
), only in the T cell are the levels of membrane CD26 under tight cellular regulation with expression upregulated upon cell activation. CD26 has been shown to have dipeptidylpeptidase IV activity (DPPIV, EC 3.4.14.5) in its extracellular domain (
5
,
6
) and the costimulatory potential appears to be partially dependent upon this enzyme activity (
7
) which can cleave amino terminal dipeptides with proline, and less effectively, alanine in the penultimate position (
8
). Although a substrate of relevance to T cell activation has not yet been identified, other substrates, including the neuropeptide substance P, may be processed in vivo by DPPIV/CD26 (
9
).
CD26 not only marks the activated state but is itself involved in the signal transducing process: crosslinking of CD3 and CD26 results in enhanced T cell activation in the absence of antigen-presenting cells (
10
). It is unlikely that CD26 is directly involved in transducing the activation signal across the T cell membrane since it has only a very short cytoplasmic region of 6 amino acids (
11
). The protein tyrosine phosphatase, CD45RO, has been shown to associate with CD26 and may provide a putative mechanism for the costimulation (
12
). Other associations include the strong binding of Adenosine Deaminase Type I (ADA-1) to CD26 (
13
). This may be of particular importance since ADA activity helps regulate the early stages of signal transduction in T lymphocytes (
14
). That the costimulatory potential of CD26 occurs extracellularly has been confirmed by showing that a soluble recombinant CD26 (rsCD26) representing the extracellular domain can enhance the T cell-mediated reaction to recall antigens (
15
). Reinforcing the proposal that soluble CD26 is costimulating, it has been found that in the absence of recall antigen, the rsCD26 has no effect upon the proliferative response. A natural form of soluble DPPIV/CD26 can be identified in normal human serum. The levels of this naturally-occurring soluble DPPIV influenced the level of reactivity of T cells to recall antigens (
15
).
SUMMARY OF THE INVENTION
We have now isolated soluble DPPIV/CD26 from human serum and have unexpectedly determined that while the serum form shares similar enzymatic and antigenic properties with the membrane form, in several biochemical aspects there are distinct differences. In particular, the soluble form has a molecular weight of 175 kDa, and it does not bind ADA-1. Nevertheless, it retains the ability to costimulate the T lymphocyte response to the recall antigen, tetanus toxoid. Furthermore, N-terminal sequencing of the resulting peptides after tryptic digestion suggested structural disparity between membrane CD26 and soluble serum DPPIV. Accordingly, we suggest that although 105 kDa membrane type CD26 may be found in the serum in small amounts, the majority of serum DPPIV activity is provided by a novel peptidase structurally distinct from DPPIV/CD26.
Thus, in one aspect the invention features a substantially purified glycoprotein, a monomer of the glycoprotein having a molecular weight of approximately 175 kDa, of which approximately 130 kDa is the unglycosylated moiety. The glycoprotein of the invention has an antigenic determinant or determinants immunologically cross-reactive with determinants of CD26, is capable of expressing functional dipeptidylpeptidase IV (DPPIV) activity, but is not capable of binding Adenosine Deaminase Type-1 (ADA-1). Preferably, the glycoprotein of the invention exists in the form of a trimer and contains N-linked glycosylation but not O-linked glycosylation.
By “substantially pure” is meant a polypeptide or protein which has been separated from biological macromolecules (e.g., other proteins, carbohydrates, etc.) with which it naturally occurs. Typically, a protein or polypeptide of interest is substantially pure when less than 25% (preferably less than 15%) of the dry weight of the sample consists of such other molecules.
The invention furthermore features the unglycosylated moiety of the above glycoprotein as a substantially purified protein having a molecular weight of approximately 130 kDa.
Also featured is a therapeutic composition containing the DPPIV/CD26 glycoprotein of the invention in a pharmaceutically acceptable carrier (e.g., saline or any aqueous or nonaqueous substance which is suitable for injection). Such a therapeutic composition can be used in a method for modulating the immune response of a patient (e.g., enhancing the immune response of an immunosuppressed patient) by administering the composition to the patient by an appropriate means. It is expected to be particularly useful for the treatment of immunosuppression in a patient infected with human immunodeficiency virus (HIV) and having the acquired immune deficiency syndrome (AIDS) or AIDS-related complex, but may also be used where the patient's immune system is depressed as a result of treatment with an immunosuppressive compound, or acquired immunodeficiency of undetermined etiology, or congenital immunodeficiency, such as autoimmune diseases. The soluble, naturally occurring high molecular weight form of DPPIV/CD26 disclosed herein will be particularly useful because the type and extent of glycosylation is the naturally occurring pattern. Therefore, the high molecular weight form should have a longer half life than any soluble recombinant form of CD26.
The compounds of the invention, when combined with a pharmaceutically acceptable carrier, are also useful as vaccine adjuvants, to be administered to an individual vaccine in conjunction with (i.e., immediately before, after, or along with) a vaccine antigen in order to enhance the immune response produced by such antigen. Examples of vaccine antigens which may be used with the adjuvant of the invention include those containing chemically inactivated or genetically engineered viral or bacterial products, such as diptheria or pertussin toxoid or recombinant viral proteins, and those containing live but attenuated virus or bacteria.
Fragments of serum soluble DPPIV/CD26 can be assayed for costimulatory activity. One such assay would include the following steps: (a) contacting a lymphocyte with a candidate fragment of serum soluble DPPIV/CD26, and (b) determining whether the fragment increases the rate of proliferation of the lymphocyte in response to antigenic stimuli, such increase being an indication of costimulatory activity.
The invention also features nucleic acid encoding 175 kDa DPPIV/CD26, which is useful for transferring expression of the 175 kDa form, either in vivo or in vitro, to cells lacking such capability.
In another aspect the invention features antibodies, polyclonal or monoclonal, and immunoreactive fragments and derivatives thereof, that bind specifically to the 175 kDa form but not to the 105 kDa form of DPPIV/CD26. An individual such antibody recognizes a unique epitope on the 175 kDa form of DPPIV. As used herein, the phrase “unique epitope” refers to any epitope on the 175 kDa form of DPPIV/CD26 that is not found on the 105 kDa form. The antibodies, fragments and derivatives of the invention are capable of differentiating these two forms of DPPIV/CD26 and are, therefore, useful reagents for identifying, quantifying and/or purifying activated T lymphocytes and also for diagnosis or for monitoring the course of various immune diseases or disorders. For example, an antibody of the invention can serve as a diagnostic ag
Duke-Cohan Jonathan S.
Morimoto Chikao
Schlossman Stuart F.
Dana-Farber Cancer Institute Inc.
Fish & Richardson P.C.
Nolan Patrick J.
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