Heterofunctional cellular immunological reagents, vaccines containing same and methods for the use of same

The present invention relates to a heterofunctional cellular immunological reagent comprising at least two T cell specific binding ligands covalently linked together, wherein one of the T cell specific binding ligands binds to a specific class or subclass of T cells and another of the T cell specific binding ligands is an antigen associated with disease or a causative agent of disease, or epitope thereof. The present invention also relates to vaccines containing the heterofunctional cellular immunological reagents and methods for the use of the same.

FIELD OF THE INVENTION 
The present invention relates to a heterofunctional cellular immunological 
reagent comprising at least two T cell specific binding ligands covalently 
linked together, wherein one of the T cell specific binding ligands binds 
to a specific class or subclass of T cells and another of the T cell 
specific binding ligands is an antigen associated with disease or a 
causative agent of disease, or epitope thereof. The present invention also 
relates to vaccines containing the heterofunctional cellular immunological 
reagents and methods for the use of the same. 
BACKGROUND OF THE INVENTION 
In cell mediated immunity, a disease causing agent, such as a virus, is 
engulfed by a specialized cell called the antigen presenting cell 
(hereinafter "APC"). The APC breaks up the virus and fragments the 
antigenic determinants of the virus, i.e., viral specific polypeptides, 
into polypeptide fragments. These fragmented antigenic determinants are 
then transported to the cell surface of the APC. At this time, the APC 
also produces or modifies the major histocompatability complex molecules 
Class I and Class II (hereinafter "MHC Class I" or "MHC Class II", 
respectively), which are heavily involved in cell mediated immunity and 
which are produced within and transported to the surface of the APC. MHC 
Class I molecules specifically bind to cytotoxic/suppressor T cells (Tc/s) 
and MHC Class II molecules specifically bind to helper/accessory T cells 
(Th). The MHC molecules contain at least two binding sites, i.e., an 
antigen binding site known as agretope binding site, which is highly 
variable between MHC molecules for different agretopes, and a site which 
binds to the T cell, i.e., the T cell specific binding ligand, which is 
highly conserved (see Bjorkman, P. J. et al, Nature, 329: 506 (1987) and 
Bjorkman, P. J. et al, Nature, 329: 512 (1987)). 
T cells are activated by the combination of (1) the binding of the 
fragmented antigenic determinants, present on the surface of the APC, to 
the surface of the T cells and (2) the binding of the highly conserved 
region of the MHC molecules, present on the surface of the APC, to the 
surface of the T cells. Usually, binding by the fragmented antigenic 
determinants or the highly conserved region of the MHC molecules alone to 
the surface of the T cells does not give rise to activation of the T cells 
since to do so would give rise to an unregulated and indiscriminate 
polyclonal activation of most, if not all, T cells and could result in 
pathogenic conditions. The binding of the MHC molecules to the surface of 
the T cells is in part mediated through the agretope, i.e., the binding of 
the antigen to the MHC molecules acts as a signal to the MHC molecules to 
bind to the surface of the T cells (see Bjorkman, P. J., Nature, 229: 506 
(1987)). In addition, the MHC molecule contains recognition sites so that 
unless the APC and the T cell contain the same MHC molecules with the same 
genetic composition, they are recognized as "not-self" and the desired 
interaction cannot successfully occur. The resulting activated T cells can 
then recognize the disease causing or associated agent, e.g., virus 
infected cell, tumor cell, etc., in the bloodstream or elsewhere, as 
foreign and acts to kill such. This gives rise to cell mediated immunity 
to the disease caused by, e.g., the virus, without any antibody, or 
humoral immunity, involvement. 
The APC, typically a macrophage, also produces and releases Interleukin 1 
(hereinafter "IL-1") as a consequence of the interaction and processing of 
the antigen. IL-1 interacts with the T cell as a part of the activation 
process of the T cell. IL-1 causes activated T cells to produce 
Interleukin-2 (hereinafter "IL-2"). However, as with most hormones, IL-1 
activity is generalized, i.e., it is not specific to a particular antigen 
but, rather, is involved in invoking a generalized inflammatory response. 
Since MHC molecules are very large and highly polymorphic, antigenicity 
problems arise when administering such to a subject. Further, there is a 
high variability of agretopes and MHC molecules. Thus, it is difficult to 
isolate an appropriate MHC molecule for a disease causing or associated 
agent of interest so as to be able to form a complex thereof which can 
thereby activate T cells specific to a disease of interest. 
In an embodiment of the present invention, the above-discussed problem is 
overcome by employing only a portion of the MHC molecules which bind to T 
cells, i.e., the highly conserved region thereof, and covalently linking 
such to an antigen associated with disease or causative agent of disease, 
or epitope thereof of interest, thereby forming a heterofunctional 
cellular immunological reagent and avoiding the necessity of isolating 
suitable or using large and polymorphic, MHC molecules. Further, in some 
cases, one of the reasons for a failure to respond to an antigen is a lack 
of antigen processing and/or appropriate MHC molecules. The 
heterofunctional cellular immunological reagent of the present invention 
overcomes this problem. 
The clinical and industrial immunologists working in AIDS have not focused 
on the correlation of cell mediated immunity and disease since most of 
their assays are based on humoral immunity mechanisms. Cell mediated 
immunity, because of its slow reactions, the requirement for a living cell 
derived from an intact host and the MHC restriction inherent in the system 
have deflected attention away from cell mediated immunity. Cell mediated 
immunity has remained, therefore, somewhat of a "black box" with inputs 
and outputs defined but little understood in the way of internal 
mechanisms. 
The rapidity of the re-activation of AIDS or herpes viruses indicates that 
re-activation cannot be a result of the breakdown of humoral response 
mechanisms. That is, re-activation occurs in a short number of days while 
serum antibodies are still abundant. In part because of the above 
circumstantial evidence in humoral response and in part because of other 
evidence in cellular mechanisms, a breakdown of cell mediated response 
mechanisms is implicated in re-activation of these disease causing agents. 
In the case of tuberculosis (hereinafter "TB"), another disease where 
cellular immunity is paramount, IL-2, also known as T cell growth factor, 
can restore the in vitro cellular immune response to the mycobacterium. 
Since IL-1 is available in subjects afflicted with TB, a defect in the 
stimulation of IL-2 production by the cells implies a failure of the APC 
presentation or recognition process. 
Exogenously provided IL-2 can restore, at least in part, in an in vitro 
assay system with AIDS patients, cell mediated immunity activity to Human 
Immunodeficiency Virus (hereinafter "HIV"). This suggests that with AIDS 
there is also a deficiency in IL-2 production, even though IL-1 production 
is above normal. AIDS infected patients also have either poor or 
ineffective antibody dependent cellular cytotoxicity (hereinafter "ADCC"), 
antibody complement cytotoxicity (hereinafter "ACC") or natural killer 
(hereinafter "NK") activity, even at early stages before any clinical 
signs of AIDS related complex (hereinafter "ARC") or AIDS. 
In an embodiment of the present invention, a vaccine for diseases, such as 
AIDS, is provided which specifically stimulates cellular immunity to 
diseases, such as AIDS. 
There are currently a series of in vitro assays for cell mediated immunity 
which use cells from the host both as the substrate cell that initiates or 
stimulates the reaction for which the assay has been developed and as the 
target to assess cell mediated immunity. These in vitro assays include the 
cytotoxic T lymphocyte assay (hereinafter "CTL"); lymphoproliferative 
assays, e.g., tritiated thymidine incorporation; the protein kinase 
assays, the ion transport assay and the lymphocyte migration inhibition 
function assay (hereinafter "LIF") (Hickling, J. K. et al, J. Virol., 61: 
3463 (1987); Hengel, H. et al, J. Immunol., 139: 4196 (1987); 
Thorley-Lawson, D. A. et al, Proc. Natl. Acad. Sci. USA, 84: 5384 (1987); 
Kadival, G. J. et al, J. Immunol., 139: 2447 (1987); Samuelson, L. E. et 
el, J. Immunol., 139: 2708 (1987); Cason, J. et al, J. Immunol. Meth., 
102: 109 (1987); and Tsein, R. J. et al, Nature, 293: 68 (1982)). These 
assays are disadvantageous in that they may lack true specificity for cell 
mediated immunity activity, they require antigen processing and 
presentation by an APC of the same MHC type, they are slow (sometimes 
lasting several days), and some are subjective and/or require the use of 
radioisotopes. 
In an embodiment of the present invention, a diagnostic assay for cell 
mediated immunity is provided which overcomes the above-described problem. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a heterofunctional 
cellular immunological reagent which specifically interacts with the 
cellular immune system. 
Another object of the present invention is to provide a vaccine for the 
prevention or treatment of disease by stimulating cellular immunity to the 
disease using the heterofunctional cellular immunological reagent. 
Still another object of the present invention is to provide a method of 
prevention or treatment of disease by stimulating cellular immunity to the 
disease using the heterofunctional cellular immunological reagent. 
A further object of the present invention is to provide a method of 
diagnosis of disease by assaying for the presence of T cells, which are 
active against the disease, using the heterofunctional cellular 
immunological reagent. 
In one embodiment of the present invention, the above-described objects 
have been met by a heterofunctional cellular immunological reagent 
comprising at least two T cell specific binding ligands covalently linked 
together, wherein one of the T cell specific binding ligands binds to a 
specific class or subclass of T cells and another of the T cell specific 
binding ligands is an antigen associated with disease or a causative agent 
of disease, or epitope thereof. 
In a second embodiment, the above-described objects of the present 
invention have been met by a vaccine for the prevention or treatment of 
disease comprising, as an active ingredient, a pharmaceutically effective 
amount of a heterofunctional cellular immunological reagent and a 
pharmaceutically acceptable carrier or diluent. 
In a third embodiment, the above-described objects of the present invention 
have been met by a method of prevention of disease comprising 
administering the vaccine to a disease susceptible subject. 
In a fourth embodiment, the above-described objects of the present 
invention have been met by a method of treatment of disease comprising 
administering the vaccine to a subject afflicted with the disease. 
In a fifth embodiment, the above-described objects of the present invention 
have been met by a method of diagnosing disease comprising assaying for 
the presence of T cells in a subject, which are active against the 
disease, using the heterofunctional cellular immunological reagent. 
DETAILED DESCRIPTION OF THE INVENTION 
As discussed above, in one embodiment of the present invention, the 
above-described objects have been met by a heterofunctional cellular 
immunological reagent comprising at least two T cell specific binding 
ligands covalently linked together, wherein one of the T cell specific 
binding ligands binds to a specific class or subclass of T cells and 
another of the T cell specific binding ligands is an antigen associated 
with disease or a causative agent of disease, or epitope thereof. 
As used herein, the expression "T cell specific binding ligand" refers to 
the entire molecule which binds to the surface of the T cell or only the T 
cell binding portion of the molecule, preferably only the T cell binding 
portion of the molecule. 
The particular type of T cell to which the T cell specific binding ligands 
bind is not critical to the present invention. Examples of such T cells 
include helper T cells, accessory T cells, suppressor T cells and 
cytotoxic T cells. These T cells include subclasses thereof, for example, 
subclasses of helper T cells include those helper T cells necessary for 
antibody synthesis and those necessary for cytotoxic activity. 
The particular T cell specific binding ligand which binds to a specific 
class or subclass of T cells employed is not critical to the present 
invention. The particular T cell specific binding ligand which binds to a 
specific class or subclass of T cells can be selected so as to bind to all 
mature T cells, only mature cytotoxic T cells, helper T cells, suppressor 
T cells or a specific class or subclass thereof. Examples of such T cell 
specific binding ligands include a T cell specific binding ligand which is 
also located on or binds to an APC, such as portions of MHC Classes I and 
II; portions of the Fc region of the heavy chain of immunoglobulins; 
Ia.sup.+ molecules; lymphocyte function associated molecule-3 
(hereinafter "LFA-3"); antibodies to CD-2, CD-3, CD-4 and CD-8; lectins 
such as concanvalin A, pokeweed mitogen, peanut agglutinin and 
phytohemagglutinin; lymphokines, such as IL-1 and IL-2; and other 
molecules such as d-poly-(E/K).sub.n (60:40). 
A small protein of MHC Class I, i.e., b-2-microglobulin (hereinafter 
"b-2-M"), which is found in various body fluids, such as serum, ascites 
and urine, has recently been shown to have biological properties 
indicating that such is a T cell specific binding ligand (see Nissen, M. 
H. et al, J. Immunol., 139: 1022 (1987)). That is, the addition of this 
molecule to an in vitro Cr.sup.+++ release cytotoxic assay system has an 
enhancing effect on CTL activity in both heterologous and 
homologous-systems, i.e., both human (heterologous) and murine 
(homologous) b-2-M give rise to the same biological effect in this assay 
system that uses murine cells. The sequence of b-2-M is reported in 
Gussow, D. et al, J. Immunol., 139: 3132 (1987). The following sequences 
at positions 24-58 and positions 58-80, respectively, of b-2-M are 
believed to be particularly useful T cell specific binding ligands: 
EQU CYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSK (MW=4474) 
EQU KDWSFYLLYYTEFTPTEKDEYAC (MW=3396) 
These two polypeptides are chosen to end at cysteines for several reasons. 
First, they represent a site that is probably outside of a linear epitope 
region. This is because in mature b-2-M, they are involved in the 
formation of intramolecular disulfide bonds. Second, they are chosen to 
take advantage of the cysteine to serve as a covalent linking site to the 
antigen associated with disease or a causative agent of disease, or 
epitope thereof. 
A common sequence for CD-2 and LFA-3 has recently been reported (see 
Peterson, A. et al, Nature, 329: 842 (1987); and Seed, B., Nature, 329: 
840 (1987)). CD-2, which is found on T cells, and a similar, if not 
identically derived molecule, LFA-3, which is found on macrophages, 
erythrocytes and nerve cells, are both implicated in various T cell 
receptor, ligand and modulation interactions. In particular, LFA-3 at 
positions 87-101: 
EQU KVSIYPTKGKNVLEK (MW=1956) 
or the derivatives thereof where a cysteine (c) and two glycines (gg) are 
added: 
EQU cggKVSIYPTKGKNVLEK (MW=2228.3) 
EQU KVSIYPTKGKNVLEKggc (MW=2228.3) 
or at positions 42-58: 
EQU KTSAKKKIAQFRKEK (MW=2043) 
or the derivatives thereof: 
EQU KTSAKKKIAQFRKEKggc (MW=2314) 
EQU ccgKTSAKKKIAQFRKEK (MW=2314) 
are believed to be useful T cell specific binding ligands. 
Based upon the charged nature of these polypeptides, in addition to their 
possible direct use as a T cell specific binding ligand, a corresponding 
acidic polypeptide from within another region of LFA-3 is believed to be 
useful as a T cell specific binding ligand (see Breitmeyer, J. B., Nature, 
329: 760 (1987)). That is, the carboxyl ultimate or penultimate sequence 
of LFA-3: 
EQU SRHRYALIPIPLAVITTCIVLYMNGIL (MW=3520) 
or the derivatives thereof wherein an internal cysteine is replaced by 
amino-butyric acid (Abu) and/or an internal methionine is replaced by 
nor-leucine (Nle): 
EQU SRHRYALIPIPLAVITTAbuIVLYMNGIL (MW=3502) 
EQU SRHRYALIPIPLAVITTAbuIVLYNleNGIL (MW=3474) 
or the derivative thereof: 
EQU cggSRHRYALIPIPLAVITTAbuIVLYNleNGIL (MW=3746) 
As discussed above, these derivatives contain amino-butyric acid (Abu) and 
nor-leucine (Nle). The reason for the former substitution is to avoid the 
possibility of forming homofunctional conjugates by removing a source of 
sulfhydryl. The reason for the latter substitution is to remove a labile 
methionine which can cleave the peptide bond and form a homocysteine 
terminated polypeptide. 
Antibodies to CD-2, CD-3, CD-4 and CD-8 are well known in the art (see 
Kung, P. et al, Proc. Natl. Acad. Sci. USA, 77: 4914 (1980)). 
In cases where the sequence of the T cell specific binding ligands are not 
known, such as for antibodies to CD-2, CD-3, CD-4, CD-8, lectins and Ia+ 
the sequence must be determined in whole or part. It is possible to 
determine a theoretical sequence by determining the nucleotide sequence of 
the anti-sense nucleotide sequence and reading in reverse direction along 
the double stranded DNA backbone and preparing an anti-sense polypeptide 
(see Smith, L. R., J. Immunol., 138: 7 (1987)) or by using the computer 
technology disclosed in U.S. Pat. No. 4,704,692. 
Lectins, such as concanvalin A, are well known to be multivalent and to 
possess specific binding sites for their ligands (see Edelmann, G. M. et 
el, Proc. Natl. Acad. Sci. USA, 69: 2580 (1972)). In addition, it is well 
known that they cause a specific general activation of T cells and alter 
the pathway of events (see Zimmerman, D. H. et al, J. Immunol., 111: 1326 
(1973)). One such T cell specific binding sequence derived from 
concanvalin A is at position 80-110: 
EQU LNDVLPEWVRVGLDSASTGLYKETNTILSWS (MW=4040) 
(see Wang, J. L. et al, J. Biol. Chem., 250: 1490 (1975) and Edelmann, G. 
M. et al, Proc. Natl. Acad. Sci. USA, 69: 2580 (1972)). 
It is well known that IL-1 has activity on several types of T cells, i.e., 
helper T cells and suppressor T cells, and is produced by the APC. 
Nencioni, L. et al, J. Immunol., 139: 800 (1987) have described the 
following T cell specific binding sequence for IL-1 at positions 163-171: 
EQU VQGEESNDK (MW=1149) 
or the derivative thereof: 
EQU VQGEESNDKggc (MW=1420) 
Similarly, IL-2, produced by one type of T cell, i.e., T helper cells, 
interacts with receptors on the same and other T cells, i.e., Th and Tc/s 
cells (see Altman, A. et al, Proc. Natl. Acad. Sci. USA, 81: 2176 (1984)). 
IL-2 is reported to have an effect on immune antibody responses. IL-2 is 
believed by the present inventors to be useful as a T cell specific 
binding ligand, particularly at positions 34-39: 
EQU LEHLLL (MW=827) 
and the derivative thereof: 
EQU cggLEHLL (MW=1098) 
since these polypeptides compete with IL-2 is a binding bioassay (see 
Reiher, W. E. et al, Proc. Natl. Aced. Sci. USA, 83: 9188 (1987)). 
Especially, IL-2 at positions 18-32: 
EQU TNSAPTSSSTKKTQL (MW=1802) 
The amino acids "TSS-T" appears to be highly conserved from a variety of 
sources (see Kohtz, D. S. et al, J. Virol., 62: 659 (1988)). Thus, the 
following derivatives of the above sequence found in retroviral protein 
sequences, which contains the TSS-T structure, may be employed as a T cell 
specific binding ligand: 
EQU TNSAPTSSSTKKTLggc (MW=2071) 
EQU AbuggTNSAPTSSSTKKTQL (MW=2055) 
Liu, F.-T. et al, Biochem, 18: 690 (1979) describe an 
inhibitory/suppressive haptenic carrier for induction of antibody 
production that is a polymer of D-glutamic acid and lysine, referred to as 
poly-(D-GL) (60:40) or, using the present nomenclature for amino acids, 
referred to as d-poly-(E/K).sub.n (60:40). In light of current knowledge 
of T cell receptors, it is believed by the present inventors that this 
polymer of D-glutamic acid and lysine encompasses a T cell binding ligand 
for the suppressor T cell and thus is believed to be useful in the present 
invention. 
It is well known that both MHC Class I and II molecules are composed of 
multiple chains that are involved in multiple binding to each other, other 
membrane associated entities, antigens, agretopes, epitopes, and T cell 
receptors. One such molecule, b-2-M discussed above (Gussow, D. et al, J. 
Immunol., 139: 3132 (1987)), is a small protein associated with MHC Class 
I. The above-mentioned sequences of b-2-M can be used for preparing 
trivalent heterofunctional cellular immunological reagents of the present 
invention. Similarly, sequences of LFA-3 or Ia+ are useful for preparing 
trivalent heterofunctional cellular immunological reagents of the present 
invention when one wants to involve both APC and T cells, in conjunction 
with the amino terminal polypeptide of b-2-M at positions 1-21: 
EQU INRTPKINVRSRHPAENGKSN (MW 2749) 
or the derivatives thereof: 
EQU cggINRTPKINVRSRHPAENGKSN (MW3020) 
EQU INRTPKINVRSRHPAENGKSNggc (MW3020) 
Since immunogenic for an antibody response, the following polypeptides of 
human chorionic somatotropin (hereinafter "HcS") at positions 166-174 may 
serve as a control to discriminate between those effects that are solely 
dependent upon the antigen epitope and those that also need the 
contribution of the ligand functional binding to a T cell which these HcS 
polypeptides do not (see Antioni, G. et al, Mol. Immunol., 22: 1337 
(1985)): 
EQU FRKDMDKVE (MW=1321) 
and the derivatives thereof: 
EQU FRKDNleDKVE (MW=1293) 
EQU FRKDNleDKVEggc (MW=1565) 
EQU AbuggFRKDNleDKVE (MW=1547) 
The T cell specific binding ligand or the T cell binding portion thereof or 
control polypeptide is commercially available or customized synthesized 
(Applied Biosystems (Foster City, Calif.), Biosearch (San Rafel, Calif.) 
Cambridge Research Biochemicals (Cambridge U.K.), Bachem Inc. (Torrance, 
Calif.), Serva (Westbury, N.Y.) or obtained from an appropriate natural 
source. 
The T cell specific binding ligand is stored as a dry powder in a 
dessicated environment at -20.degree. to -70.degree. C. 
In the following examples, all media and solutions are made up in freshly 
prepared glass distilled water or water of at least the same or greater 
quality unless otherwise indicated. These examples are provided for 
illustrative purposes only and are in no way intended to limit the scope 
of the present invention.

SYNTHESIS EXAMPLE 1 
Isolation of the b-2-M T Cell Specific Ligand 
The following example describes isolating and determining the T cell 
specific binding portion of a protein, i.e., b-2-M, whose amino acid 
sequence and nucleotide sequence are known. The approach described herein 
is also useful for a wide variety of other T cell specific binding 
ligands, e.g., lectins such as concanvalin A, pokeweed mitogen, peanut 
agglutinin and phytohemagglutinin; antibodies including those for cell 
surface proteins, such as CD-2, CD-3, CD-4, CD-8, etc.; as well as surface 
proteins such as LFA-3 and Ia+, where all of the sequences may not be 
known. 
In this example, b-2-M extracted from cultured human B cells is purified 
by, e.g., affinity chromatography as described in Lerch, P. G. et al, Mol. 
Immunol., 23: 131 (1986). 
More specifically, the murine monoclonal antibody (anti-human b-2-M) 
producing hybridoma BBM.1 (ATCC No. HB-28) is grown in RPMI 1640 media 
containing 5.0 to 10% (v/v) fetal bovine serum. When 100 ml of cells at a 
concentration of 5.times.10.sup.5 cells/ml are obtained, the cells are 
collected by centrifugation at 500.times.g at 15.degree. to 25.degree. C. 
for 5 min and resuspended in 50 ml of 0.01M potassium phosphate buffer (pH 
7.0) containing 0.15M NaCl (hereinafter "PBS"), centrifuged as described 
above and resuspended in 50 ml of the same buffer. 
Each of 15-25 (10-14 week old) Balb/c female mice, which have been 
inoculated 5.0 to 30 days, preferably 10 to 15 days, before with 0.5 ml of 
pristane (Serva, Westbury, N.Y.), is injected intraperitoneally with 0.1 
to 1.0 ml of the resulting BBM.1 cells (0.2.times.10.sup.6 to 
1.times.10.sup.6 cells/ml), preferably 0.5 ml (0.3.times.10.sup.6 
cells/ml). The inoculated mice are observed every other day for the next 
10 days and starting on day 10, the mice are examined and ascites fluid 
collected by the "tapping", i.e., penetration, of the abdominal cavity, 
not more than every other day, with a 16-18 gauge needle (B bevel) and 
allowing the ascites fluid to drain into a sterile 15 ml centrifuge tube. 
After storage overnight at 4.degree. C. to allow the fluid to clot, the 
clot is removed by centrifuging the sample at 1500.times.g at 2.degree. to 
8.degree. C. for 15 min and the clear straw to reddish colored ascites 
fluid is collected. The collected fluid is pooled into 50 ml centrifuge 
tubes and stored at -20.degree. to -70.degree. C. until all of the mice 
that are being so tapped expire. All of the thus collected fluid from one 
group of mice, inoculated at the same time with the same lot of cells, is 
thawed, pooled, centrifuged as described above and aliquoted into 5.0 ml 
samples and frozen until used. Approximately 50 to 150 ml of fluid is 
collected depending upon the properties of the particular hybridoma 
employed, such as cell viability, growth rate and rate of production; the 
number of mice; and the overall efficiency of inoculation, collection and 
harvesting of fluid from the clotted material. 
The antibodies are purified from the fluid (serum, ascites, tissue culture 
fluid) by precipitation and separation of the antibodies from many of the 
other fluid proteins. For example, the antibodies can be selectively 
precipitated by ammonium sulfate as follows. 
The thawed fluid volume is recorded and the fluid is gently stirred while 
cooling to 4.degree. C. in an ice bath. If serum is used, an equal volume 
of cold distilled or deionized water is slowly added. Then, while 
continually stirring, solid crystalline enzyme grade ammonium sulfate 
(Life Technologies, Inc., Gaithersburg, Md.) is added to an amount 
calculated as follows (total in g=volume in ml.times.0.706 
g/ml.times.0.4). The mixture is then stirred for at least 1.0 hours and 
centrifuged at 3000.times.g at 2.degree. to 8.degree. C. for 45 min to 
separate the precipitated protein from the soluble material. Next, the 
precipitated protein is resuspended in a minimal volume, typically about 
1.0 to 10% of the starting sample, using, e.g., PBS, and dialyzed against 
the same buffer at 4.degree. C. for a minimum of 2 hours per buffer change 
and a minimum of 3 buffer changes, wherein the volume ratio of buffer to 
sample is at least 50:1. Then, the sample is clarified by centrifugation 
at 3000.times.g at 2.degree. to 8.degree. C. for 15 min and/or by 
ultrafiltration (0.2 .mu.m filter size) before use or storage. If stored 
for any significant time, the sample is again clarified before use. If a 
fatty-like pellicle exists floating on top of the fluid phase, it can 
interfere with some of the subsequent steps and should be removed and 
discarded by either collection using a syringe and needle below the 
pellicle or by passage over a glass wool fiber filter pad. 
More specifically, if 100 ml of ascites or serum is used, 100 ml of water 
is added along with 56.48 g of ammonium sulfate. The precipitated 
antibodies are resuspended in 5.0 ml of PBS and another 5.0 ml of PBS is 
used to wash the material into the dialysis bag. After dialysis against 
1.0 liter of 0.01M potassium phosphate buffer (pH 7.0) for 18 to 72 hours 
and using at least 3 buffer changes of at least 1.0 liter each, the volume 
is about 15 ml. (Note, different buffers are employed for dialysis 
depending on the particular needs, i.e., ion exchange chromatography with 
a DE-52 (Whatman, Clifton, N.J.) column usually employs 0.001 to 0.05M 
potassium phosphate buffer (pH 6.5 to 7.5), preferably 0.01M potassium 
phosphate buffer (pH 7.0); or fast pressure liquid chromatography 
(hereinafter "FPLC") usually employs 0.01M Tris-HCl buffer (pH 7.5 to 
8.5), preferably 0.02M Tris-HCl buffer (pH 8.0)). 
The resulting material is applied to a DE-52 column (2.5.times.10 cm), 
eluted with, e.g., 0.01M potassium phosphate buffer (pH 7.0) and 2.0 ml 
fractions collected. The bulk of the antibodies are collected in fractions 
5 to 15 for a total volume of 20 ml with an average protein content of 
&gt;5.0 mg/ml (based on an A.sub.280 of 1.5 for 1.0 mg/ml). 
Alternatively, polyethylene glycol (PEG-6000) (Sigma Chemical Co., St. 
Louis, Mo.) can be used to precipitate antibodies from sera, ascites and 
is most useful with large volumes of dilute protein solutions, such as 
tissue culture fluid. 
More specifically, the volume is recorded and the material cooled to 
4.degree. C. While gently stirring, fine granular PEG-6000 is added to 
bring the final concentration to the desired % (w/v). For IgM, the final 
concentration of PEG-6000 is about 5.0% (w/v). For IgG, the final 
concentration of PEG-6000 is about 12% (w/v), although in some cases up to 
20% (w/v) may be required. The material is left to stir for at least 1.0 
hour at 2.degree. to 8.degree. C. after all of the PEG-6000 is dissolved. 
Then, the material is centrifuged at .gtoreq.1500.times.g at 2.degree. to 
8.degree. C. for 30 min and the precipitated and soluble material are 
separated by decanting. Next, the precipitate is dissolved in a minimal 
volume (1.0% (v/v) of sample volume) of PBS, dialyzed and fractionated as 
described above. 
In another alternative, caprylic (octanoic acid) treatment can be carried 
out to purify antibodies from clotted sera, plasma ascites or tissue 
culture fluid. 
More specifically, concentrated acetic acid is slowly added in small 
amounts thereto at 4.degree. C. with gentle stirring so as to adjust the 
pH to 4 to 5. Then, caprylic acid (octanoic acid) is added slowly with 
stirring in an amount of, e.g., 3.3 ml per 100 ml of ascites or serum, for 
at least 1.0 hour at 2.degree. to 8.degree. C. and the material is 
centrifuged at 3000.times.g at 2.degree. to 8.degree. C. for 30 min to 
separate precipitated non-immunoglobulins and soluble antibodies. The 
precipitate is discarded, the pH of the supernatant is adjusted to 7.0 
with 1.0M sodium phosphate buffer (pH 7.5), the supernatant is dialyzed 
against PBS, or 0.01M potassium phosphate buffer (pH 7.0), if DE-52 
chromatography as described above is to be carried out. 
To a solution of 0.1 to 20 mg/ml, preferably 10 mg/ml of antibody, purified 
as described above, is added an equal volume, preferably 5.0 ml, of 
freshly washed Affi-Gel-10 (BioRad, Richmond, Calif.) in 0.01M potassium 
phosphate buffer (pH 7.0). Then, the tube or container with the protein 
and gel is sealed and placed at 2.degree. to 8.degree. C. in a rotating 
capped vessel overnight in order to allow the coupling of the antibody to 
the gel matrix. The next day the liquid is decanted off and the gel is 
washed at least three times with a 10 fold volume of cold PBS. The settled 
gel is resuspended in an equal volume of 0.1M ethanolamine in PBS, stirred 
for 1 hour at 4.degree. C., washed extensively with PBS and then 
"stripped" with an agent, such as a chaotropic buffer, e.g., 2.8M 
MgCl.sub.2, which can be used to elute the T cell specific binding ligand, 
i.e., b-2-M, from the bound antibody. Next, the gel is re-equilibrated in 
PBS, and finally stored at 4.degree. C. in PBS containing 0.01M EDTA and 
0.1% (w/v) NaN.sub.3. For initial use and after storage for long periods 
of time, i.e., greater than 1 week, the gel is first poured into a column 
(1.0 to 1.5.times.2.5 to 5 cm) which is washed with 50 ml each of PBS, 
2.8M MgCl.sub.2 and PBS. 
Next, the T cell specific binding ligand b-2-M is prepared from cultured 
human B cells (HEL 92.1.7) (ATCC No. TIB-80). More specifically, HEL 
92.1.7 cells are cultured in a suspension culture using RPMI 1640 
containing 10% (v/v) fetal bovine serum. Approximately 1000 ml of cells, 
at a density of about 5.times.10.sup.5 cells/ml are collected. The cells 
are then extracted for b-2-M as described by Lerch, P. G. et al, Mol. 
Immunol., 23: 321 (1986). 
Alternatively, the cells are suspended in 10 to 20 ml of cold 3.0M KCl and 
after allowing to sit for a brief time, the treated cells are centrifuged 
at 3000.times.g at 2.degree. to 8.degree. C. for 30 min so as to pellet 
and separate the solublized surface membrane released proteins from the 
insoluble material. To the solubilized membrane proteins is added a 1.0% 
(v/v) of Triton X-100. Then, the material is dialyzed against 3 changes of 
1.0 liter each of cold PBS. After dialysis, the sample is clarified by 
centrifugation at 10,000.times.g for 30 min. The preparation may be 
filtered using a 0.2 .mu.m filter if desired. 
Thereafter, the sample is applied to the anti-b-2-M affinity column 
prepared as described above, eluted with 100 ml of PBS or more until the 
absorbance level in the eluent, as monitored by absorption at A.sub.280, 
is less than 0.1% of the starting sample or is undetectable. Then, the T 
cell specific binding ligand, i.e., b-2-M is eluted with 2.8M MgCl.sub.2, 
at a flow rate of 1 to 20 ml/hour. Individual fractions having a volume of 
0.1 to 2.0 ml are collected depending upon the sample, buffer and column. 
Size exclusion (molecular sieve) and/or desalting (buffer exchange) 
chromatography with appropriate sizing characteristics for the T cell 
specific binding ligand and buffer is carried out on the peak protein 
containing fractions from the affinity column described above to reduce 
the salt content from the 2.8M MgCl.sub.2 and to separate aggregated 
material from native material. Neutral, non-dissociating buffers, e.g., 
PBS, or other saline buffers with pH ranges of 6.0 to 9.6 such as, 
Tris-HCl, 0.05M sodium barbital, 0.05M sodium borate or 0.1M sodium 
bicarbonate can be employed for this chromatography. In some cases, 
dissociating buffers, e.g., 5.0M guanidine HCl, 8.0M urea, 0.1 to 2.0% 
(v/v) detergents, such as Triton X-100 can be employed for this 
chromatography, especially for removal of aggregates. Buffers which are 
volatile for ease in lyopholization such as, 0.1 to 1.0M ammonium formate 
or 0.1 to 1.0M ammonium acetate can also be employed. Nonvolatile buffers 
are preferably employed if lyopholization is not required. The sample 
volume is about 1.0% (v/v) or less of the bed volume, the void position is 
about 0.3 to 0.4 of the bed volume, the internal volume is about 1.0 to 
1.2 of the bed volume and the optimal fraction size is about 1.0% of the 
column. Collection of fractions begins upon application of the sample. The 
flow rate is as specified by the manufacture, preferably using mid-point 
to lower values. 
Enzyme digestion of the resulting purified T cell specific binding ligand 
is then carried out to determine the T cell specific binding portion 
thereof. More specifically, 2.0 mg/ml of the purified T cell specific 
binding ligand is dissolved in an appropriate enzyme digestion buffer, 
such as 0.1M ammonium acetate (pH 7.0), and to 1.0 ml thereof is added 50 
.mu.l of a proteolytic enzyme, such as trypsin, chymotrypsin, thermolysin, 
proteinase K, Staphylococcus aureus protease, Submaxilaris protease, 
subtilisin, or clostripian, to achieve a weight ratio of enzyme to 
substrate of 1:50. For example, 0.1 ml of purified b-2-M at 20 mg/ml in 
PBS is added to 0.9 ml of 0.1M ammonium acetate (pH 7.0). Then, 50 .mu.l 
of a 2.0 mg enzyme solution in water is added. Incubation is carried out 
at about 37.degree. C. for time intervals of about 10 to 120 min. At this 
point, the reaction in, e.g., a 13.times.100 glass test tube capped 
loosely with a marble, is terminated by immersion of the tube to a depth 
of 2.0 to 4.0 cm in a boiling water bath for 5 min. The terminated 
reaction sample is then lyophilized, or in some cases 100 .mu.l thereof is 
added to 10 ml of PBS, and assayed directly. The latter is carried out at 
the initial stages when it is not necessary to separate the digested 
polypeptides, i.e., it is only necessary to determine an allowable 
reaction condition for limited proteolysis to yield digested polypeptides. 
In this case, 6 replicate sets of reactions for each enzyme are prepared 
and termination is carried out after 0, 10, 20, 30, 60 and 120 min of 
incubation. Trypsin digestion is also performed on native and 
citraconylated T cell specific binding ligands to distinguish between 
lysine and arginine sensitive sites. Note, citraconylated lysines are 
resistant to trypsin hydrolysis. 
More specifically, multiple preparations are separately prepared as follows 
with each enzyme and time point. 0.1 ml of a purified b-2-M preparation 
containing 20 mg/ml protein in PBS is added to 0.1M of ammonium 
bicarbonate (pH 7.0) and then 0.05 ml of a 1.0 mg/ml enzyme solution of 
trypsin, or proteinase K, or chymotrypsin, or thermolysin, Staphylococcus 
aureus protease or clostripain or Submaxilaris protease or subtilisin is 
added. For each time series, 6 identical preparations, in 13.times.100 mm 
glass test tubes covered with a glass marble that fits over the tube top, 
are prepared and the reaction terminated by immersion of the tube to a 
depth of over 3.0 cm in a boiling water bath for 5 min after 0, 10, 20, 
30, 60 and 120 min of incubation. As discussed above, if comparable 
reactions (for trypsin only) are carried out with or without citraconic 
anhydride treated b-2-M polypeptide, only the arginine sites are sensitive 
to cleavage since the lysine sites are protected. 
In some cases, selective chemical hydrolysis using, for example, CNBr to 
cleave methionines is employed in formic acid. Other cleavage sites with 
other agents or conditions have been reported (see Fontana, A., in Meth. 
in Enzymol., 25: 419 (1972); Ozols, J. et al, J. Biol. Chem., 252: 5986 
(1977); and Hunziker, P. E. et al, Biochem. J., 187: 515 (1980)). 
Amino and carboxyl terminal amino acids are determined using either 
appropriate proteases as recommended by the manufacturer (Pierce 
Chemicals, Rockford, Ill.) or by a system for automated sequential 
degradation, separation and analysis, i.e., a "polypeptide sequencer" 
(Applied Biosystems, Foster City, Calif.). Also, it is often the case that 
the polypeptide will be analyzed for total amino acid content after 
hydrolysis for 24, 48 and 72 hours in boiling 6.0N HCl or using other 
appropriate means as recommended by the manufacturer of an "amino acid 
analyzer" (Beckman, Palo Alto, Calif.; and Applied Biosystems, Foster 
City, Calif.). 
To detect a reactive polypeptide derived from the purified T cell specific 
binding ligand, a standard competition inhibition immunoassay is performed 
wherein the test specimen, e.g., enzyme-digested sample, is incubated at 
several dilutions with the ligand binding species prior to the indicator 
being incubated with a labeled indicator. Normally, replicates are carried 
out at at least 3 different dilutions, usually over 3 ten fold log, i.e., 
1/10, 1/100, and 1/1000. With very concentrated or high affinity T cell 
specific binding ligand, higher dilution levels are employed, such as 
1/10,000, 1/100,000, up to 1,000,000 or even greater. 
The labelled indicator in this case is b-2-M that is coupled with an 
appropriate molecule such as Biotin-N-hydroxysuccimide ester (Biotin-NHS) 
(Pierce Chemical, Rockford, Ill.). Other labelled indicators include 
radioisotopes, fluorescent dyes or common color developing enzymes, such 
as horseradish peroxidase, can be employed in place of Biotin. The 
competition can be carried out with a monoclonal antibody or other ligand 
binding species, such as a T cell membrane. In the former case, after the 
appropriate pre-incubation of the diluted enzymatic or chemically digested 
materials and the Biotin-b-2-M conjugate at the optimal dilution, which is 
determined previously for that particular lot, the mixture of materials is 
reacted with the T cell or immobilized monoclonal antibody. Typically with 
a 1.0 mg/ml solution of b-2-M and an indicator/protein ratio of 2.0 the 
dilution will be 1:10,000. Immobilized monoclonal antibody can be employed 
if properly selected to recognize the same ligand as the T cell membrane 
component, i.e., if it competes with the T cell for binding to the native 
ligand. The use of non-living material is preferable for a number of 
reasons, including less dependence on sensitive critical and sometimes 
unavailable living material, ease of use and control of conditions. 
After incubating the mixture of the competing species and the immobilized 
material (cells or antibody coated microplates), for example, at 
37.degree. C. for 120 min for monoclonal antibody coated microplates, or 
at 2.degree. to 8.degree. C. for 30 min for T cells, the cells or 
microplates are washed extensively with PBS, and the presence of the bound 
labeled species detected, directly if possible or developed as required. 
If development is required, a conjugate of avidin and either horseradish 
peroxidase or fluorescein is used at a dilution of 1:250 to 1:5000 and 
incubated as described above for the Biotin-derivative, and washed and 
processed as required to detect its presence. 
Once a enzyme system is identified that digests the T cell specific binding 
ligand but does not substantially alter its activity, it is verified that 
the reactive polypeptide is indeed a polypeptide by a variety of 
techniques, such as RP-HPLC, high voltage electrophoresis and ascending 
thin layer chromatography. 
For RP-HPLC, a C-18 column (Vydac 15 to 20 .mu.m 300 .ANG. pore size 
30.times.5 cm) can be used. In this case, a gradient of a mixture of 
aqueous triethylammonium phosphate ("TEAP") (pH 2.25) and 60 or 70% (v/v) 
acetonitrile with 0.1% (v/v) aqueous trifluoroacetic acid (hereinafter 
"TFA") in TEAP can be used as an elution buffer. A flow rate of 2.0 ml/min 
for an analytical column and 80 to 120 ml/min for a preparative column can 
be employed. Polypeptides are detected by monitoring absorbance at 
A.sub.220 (see Hoeger, C. et al, Biotechniques, 2: 134 (1987)). 
For high voltage electrophoresis, two-dimensional separations using a solid 
support, such as Whatman 3MM paper, is carried out. A total of 100 to 400 
.mu.g of enzyme digested material is loaded onto the support by multiple 
additions and drying of 5.0 to 10 .mu.l samples of enzyme digested 
material onto a spot in the corner, inside 1.0 cm in each dimension, of 
23.times.23 cm paper. High voltage electrophoresis is carried out using a 
buffer system of pyridine: acetic acid: water (25:1:225 (v/v/v)) at 2.0 kV 
and 35 to 60 mA for 80 min at 2.degree. to 8.degree. C. After removing and 
drying, the support is rotated 90 degrees and then equilibrated in the 
solvent described below for thin layer chromatography (TLC) and 
chromatographed in the same manner. After drying, polypeptides are 
detected by the use of 0.0215% (w/v) of a commercial Ninhydrin Spray or 
fluorescamine dye (Hoffmann-LaRoche, Nutley, N.J.) in acetone and UV 
light. 
For ascending TLC, 3.0 .mu.l of lyopholized enzyme digested material is 
dissolved in 10% (v/v) acetic acid at 2.0 mg/ml. Among the useful 
commercially available TLC plates are those available from E. Merck 
(Darnstadt, West Germany). The sample (3.0 .mu.l) is applied to the plates 
which are placed above an atmosphere saturated with a solvent system of 
pyridine:n-butanol:acetic acid:water (50:75:15:60 (v/v/v/v)) at 4.degree. 
C. After allowing to equilibrate for 2 hours, the plates are introduced 
into the solvent to a depth of about 1.0 cm and allowed to develop 
overnight. Usually the solvent front advances up about 18 cm. The plates 
are then removed and dried at room temperature. After drying, the 
polypeptides are detected as described above. The polypeptides so detected 
are then eluted from the plates with 0.07% (v/v) ammonium hydroxide, 
followed by competitive inhibition analysis (if an appropriate dilution is 
allowable with the buffers or else lyopholized first to concentrate such). 
The separated polypeptide may be fixed onto the plates (or paper if 
appropriate) with 2.0% (v/v) glutaraldehyde for 15 min, "blocked" with a 
mixture of 2.0% (v/v) serum of the same species as the enzyme conjugated 
antibody to be used later and 2.0 to 10% (v/v) bovine serum albumin in 
0.1M Tris (pH 8.0) containing 0.15M sodium chloride. Then, the fixed 
polypeptide is analyzed for reactivity by reaction with about 0.1 .mu.g 
of, e.g., Biotin- or enzyme-, such as horseradish peroxidase or alkaline 
phosphatase, conjugated antibody by incubation therewith for 1 to 18 hours 
at 4.degree. to 37.degree. C., washed with the same buffer and then 
developed with a precipitating color as per standard Western blot 
immunological procedures (Biotech, Rockville, Md.). 
Alternatively, if the labelled indicator that the polypeptide reacts with 
is an antibody, the unlabelled antibody is dissolved in freshly prepared 
0.15M sodium carbonate buffer (pH 9.5) at a concentration generally of 
about 0.1 to 10 .mu.g/ml, and then added in an amount of 100 to 200 
.mu.l/well of microtiter plates (Immunlon Dynatech, Alexandria, Va. or BD, 
Oxnard, Calif.). This coated antibody, while reactive with the parent 
molecule and derivatives thereof, is of a different specificity than that 
of the labelled antibody. Thus, it does not compete, interfere or 
stimulate the binding of the other antibody but, rather, only serves as a 
means of attachment for the ligand that the other monoclonal antibody 
binds. The plates are covered for 2 hours at room temperature, washed 3 to 
5 times with 0.01 to 5.0% (v/v) bovine serum albumin (hereinafter "BSA") 
along with 0.1% (v/v) Tween-20, or other mild detergent such as, Triton 
X-100, in PBS at 4.degree. C. If necessary, 0.01% (v/v) PVP and/or 5.0 to 
20 mg/ml of sugar, such as dextrose or sucrose are added for stability in 
the last few washes, dried and stored in a dark moisture free environment 
4.degree. C. In addition, if necessary, the wells may be fixed with 
agents, such as 0.25 to 2.0% (v/v), preferably 0.25% (v/v) of 
glutaraldehyde prior to blocking with BSA. 
An appropriately diluted sample of polypeptide (1:100 to 1,000,000) in PBS 
containing 0.1% detergent, e.g., Tween-20, is added along with diluted 
animal sera of the same type as that of the detection conjugate 
(enzyme:antibody) and incubated at 37.degree. C. for 30 min to 24 hours. 
Then, the wells are washed 5 times with PBS and an appropriate dilution of 
the detection conjugate (1/1000 to 100,000) in the same buffer is added 
incubated and washed as described above. Next, substrate is added and 
detection is carried out as described above. 
In addition, the amino acid composition of the final product is determined 
and then compared with the predicted amino acid composition. A deviation 
greater than 5 to 10% is investigated in order to verify that the product 
is what is expected, and that the deviation is due to the hydrolysis 
condition used to determine the enzyme digest. 
The particular antigen or epitope thereof which is associated with disease 
is also not critical to the present invention. Examples of such antigens 
include allergens, such as cat dander antigens, dust mite fecal antigens 
and food allergens such as wheat glutenin; or self-derived antigens, such 
as epidermal growth factor (hereinafter "EGF"), which is a breast tumor 
cell specific marker or carcinoebryonic antigen (hereinafter "CEA") or 
prostate acid phosphotase (hereinafter "PAP"), which are associated with 
colorectal carcinoma and prostate tumors, respectively; cell surface 
antigens; or antigens associated with auto-immunity such as diabetes, 
Rheumatoid arthritis and thyroiditis. 
In addition, the particular causative agent of disease to which the antigen 
or epitope thereof is associated, is also not critical to the present 
invention. Examples of causative agents of disease include prions; 
viruses, such as HIV, Herpes Simplex Virus (hereinafter "HSV"), Epstein 
Bar Virus (hereinafter "EBV"), cytomegalo virus (hereinafter "CMV"), human 
B lymphotropic virus (hereinafter "HBLV"), varicella zoster virus 
(hereinafter "VZV"), adenovirus and hepatitis B virus: bacteria, such as 
streptococcus, diptheria, mycobacterium and troponema; fungi, such as 
candida; protozoa, such as giardia; and parasites, such as plasmodium, 
ascaris and leishmania. 
Furthermore, the particular disease to which prevention, treatment or 
diagnosis is desired is not critical to the present invention and can 
include any disease associated with the above-described antigens or 
epitopes thereof. 
Recently, several polypeptides have been reported that are able to generate 
antibodies that react with a prion protein having the following sequences 
at positions 90-102, 15-40 and 220-223, respectively (see Barry, R. A. et 
al, J. Immunol., 140: 1185 (1988)): 
EQU GQGGGTHNQWNKPGGC (MW=1960) 
EQU MWTDVGLCKKRPKPGGWNTGGSYRYPGGC (MW=3685) 
EQU CGGKESNAYYDGRRSSA (MW=2109) 
Chapman, M. D. et al, J. Immunol., 140: 812 (1988) describe, for the cat 
dander antigen, referred to as Fel d I, an antigenic sequence at positions 
1-33: 
EQU GITPAVKRDVDLFLTGTPDEYVEQVAQYKAPDV (MW=4213) 
or the derivative thereof: 
EQU GITPAVKRDVDLFLTGTPDEYVEQVAQYKAPDVc (MW=4331) 
A sequence that is shared by the early region protein E1b of adenovirus 
type 12 and gliadin, a wheat glutenin protein, is believed to have 
important implications in coelaic disease (see Karagiannis, J. S. et al, 
Lancet, I: 884 (1987) and Kagnoff, M. F. et al, J. Exp. Med., 160: 1544 
(1984)). That is, in gliadin at positions 211-217: 
EQU FRPSQQN (MW=983) 
and the derivative thereof: 
EQU FRPSQQNggC (MW=1255) 
The above epitope shared in common between adenovirus and gliadin can be 
considered illustrative of an association of autoimmune, allergic and 
infectious conditions. Another epitope associated with autoimmune and 
infectious conditions is that of mycobacterium purified protein derivative 
(PPD). This epitope has implications in adjuvant induced arthritis in 
animals (see Lider, O. et al, Proc. Natl. Acad. Sci. USA, 84: 4577 
(1987)). 
Epitopes of myelin basic protein (MBP) and collagen (see Ellerman, K. E. et 
al, Nature, 331: 265 (1988); Lider, O. et al, Science, 239: 181 (1988); 
and Kakimoto, K. et al, J. Immunol., 140: 78 (1988)) are illustrative of 
epitopes involved in autoimmune encephalomyelitis and arthritis, 
respectively. Thus, epitopes of these proteins can be determined and 
employed in the present invention. 
Examples of antigens or epitopes thereof of HIV which are associated with 
humoral immunity and thus which can be employed in the present invention 
include the envelope proteins of HIV, such as gp120 and gp41. 
HIV gp41 has the following antigenic sequence at positions 594-605 (see 
Banapour, B. et al, J. Immunol., 239: 4027 (1987)): 
EQU G(I/M)WGCSGK(L/H)(I/L)C 
Since genetic variants exist which have substitutions at certain 
non-critical positions, these non-critical positions have been indicated 
by a "/" along with the possible amino acids for these positions enclosed 
by a "()". Thus, for example, a derivative of the above antigenic 
sequences of HIV gp41 can be as follows: 
EQU GIWGAbuSGKLIC (MW=1389) 
Some animal experiments demonstrate that the following adjacent region of 
HIV gp41 at positions 609-620: 
EQU CTTAVPWNASWS (MW=1700) 
while immunogenic in animals, may not be as immunogenic in humans (see 
Gnann, J. W. et al, J. Infect. Dis., 156: 261 (1987)). The failure to be 
immunogenic can be the result of one or more defects. One such defect can 
be a failure to recognize an agretope due to the lack of an appropriate 
receptor. Similarly, the corresponding T cell may not be present for the 
desirable T cell class, e.g., helper T cells which recognizes the epitope 
are desired but, only cytotoxic T cells that recognize the epitope are 
present. Thus, other polypeptides including both regions, such as shown 
below are believed to be useful for the synthesis of heterofunctional 
cellular immunological reagents of the present invention. 
EQU G(I/M)WGCSGK(L/H)(I/L)CTTAVPWNASWS 
or in one form: 
EQU LGLWGCSGKLICTTAVPWNASWS (MW=3118) 
or the derivatives thereof: 
EQU cggLGLWGCSGKLICTTAVPNASWS (MW=3389) 
EQU LGLWGCSGKLICTTAVPWNASWSggc (MW=3389) 
If the sensitive sites of the internal cysteines are substituted with 
amino-butyric acid (Abu) the epitope has the sequence shown below: 
EQU LGLWGAbuSGKLIAbuTTAVPNASWSggc (MW=3353) 
Another HIV envelope protein is gp120. One of the epitopes thereof 
associated with humoral immunity includes the sequence at positions 
108-119 (see Gnann, J. W. et al, J. Infect. Dis., 156: 261 (1987)): 
EQU ILSLWDQSLKPC (MW=1690) 
Chanh, T. C. et al, EMBO J., 5: 3065 (1986); Chanh, T. C. et al, Eur. J. 
Immunol., 16: 1465 (1986); Kennedy, R. C. et al, J. Biol. Chem., 262: 5769 
(1987); and Kennedy, R. C. et al, Science, 231: 1556 (1986)) describe the 
same epitope using their sequence position nomenclature at positions 
503-532: 
EQU VAPTAKRVVQRKRAVGIGALFLGFLGAG (MW=3340) 
or the derivative thereof: 
EQU VAPTAKRVVQRKRAVGIGALFLGFLGAGggc (MW=3611) 
One of the first synthetic polypeptides shown to be immunogenic, in terms 
of antibody generation, for HIV was that for the gp41 protein at positions 
735-752: 
EQU (R/D)RPEGIEEEGGERDRDR(S/G)C 
or one form thereof: 
EQU RRPEGIEEEGGERDRDRSC (MW=2570) 
(see Kennedy, P. C. et al, Science, 231: 1556 (1986)) and is believed to 
therefore be a useful polypeptide sequence in whole or in part. 
Another one of the HIV proteins, gag p17, at positions 92-109: 
EQU IDVKDTKEALEKIEE (MW=2438) 
or the derivatives thereof: 
EQU abuggIDVKDTKEALEKIEE (MW=2691) 
EQU IDVKDTKEALEKIEEggc (MW=2709) 
due to its similarity to thymosin alpha, has been discussed as a candidate 
for immunization to generate humoral antibodies (see Sarin, P. et al, 
Science, 232: 1135 (1986)). The sequence relationship to thymosin alpha 
strongly suggests the need and relevance to cellular immunity since 
thymosin alpha is considered to be an immune system hormone or 
immunomodulator. 
Other HIV gag polypeptides from both the p17 and p24 region described by 
Gnann, J. W. et al, J. Infect. Dis., 156: 261 (1987) are believed to be 
useful in the present invention. These derivatives are important since 
some animals can recognize and make antibodies to these epitopes and serum 
antibodies are neutralized in an in vitro assay using these epitopes (see 
Ho, D. D. et al, J. Virol., 61: 2024 (1987); and Satin, P. et al, Science, 
232: 1135 (1986)). 
As discussed above HSV EBV CMV, HBLV and VZV antigens are also believed to 
be useful in the present invention. For example, Zweig, M. et al, J. 
Virol., 51: 340 (1984) describe for HSV gC, the following antigenic 
sequence at positions 128-139: 
EQU DRRDPLARYGSR (MW=1659) 
or the derivative thereof: 
EQU DRRDPLARYGSRggc (MW=1932) 
Bosch, D. L. et al, J. Virol., 61: 3607 (1987); and Weijer, W. J. et al, J. 
Virol., 62: 501 (1988) describe for HSV gD, the antigenic sequence at 
positions 1-30, especially at positions 9-21 and in particular, the amino 
acids at positions 10, 16 and 20: 
EQU KRALADASLKMADPNRFRGKDLPVLDQLTD (MW=3888) 
or the derivatives thereof: 
EQU KRALADASLKMADPNRFRGKDLPVLDQLTDc (MW=4009) 
Kinchington, P. R. et al, J. Virol., 62: 802 (1988) describes for the major 
DNA binding protein of VZV, the antigenic sequence at the carboxyl 
terminus: 
EQU PIKHNGITMEMI (MW=1602) 
or the derivatives thereof: 
EQU PIKHNGITNleENleIggc (MW=1817) 
EQU AbuggPIKHNGITNleENleI (MW=1799) 
Oba, D. E. et al, J. Virol., 62: 1108 (1988) describe for EBV gp85, the 
antigenic sequence at positions 518 to 533: 
EQU CSLEREDRDAWHLPAYK (MW=2467) 
The surface antigen protein of hepatitis B virus has the following 
antigenic region at positions 144-159 (see Pfaff, E. M. et al, EMBO J., 1: 
869 (1982)): 
EQU LRGDLQVLAQKVARTL (MW=2079) 
or the derivative thereof: 
EQU LRGDLQVLAQKVARTLggc (MW=2350) 
or using the nomenclature of others at positions 139-158 (see Bhatnagar, P. 
K. et al, Proc. Natl. Acad. Sci. USA, 79: 4400 (1982)): 
EQU CTKPTDGNCTCIPIPSSWAF (MW=2573) 
or the derivative thereof: 
EQU CTKPTDGNAbuTAbuIPIPSSWAF (MW=2537) 
The preS-2 region of hepatitis B virus is also antigenic, particularly at 
positions 99-121 (see Jolivet, M. E. et al, Infect. & Immunol., 55: 1498 
(1987), and Aubibert, F. M. et al, Infect. & Immunol., 45: 261 (1984): 
EQU DYQGMLPVCPLIPGSSTTSTGPC (MW=2731) 
or the derivative thereof: 
EQU DYQGNleLPVAbuPLIPGSSTTSTGPC (MW=2685) 
Bacterially important epitopes are described in Beachley, E. H. et al, J. 
Exp. Med., 166: 647 (1987), e.g., the streptococcal epitopes of the 
approximately first 10 amino terminal region of the M protein of three 
strains of streptococci and a polypeptide containing these three different 
amino terminal sequences: 
EQU TVTRGTISDPRVFPRGTVENPVATRSQTDTSEKc (MW=4301) 
or a derivative thereof where the lysine, which contains a potential 
reactive group, is substituted by glycine, which, since it is adjacent to 
the added cysteine not found in other variants at this location, suggests 
that it is outside of the primary epitope: 
EQU TVTRRGTISDPRVFRRGTVENPVATRSQTDTSEGc (MW=4230) 
Jolivet, M. E. et al, Infect. & Immunol., 55: 1498 (1987) describe the 
following antigenic sequence for diphtheria toxin at positions 186-201: 
EQU CAGNRVRRSVGSSLKC (MW=1945) 
or the derivative thereof when the internal cysteine is replaced by 
amino-butyric acid and two alanines are added: 
EQU aaAbuAGNRVRRSVGSSLKC (MW 2123) 
The circumsporozoite stage protein of plasmodium, including but not limited 
to such species as falciparum, knowlensi, etc. referred to as CSP-1, has 
an internal repetitive sequence depending upon the species (see Lise, L. 
D. et al, Infect. & Immunol., 55: 2658 (1987); Ballou, W. R. et al, 
Science, 228: 991 (1985); Jolivet, M. E. et al, Infect. & Immunol., 55: 
1498 (1987); Patarroyo, M. E. et al, Nature, 328: 629 (1987); and Good, M. 
F. et al, Science, 235: 1059 (1987)). The following antigenic sequences of 
CSP-1 are believed to be useful epitopes in the present invention: 
EQU NANPNANPNANPNANPNAC or (NANP).sub.4 C (MW=1994) 
EQU Y(QAQGDGANAGQP).sub.2 C (MW=2925) 
EQU c(QAQGDGANAGQP).sub.2 y (MW=3106) 
Other sequences of the CSP-1 used for vaccines include the repetitive 
sequence: 
EQU [(NANP).sub.15 (NVDP)].sub.2 (MW=15047) 
as well as the following two sequences at positions 103-116 and 323-349, 
respectively: 
EQU EKLRKPKHKKLKQP (MW=1992) 
EQU NNEEPSDKHIEQYLKKIKNSISTEWSPC (MW=3849) 
(see Good, M. F. et al, Science, 235: 1059 (1987)). The latter sequences 
have been used for generation of an immune response either directly or 
indirectly by "helper T cell epitopes". The last sequence was shown to be 
restricted by the I-A and or H-2 MHC genes of certain phenotypes. 
The amino terminal polypeptide sequence of another plasmodium protein, 
i.e., the 35 kd protein, having the following sequence, has been used for 
humoral responses (see Patarroyo, M. E. et al, Nature, 328: 629 (1987)): 
EQU WGGPANKKNAG (MW=1237) 
or the derivatives thereof: 
EQU abuggWGGPANKKNAG (MW=1622) 
EQU WGGPANKKNAGggc (MW=1641) 
Other polypeptides from malaria proteins which can be employed include 
those described in Etlinger, H. M. et al, J. Immunol., 140: 626 (1988); 
Sadoff, J. C. et al, Science 240: 336 (1988); Richars, R. A. et al, Infect 
& Immunol., 56: 682 (1988); Richman, S. J. et al, Proc. Natl. Acad. Sci. 
USA, 85: 1667 (1988); Weiss, W. R. et al, Proc. Natl. Acad. Sci. USA, 85: 
573 (1988); Good, M. F. et al, J. Immunol., 140: 1645 (1988); Brake, D. A. 
et al, J. Immunol., 140: 1989 (1988); and Good, M. F. et al, Proc. Natl. 
Acad. Sci. USA, 85: 1199 (1988). 
Oba, D. E. et el, J. Virol., 62: 1108 (1988) also describe the control 
polypeptide from human C9, a complement series protein, at positions 
399-413: 
EQU CLIDDVVSLIRGGTRK (MW=2015) 
As another control, epitopic sequences for the foot and mouth disease virus 
(hereinafter "FMDV") can be used, particular VP-1 having the following 
sequence at positions 141-160 (see Bittle, J. L. et al, Nature, 298: 30 
(1983)) to which cysteine is added at the amino acid: 
EQU cVPNLRGDLQVLAQKVARTLP (MW=2652.2) 
This epitope is of a similar size and composition to the above-described 
polypeptides and can serve as a control sequence for either replacement of 
the antigen associated with disease or a causative agent of disease, or 
epitope thereof or the other T cell specific binding ligand. Because of 
the addition of cysteine to the amino terminal, it can be linked by 
several mechanisms that utilize sulfhydryl linkages. 
Still another control sequence is the following sequence of cytochrome c, 
believed to be representative of a non-pathogenic or non-disease 
condition, at positions 81-104 (see Fox, B. S. et al, J. Immunol., 139: 
1578 (1987)): 
EQU IFAGIKKANERAELIAYLKQATKC (MW=3094) 
The antigen associated with disease or a causative agent of disease, or 
epitope thereof or control polypeptide is commercially available or 
customized synthesized (Applied Biosystems, Foster City, Calif.), 
Biosearch (San Rafel, Calif.) Cambridge Research Biochemicals (Cambridge, 
U.K.), Bachem Inc. (Torrance, Calif.), Serva (Westbury, N.Y.) or obtained 
from the native source. 
The antigen associated with disease or a causative agent of disease, or 
epitope thereof is stored as a dry powder in a dessicated environment at 
-20.degree. to -70.degree. C. 
The particular size of the heterofunctional cellular immunological reagent 
of the present invention is not critical thereto. Generally, the 
heterofunctional cellular immunological reagent is about 20 to 100 amino 
acids in length, preferably about 40 to 60 amino acids in length. 
The heterofunctional cellular immunological reagent of the present 
invention can be prepared by the use of bifunctional linkers. Examples of 
bifunctional linkers which can be employed in the present invention to 
covalently link the T cell specific binding ligand and antigen associated 
with disease or a causative agent of disease, or epitope thereof include 
N-succinimidyl-3-(2-pyridyldthio)propinate (hereinafter "SPDP") 
(Pharmacia, Piscataway, N.J.), which activates and allows formation of a 
bridge between two sulfhydryl groups of cysteines or a bridge between a 
derivatized (propinated-thiolyated) primary amino group and a cysteine; 
m-maleimidobenzoyl-N-hydroxy-succimide ester (hereinafter "MBS") (Pierce 
Chemical, Rockford, Ill.), which activates an amino group and then couples 
by a sulfhydryl group to a cysteine sulfydryl so as to form a disulfide 
bond between the two polypeptides; and 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (hereinafter "EDC") (Pierce 
Chemical, Rockford, Ill.), which can cross-link two polypeptides by 
sequentially activating the carboxyl group of one polypeptide and then 
adding such to an amino group of another polypeptide. 
N-isocyano-ethylmorphlin, bis-diazotized-benzidine, benzoquone and 
glutaraldehyde, which are other reagents commonly employed to link 
polypeptides, can be employed in the present invention and are available 
from Pierce Chemical, Rockford, Ill.; Eastman Kodak Chemicals, Rochester, 
N.Y.; Serva, Westbury, N.Y.; Sigma Chemical Co., St. Louis, Mo.; and E. 
Merck, Darnstadt, West Germany (see Briand, J. S. et al, J. Immunol. 
Meth., 78: 59 (1985); Kitagawa, T. et al, J. Biochem., 79: 233 (1976); 
Liu, F. T. et al, Biochem., 18: 690 (1979); Ternynck, T. et al, 
Immunochem., 14: 767 (1977); and Drevin, H. et al, J. Immunol. Meth., 77: 
9 (1985)). 
The heterofunctional cellular immunological reagent of the present 
invention can also be prepared by chemical synthesis or by using 
recombinant DNA techniques, i.e., where the nucleotide sequence of the T 
cell specific binding ligand and antigen associated with disease or 
causative agent of disease or epitope thereof are adjacent to each other 
and inserted into an appropriate expression vector so that a single 
molecule is synthesized recombinantly. 
In order to prepare the heterofunctional cellular immunological reagent of 
the present invention, it is first necessary to analyze the sequences that 
are to be covalently linked so as to determine what amino acids can be 
substituted with a stable (or less reactive) amino acid, such as 
substitution of nor-leucine for methionine, or amino-butyric acid for a 
cysteine, in the chemical synthesis of the polypeptide. If one of the 
desired sequences contains a cysteine and a substitution with 
amino-butyric acid is not practical, this polypeptide can be linked using 
MBS or EDC. Substitution of the cysteine with amino-butyric acid is 
practical if it is not essential that the cysteine be linked by a 
disulfide bond to another cysteine or another reactive group. If a 
cysteine is at the amino or carboxyl terminal of the polypeptide it is 
less likely to be in the T cell specific ligand binding portion or an 
epitope. Therefore, conjugation at this site can be carried out using MBS 
or SPDP as a bifunctional linker group. 
Depending upon the synthetic reactions or other conditions, protection of 
other reactive groups, such as the epsilon amino of lysine, or groups of 
arginine, histidine and the carboxyl or derivatives thereof of aspartic 
and glutamic, may not be necessary. In this case one can proceed directly 
to the linking of the T cell specific binding ligand and antigen 
associated with disease or a causative agent of disease, or epitope 
thereof so as to produce the heterofunctional cellular immunological 
reagent without prior derivatization for protection. 
Protection, such as the temporary blockage of the epsilon amino of lysine 
is accomplished by pretreatment of the polypeptide with citraconic 
anhydride, maleic anhydride or other similar acid anhydrides that will 
reversibly displace the H on the amino groups. After the construction of 
the heterofunctional cellular immunological reagent, the acid group added 
to the amino when the H is displaced is in turn displaced by acid 
treatment. 
SYNTHESIS EXAMPLE 2 USING SPDP 
The following example describes the production of a heterofunctional 
cellular immunological reagent comprising an HIV epitope covalently linked 
using SPDP to the T cell specific binding ligand portion of b-2 -M. 
3.4 mg (2.0 .mu.mole) of the derivative of the HIV gp41 epitope: 
EQU AbuTTAVPWNASWS (MW=1682) 
is dissolved in 1.0 ml of 0.1M sodium phosphate buffer (pH 7.5) at 
15.degree. to 25.degree. C. in a conical stirring reaction vessel. To this 
solution is added 0.1 ml of a freshly prepared SPDP solution (20 .mu.mole) 
(6.4 mg/ml of SPDP dissolved in ethanol or DMSO). (Note, if the water 
soluble forms of substituted SPDP are available they should be employed 
since this avoids the undesirable use of organic solvents such as DMSO or 
ethanol.) The reaction mixture is stirred for 0.5 hours at 15.degree. to 
25.degree. C., after which time the material is chromatographed using a 
desalting column, such as Bio-gel P2, P4 P6 or P10 (BioRad, Richmond, 
Calif.) or the Sephadex equivalent. In this example, preferably P-2, P-4 
or Sephadex G-10 (Pharmacia, Piscataway, N.J.) is used. The column's 
internal dimensions are 1.5.times.75 cm (although dimensions of 0.9 to 
2.5.times.50 to 100 cm are also satisfactory). 
Elution is carried out using 0.05M sodium phosphate buffer (pH 7.0) 
containing 0.15M NaCl, at a flow rate of 10 to 25 ml/hr. (If the material 
is to be lyopholized, an acceptable alternative buffer which can be used 
is 0.05 to 0.1M ammonium acetate buffer (pH 7.0).) 
About 100 or so equal fractions (between 0.5 and 1.0 ml in size for the 
preferred column) are collected. Then, the column is eluted with at least 
100 ml more of the same buffer. The elution profile is monitored by 
recording the absorbance at an appropriate wavelength, typically between 
A.sub.210 and A.sub.290. Under ideal conditions of gel size, flow rate, 
buffer, conformation of polypeptides, etc., there may be some resolution, 
especially on the leading edge, of substituted and non-substituted 
polypeptides. If such desirable resolution occurs, then a skewed selection 
of the first peak is desired. The main peak (normally 2 to 3 1.0 ml 
fractions, but on occasion fractions 4 to 6 1.0 ml fractions) that 
represent the bulk of the first peak containing the substituted 
polypeptide are then pooled. A typical expected yield of the 
SPDP-polypeptide is 50-75%. 
The resulting species is a 2-pyridyl-dithio-propinate-polypeptide between 
the carboxyl of the propinate and an amino group in the polypeptide, 
wherein the N-hydroxysuccimide of the SPDP is displaced by the amino group 
of the polypeptide. 
Next, the T cell specific binding ligand portion of b-2-M: 
EQU KDWSFYLLYYTEFTPTEKDEYAC (MW=3400) 
which contains a cysteine at the carboxyl terminal is employed. To insure 
that this polypeptide does not form any polymeric disulfides, 6.8 mg of 
this polypeptide, in 1.0 ml of 0.1M sodium phosphate buffer (pH 7.0), is 
reduced in the presence of 10 mM dithiothretiol (hereinafter "DTT") 
(freshly prepared by dissolving 15.4 mg of DTT in 1.0 ml water and then 
adding 100 .mu.l to the above polypeptide solution). After 45 min at room 
temperature, the reduced polypeptide is separated from the DTT and other 
products by fractionation on a P4 or P-6 column (BioRad, Richmond, 
Calif.). The peak fraction of the polypeptide is separately pooled. 
Next, estimated equal molar portions of each polypeptide are mixed in a 
reaction conical stirring vessel and the reaction is allowed to proceed 
for 2 hours at 15.degree. to 25.degree. C. Under these conditions, the 
reduced sulfhydryl of the cysteine in the b-2-M polypeptide preferentially 
reacts with the 2-pyridyl-propinate-disulfide of the HIV polypeptide, the 
2-pyridyl is displaced and a new disulfide is formed which results in a 
dipeptide bridge with a propinate residue. If desired, a chelating agent, 
such as 0.001 to 0.01M EDTA, may be added to reduce side reactions with 
contaminating heavy metals that may sometimes be introduced by, e.g., 
contaminated water, buffers, etc. 
Then, the reaction is terminated by separation of the products and 
reactants on a molecular sieve and desalting column such as G-10 or G-25. 
In this example, a matrix, buffer and flow rate allowing separation of 
1.2, 3.2 and about 5 kD materials is desired and either a P-6 or G-10 
column is the preferred choice. The sample volume is approximately 5.0 ml 
and a column of 2.5 to 5.0 cm diameter is used. In addition, it is 
desirable to add stabilizers, such as 0.15M sodium chloride, 0.001M EDTA 
and often 0.01% (w/v) polyethylene glycol 6000 in 0.1M glycine buffer (pH 
6.0), along with a bacteriostatic agent, such as 1000 units of penicillin 
or 10 .mu.g/ml of streptomycin, at this stage. Further, the pH of the 
elution buffer can be reduced to pH 6.5 for stabilization and storage. 
Alternatively, RP-HPLC separation or high voltage electrophoresis can be 
carried out as described above for separation and so as to determine the 
purity thereof. If the elution buffer used has a reduced pH, such as 5.2 
or 7.0, it is desirable to use RP-HPLC. 
The resulting heterofunctional cellular immunological reagent is set forth 
below: 
##STR1## 
SYNTHESIS EXAMPLE 3 USING MSB 
The following example describes the production of a heterofunctional 
cellular immunological reagent comprising an HIV epitope covalently linked 
using MSB to the T cell specific binding ligand portion of LFA-3. 
6.7 mg (2.0 .mu.mole) of the nor-leucine, amino-butyric acid form of the T 
cell specific binding ligand portion of LFA-3 (see Breitmeyer, J. B., 
Nature, 329: 760 (1987) and Seed, B., Nature, 329: 840 (1987)): 
EQU SRHRYALIPIPLAVITTCIVIYMNVL (MW=3431) 
or the derivative thereof: 
EQU SRHRYALIPIPLAVITTAbuIVIYNleNVL (MW=3385) 
is dissolved in 1.0 ml of PBS. To this solution is added 50 .mu.l of a MBS 
solution (4.0 .mu.mole) (24.8 mg/ml of MBS in dimethylformamide) in a 
conical reaction vessel. The reaction mixture is continuously stirred for 
30 min at room temperature. Then, the reaction is terminated as described 
below. 
The products and reactants are separated by desalting on an appropriate 
column as described above, preferably, on a P-4 column (0.9.times.40 cm) 
at 4.degree. C. using, as the elution buffer, 0.1M potassium phosphate 
buffer (pH 6.0) or 0.1M sodium phosphate buffer (pH 6.0). The pool of the 
relevant fractions containing the desired product is stored at 4.degree. 
C. until use (normally within 24 hours). The typical yield of the 
MBS-polypeptide is 3.0 to 4.5 mg. The resulting polypeptide is an amino 
substituted polypeptide derivative of benzoic acid. 
To the resulting polypeptide, estimated as 3.1 mg in 3.0 ml of 0.1M sodium 
phosphate buffer (pH 6.0) is added, 3.0 ml, containing 5.2 mg (2.0 
.mu.mole) of the following HIV gp41 epitope dissolved in 0.05 to 0.1M 
sodium phosphate buffer (pH 6.4) containing 0.15M sodium chloride and 
0.01M EDTA: 
EQU RRPEGIEEEGGERDRDRSC (MW=2570) 
It is important that the above buffer is deoxygenated by at least 3 
repetitive cycles of vacuum-bleeding prior to use to minimize dissolved 
gases. Alternatively the buffer can be prepared from freshly boiled 
distilled or deionized water that is then stored in stoppered air-tight 
containers. 
The reaction is allowed to continue for 3 hours at room temperature and 
then is terminated by the addition of 2-mercaptoethanol to a final 
concentration of 1.0 mM (60 .mu.l of a 0.1M solution of 2-mercaptoethanol) 
followed by the addition of N-ethylmaleimide to a final concentration of 
2.0 mM (60 .mu.l of a 0.2M solution of N-ethylmaleimide). 
Next, the product is purified as described above and stored at pH 6.0 to 
6.5 in the presence of preservatives and other stabilizing agents, such as 
10-20 mg/ml of human IgG. 
The resulting heterofunctional cellular immunological reagent is set forth 
below: 
##STR2## 
SYNTHESIS EXAMPLE 4 USING CITRACONIC ANHYDRIDE AND SPDP 
The following example describes the production of a heterofunctional 
cellular immunological reagent comprising a cat dander epitope covalently 
linked using SPDP to the T cell specific binding ligand portion of 
IL-1.beta., wherein the epsilon amino lysine(s) of the T cell specific 
ligand binding portion of IL-1.beta. is protected in order to prevent 
undesirable side reactions. 
The T cell specific binding portion derivative of IL-1.beta.: 
EQU AbuggVQGEENDK (MW=1402) 
is used (see Nencioni, L. et al, J. Immunol., 139: 800 (1987)). However, as 
discussed above, it is first desirable to protect the epsilon amino lysine 
with, e.g., citraconic anhydride or alternatively, dimethyl maleic 
anhydride, to avoid undesirable side reactions. 
More specifically, 2.6 mg (2.0 .mu.mole) of the above T cell specific 
binding ligand of IL-1.beta. is dissolved in 200 .mu.l of 0.2M 
N-ethylmorpholine acetate and adjusted to a pH of 8.5 with 1.0N NaOH. 
Then, a large excess of citranoic anhydride (E. Merck, Darnstadt, West 
Germany or Pierce Chemical, Rockford, Ill.) (MW=112.08), calculated from 
the theoretical number of free amino groups (two per mole in this example, 
or a total of 4.0 .mu.mole), is added in 5 to 10 equal increments 
(typically 5.0 .mu.l) approximately every 5 min into a constantly stirring 
conical reaction vessel. The pH is monitored frequently and adjustments 
made with 1.0N NaOH to keep the pH at or slightly above pH 8.5. After 
stirring for 1 hour at room temperature, the citraconylated polypeptide is 
separated from the citraconic anhydride by chromatography on a P-2 column 
(1.5.times.25 cm) using 0.1N ammonium bicarbonate buffer (pH 8.5). At 
least 100 0.5 ml size fractions, which fractions typically represent about 
1/100 of the total column volume, are collected and monitored at A.sub.220 
to A.sub.280. The first peak containing the derivatized polypeptide is 
lyopholized and then stored dessicated if necessary, to obtain the 
polypeptide shown below: 
EQU AbuggVQGEESNDK(cit) (MW=1410) 
Then, 2.8 mg of the citraconylated polypeptide (2.0 .mu.m) is treated with 
SPDP as described above to obtain the polypeptide shown below: 
EQU SPDP-AbuggVQGEESNDK(cit) 
Next, 8.7 mg (2.0 .mu.mole) of a cat dander epitope: 
EQU GITPAVKRDVDLFLTGTPDEYVEQVAQYKAPDVc (MW=4333) 
is coupled to the SPDP-IL-1.beta. polypeptide described above. After 
coupling the IL-1.beta. T cell specific binding ligand with the cat dander 
epitope as described above to form the heterofunctional cellular 
immunological reagent, the citraconylated groups are deblocked by removing 
the citranoic group by acid treatment. This is accomplished by dialysis of 
the 3.0 to 5.0 ml pool after the P-10 or G-10 column separation of the 
final product against 2 changes of 1.0 liter of 5.0% (v/v) acetic acid for 
4 hours at 4.degree. C., followed by dialysis against 2 changes of 1.0 
liter of 0.05M glycine buffer (pH 6.5) containing 0.15M sodium chloride 
and 0.01M EDTA, for at least 2 hours and then overnight at 2.degree. to 
8.degree. C. 
The resulting heterofunctional cellular immunological reagent is shown 
below: 
##STR3## 
In a second embodiment, the above-described objects of the present 
invention have been met by a vaccine for the prevention or treatment of 
disease comprising, as an active ingredient, a pharmaceutically effective 
amount of a heterofunctional cellular immunological reagent and a 
pharmaceutically acceptable carrier or diluent. 
The pharmaceutically acceptable carrier employed in the present invention 
is not critical thereto. Examples of pharmaceutically acceptable carriers 
include proteins, e.g., human serum albumin and gamma-globulin or 
polymers, e.g., dextran or polyethylene glycol, and/or adjuvants, e.g., 
alum. A typical alum adjuvant is ALUGEL 50 (Serva Feinbiochemica, 
Heidelberg, West Germany). The amount of carrier employed is generally a 
50:50 (v/w) ratio with respect to the heterofunctional cellular 
immunological reagent of the present invention. 
The pharmaceutically acceptable diluent employed in the present invention 
is not critical thereto. Examples of pharmaceutically acceptable diluents 
include sterile 0.01 to 1.0M, preferably 0.05M glycine buffer (pH 6.5) 
containing 0.15M sodium chloride and 0.01M EDTA; tissue culture media such 
as RPMI 1640; or a physiological buffered salt solution such as Hanks 
Balanced Salt Solution (Life Technologies, Inc., Gaithersburg, Md.). The 
heterofunctional cellular immunological reagent of the present invention 
is generally diluted to a concentration of about 0.1 to 2000 .mu.g/ml, 
preferably about 2.0 to 100 .mu.g/ml. 
In a third embodiment, the above-described objects of the present invention 
have been met by a method of prevention of disease comprising 
administering the vaccine to disease susceptible subject. A disease 
susceptible subject is an individual who has not previously been exposed 
to either the disease or has been treated for such with a vaccine and who, 
because of genetic, or environmental factors such as age, sex, diet, life 
style, lodging, etc., is at an increased risk of being exposed and/or 
developing the disease. 
In a fourth embodiment, the above-described objects of the present 
invention have been met by a method of treatment of a disease comprising 
administering the vaccine to a subject afflicted with the disease. 
The particular heterofunctional cellular immunological reagent employed as 
the active ingredient in the vaccine will depend upon the disease for 
which prevention or treatment is sought. That is, for prevention or 
treatment of a particular disease, one component of the heterofunctional 
cellular immunological reagent must be an antigen associated with the 
disease or a causative agent of the disease, or epitope thereof. 
The amount of the heterofunctional cellular immunological reagent to be 
administered for prevention and/or treatment of disease will depend upon 
the age, weight and sex of the subject. Generally, the heterofunctional 
cellular immunological reagent is administered in an amount of from about 
2.0 to 100 .mu.g/70 kg of body weight, preferably about 10 to 20 .mu.g/70 
kg of body weight. 
The site and mode of administration are not critical to the present 
invention. For example the heterofunctional cellular immunological reagent 
can be administered intradermally, subcutaneously, intraperitoneally, 
intramuscularly and, also, when an adjuvant is not employed, 
intravenously. A preferable mode of administration is intradermally or 
intramuscularly. 
Multiple inoculations of the heterofunctional cellular immunological 
reagent are generally employed, with 3 to 4 weeks between the first and 
second inoculations and 6 months between the second and third 
inoculations. Subsequent boosters may also be employed if desired. 
SYNTHESIS EXAMPLE 5 
Vaccine Against Streptococcus 
The following example describes the production of a vaccine for the 
prevention of infection by streptococcus and/or treatment of a subject 
infected with the same. 
8.6 mg (2.0 .mu.mole) of the streptococcal epitope: 
EQU TVTRGTISDPRVFPRGTVENPVATRSQTDTSEKC (MW=4301) 
is dissolved in 2.0 ml of 0.1M potassium phosphate buffer (pH 7.5) 
containing 0.15M sodium chloride, reduced with 0.01M DTT for 45 min at 
room temperature and purified as described above. 
This material is then coupled to an equal molar amount of the 
SPDP-derivatized b-2-M: 
EQU SPDP-AbuYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS (MW=4473) 
which is formed by reacting 8.9 mg (2.0 .mu.mole) of b-2-M with 0.05 ml of 
24.8 mg/ml of SPDP (4.0 .mu.mole) in DMSO, for 2 hours at room 
temperature. The resulting heterofunctional cellular immunological reagent 
is shown below: 
##STR4## 
After purification, the heterofunctional cellular immunological reagent is 
equilibrated in a 0.05M glycine buffer (pH 6.5) containing 0.15M sodium 
chloride, 0.01M EDTA, and 1.0 mg/ml of alum and as such, is useful as a 
vaccine against streptococcal infection. 
In a fifth embodiment, the above-described objects of the present invention 
have been met by a method of diagnosing disease comprising assaying for 
the presence of T cells in a subject, which are active against the 
disease, using the heterofunctional cellular immunological reagent. 
Again, the particular heterofunctional cellular immunological reagent to be 
used will depend upon the disease for which diagnosis is sought. That is, 
one component of the heterofunctional cellular immunological reagent must 
be an antigen associated with the disease or a causative agent of the 
disease, or epitope thereof for which diagnosis is sought or a control, or 
non-related epitope. 
The particular assay employed to diagnose the disease, i.e., to determine 
the presence of T cells in the subject which are active against the 
disease, is not critical to the present invention. Examples of such assays 
include the lymphoproliferative assay using radioisotopes (see Cason, J. 
et al, J. Immunol. Meth., 102: 109 (1987)); or a non-isotopic 
lymphoproliferative assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 
tetrazolium (hereinafter "MTT") (Sigma Chemical Co., St. Louis, Mo.) (see 
Mosmann, T., J. Immunol. Meth., 65: 55 (1983)); assays that measure cell 
death, such as the Cr.sup.+++ release assay (see Chapters 9-18 in Manual 
Clin. Immunol., Ed. Rose, N. et al (1976) and Fund. Clin. Immunol., Ed. 
Alexander, J. W. et al (1977)); a carboxyfluorescein diacetate 
(hereinafter "CDTA") uptake assay (see Bruning, J. W. et al, J. Immunol. 
Meth., 33: 33 (1980); Hansson, Y. et al, J. Immunol. Meth., 100: 261 
(1987); Mosmann, T., J. Immunol. Meth., 65: 55 (1983); Gerlier, D. et al, 
J. Immunol. Meth., 94: 57 (1986)); a lymphocyte migration inhibition assay 
(see Chapters 9-18 in Manual Clin. Immunol., Ed. Rose, N. et al (1976) and 
Fund. Clin. Immunol., Ed. Alexander, J. W. et al (1977)); a 
phosphorylation assay (see Samuelson, L. E. et al, J. Immunol., 139: 2708 
(1987)); a delayed type hypersensitivity (DHT) skin test assay (see 
Kadival, G. J. et al, J. Immunol., 139: 2447 (1987); and Keeney, R. T. et 
al, J. Immunol. Meth., 101: 110 (1987)); or a T cell binding assay using a 
dye such as 5-[(4,6-dichlorotriazin-2-yl)amino]-fluorescein hydrochloride 
(hereinafter "DTAF") (see Kung, P. et al, Proc. Natl. Acad. Sci. USA, 77: 
4914 (1980)). 
The lymphoproliferative assay is modified for use to test cellular immunity 
to a particular disease as follows. For example, samples of blood 
circulatory lymph node or spleenic lymphocytes, which contain T cells, are 
collected using the ascetic technique, by venipuncture in sterile 
containers, from animals immunized with an antigen associated with disease 
or a causative agent of disease or an epitope thereof or the 
heterofunctional cellular immunological reagent. Often with animals, 
particularly when using mice or rats and in many cases rabbits, the 
animals are sacrificed, for example, 14 days after an inoculation by, 
e.g., cervical dislocation, and the inguinal lymph nodes and/or spleen 
teased into a single cell suspension. If whole blood is used, as in most 
cases with humans, the peripheral blood or single cell lymphocytes are 
treated, for example, by the "Ficoll-Hypaque" method (Pharmacia, 
Piscataway, N.J.) and the cells resuspended and plated into sterile 
culture microwells. Ficoll-Hypague treatment is often carried out to 
remove erythrocytes, macrophages and other cells which may interfere by 
consuming nutrients in long term (&gt;hours or days) assays. Their removal is 
desirable since this may simplify the assay and/or interpretation of the 
results. In many cases, especially with early and or rapid events, this 
separation is not necessary. 
More specifically, a 96 well Costar plate is seeded at 1.0 to 
5.0.times.10.sup.5, preferably 2.0.times.10.sup.5 cells/well in 0.2 ml of 
RPMI 1640 medium, supplemented with standard antibiotics, such as 
penicillin and streptomycin, and often 0.5 to 10% (v/v) homologous serum, 
e.g., normal mouse serum for murine cells or human AB serum for human 
cells and BSA for other types of cells. 
Next, a solution that brings both 2-mercaptoethanol to a concentration of 
0.00005M and the antigen associated with disease or a causative agent of 
disease, or an epitope thereof, to a concentration of 0.001 .mu.g/ml to 
100 .mu.g/ml, preferably 0.01 to 10 .mu.g/ml, or the heterofunctional 
cellular immunological reagent(s) at a series of concentrations, usually 
from about 0.0001 to 10 .mu.g/ml, preferably 0.01 to 1.0 .mu.g/ml, are 
added to the wells. 
For proper design, both the use of replicates and for better control, and 
more meaningful results, the use of a series of related heterofunctional 
cellular immunological reagents, such as those shown below, are tested at 
several serial dilutions usually starting at 10 .mu.g/ml. 
##STR5## 
It is desirable to test the same polypeptides but employing different 
orientations and different methods of linking, e.g., to couple the amino 
group to the carboxyl polypeptide by use of a carbodiimide activated 
carboxyl group of an amino group. 
The various microcultures are incubated from 3 to 6 days preferably 4 days 
at 37.degree. C. in a 5% CO.sub.2, 95% air mixture at 95% relative 
humidity. Then, 0.4 .mu.Ci of .sup.3 H-thymidine (New England Nuclear, 
Boston, Mass.) with a specific activity of 1.0 Ci/nmol is added to each 
well and incubation at 37.degree. C. is continued for 18 hours. The 
incubation is terminated and the samples processed to determine the amount 
of incorporation of .sup.3 H-thymidine into DNA by use of a multiple assay 
sample harvester (hereinafter "MASH"). The data is analyzed for 
reproducibility using 4 replicates, a blank with no cells, and various 
specific and non-specific heterofunctional cellular immunological reagents 
as controls. The results are expressed as the groups's mean+/-the standard 
deviation of the replicates. If less than 3 of the 4 replicates are 
allowable, i.e., x+/-S.D., the determination should be repeated. Values 
greater than +/-20% of the mean are excluded. To be significant, at least 
two different means at 2 adjacent concentrations in a series must be 
different by more than 50% of the controls. If the base line is 
significant then the data are interpreted as an inhibitory effect or a 
stimulatory effect. No effect means that the patient has no cellular 
immunity. A stimulatory effect in this assay implies helper T cell 
activity of a stimulatory nature and an inhibitory effect implies that the 
base line T cell activity is suppressed by the system. 
Typical assay conditions are selected, if possible, so that without the 
test reagent, such as the native intact antigen or epitope thereof or a 
heterofunctional cellular immunological reagent of the present invention, 
the positive and negative control have values preferably about 2 to 3 
times the background level where no cells are present. Then the positive 
control should give a value of at least 10, preferably 100 or more times 
the background level and the negative control should be the same as 
described above. In this case, for a sample to register as positive or 
significant, it should be above the first quarter percentile between the 
two controls. 
In many cases, the first determination of the presence or absence of 
cellular immunity is sufficient. The clinical interpretation of the 
consequences is often on a case by case basis. Thus, in many cases, such 
as infectious disease agents, there is a desire or need to determine or 
develop a positive cellular immunity presence. In other conditions, such 
as allergies, the cellular immunity may be misdirected causing helper T 
cells to produce excess IgE, as opposed to IgG or other classes or 
subclasses of immunoglobulins. In still other cases, diagnosis may show 
that the correct type of T cells for cellular immunity, e.g., helper T 
cells for increasing antibody production may be present but what is needed 
is an increase in the production of cytotoxic T cells for a particular 
antigen associated with disease or a causative agent of disease or epitope 
thereof. 
As discussed above, a non-isotopic lymphoproliferative assay using MTT can 
also be employed to test for cellular immunity to a particular disease 
(see Mosmann, T., J. Immunol. Meth., 65: 55 (1983); and Gerlier, A. et al, 
J. Immunol. Meth., 94: 57 (1986)). 
For the use of MTT as a replacement of .sup.3 H-thymidine for analysis of 
lymphoproliferation, the cells are processed, treated and incubated as 
with the thymidine assay up to the point where the .sup.3 H-thymidine is 
added. Instead of adding the .sup.3 H-thymidine, MTT is dissolved in 
sterile pyrogen free PBS to a concentration of MTT of 5.0 mg/ml and 
freshly filtered through a 0.2 .mu.m filter. This solution is added at a 
ratio of 10 .mu.l per 100 .mu.l of culture. The incubation is continued at 
37.degree. C. for 4 hours. Then, the incubation is terminated by 
centrifugation of the cells, aspiration of the fluid and resuspension and 
lysis of the cells. Next, the incorporated and converted MTT, in the form 
of a blue precipitate, is dissolved by the addition of 100 .mu.l of 
acid-isopropanol solution comprising 100 .mu.l of 0.04N HCl in 
isopropanol. The contents of the wells are allowed to mix by gentle mixing 
on an orbital shaker at room temperature and the absorbance at A.sub.570 
is read after 15 min to 1 hour. 
For the use of the heterofunctional cellular immunological reagent in 
cytotoxicity assays as a replacement of the MHC processing and 
presentation for analysis of cytotoxicity, the cells are processed, 
treated with the heterofunctional cellular immunological reagent for the 
first 6-9 days as with the lympholiferative assay. At this point, a 
standard Cr.sup.+++ release assay is performed. More specifically, the 
cells are washed and resuspended in 0.1 ml of fresh media. Then 0.1 ml of 
infected cells are added to effector cells at between 10 and 200% of the 
concentration of the effector cells. The infected cells are freshly 
collected the previous day from the same donor as the effector cells and 
infected overnight with the agent of disease from which the antigen of the 
heterofunctional cellular immunological reagent was derived at a 
multiplicity of infection of between 0.01 and 10, preferably 1.0. After an 
overnight infection, the cells are labelled with CrCl.sub.3, in the form 
.sup.51 Cr, and then washed 5 to 10 times until the amount of soluble 
radioactivity is less than 0.01% of the cells bound. Preferably 10,000 to 
1,000,000 cpm of such labelled infected target cells are added to the 
effector cells and then the cells incubated for 4 to 6 hours at 37.degree. 
C. Next, the cells are washed 5 times with PBS, counted and the data 
calculated using controls. 
Also, as discussed above, another assay based on CFDA uptake can be 
employed to test for cellular immunity to a particular disease (see 
Mosmann, T., J. Immunol. Meth., 65: 55 (1983); and Hansson, Y. et al, J. 
Immunol. Meth., 100: 261 (1988)). 
CFDA is advantageous in that CFDA passively traverses through the cell 
membrane and then, as a result of intracellular enzymes, is hydrolyzed by 
living cells to carboxyl fluorescence which will accumulate in living 
cells and makes such fluorescent under blue light. More specifically, 
cells are prepared and treated with an antigen associated with disease or 
a causative agent of disease or epitope thereof, or a heterofunctional 
cellular immunological reagent as described above for the 
lymphoproliferative or MTT assay. However, instead of processing as in 
those assays, the cells are washed twice with sterile Hanks Balanced Salt 
Solution and resuspended in 25 .mu.l thereof to which is added 5.0 .mu.l 
of a freshly diluted and filtered (0.2 .mu.m) CFDA solution. The CFDA 
solution is prepared as follows: 10 .mu.l of standard stock is prepared by 
acetylation of 6-carboxyfluorescein, as described by Bruning, J. W. et al, 
J. Immunol. Meth., 33: 33 (1980) (Eastman Kodak, Rochester, N.Y.) and 
stored as 10 mg/ml in reagent grade acetone in a dark bottle in the cold 
and sealed with a glass stopper. Then, 10 ml of Hanks Balanced Salt 
Solution buffered with 0.02M Tris-Cl (pH 7.4) or Hepes (pH 7.4) is added. 
After the cells are allowed to take up and hydrolyze the CFDA for a set 
period of time, usually 15 min incubation at 37.degree. C., the cells are 
washed with fresh Hanks Balanced Salt Solution and resuspended in 0.01 to 
10 mg/ml of hemoglobin to reduce background scatter and noise. The 
incorporated CFDA in the cells in the wells is determined by measuring the 
CFDA content by monitoring fluorescence with appropriate and available 
fluorometers that utilize this 96 wells plate configuration. 
The phosphorylation assay is carried out by activation of the cells with 
the heterofunctional cellular immunological reagent of the present 
invention and the incorporation of .sup.32 P into proteins from GTP is 
measured. More specifically, the cells, after a short period of treatment 
with the heterofunctional cellular immunological reagent of the present 
invention, i.e., about 5 to 60 min, are incubated with, for example, 0.001 
to 0.1 .mu.Ci of .sup.32 P gamma-labelled GTP for 15 to 20 min. Then, the 
cells are processed for determination or .sup.32 P incorporation in all of 
the proteins or in a specific protein, such as a specific internal protein 
or a specific immunoprecipitable membrane protein. 
The delayed hypersensitivity type assay (DHT assay) is conducted as 
follows: the antigen associated with disease or a causative agent of 
disease or epitope thereof, or the heterofunctional cellular immunological 
reagent, at an appropriate concentration, usually 0.001 to 10.0 .mu.g/ml, 
preferably 0.01 to 1.0 .mu.g/ml in PBS, is introduced by use of a thin 
hypodermic needle and syringe, preferably 0.05 ml with a 27 gauge, into 
the dermal layer of skin. A region is selected where changes in shape, 
coloration, thickness and other properties are easily observed and 
measured with calipers if desired. This usually means an area with little 
or no hair or pigmentation. After 24 and 48 hours, the area is observed 
and the results are recorded, with particular note of a hematoma, 
induration and shape. Often for ease of comparison, standard agents known 
to both evoke a positive DHT reaction and to be completely inert, such as 
a physiologically buffered saline solution, are included at a site 1.0 to 
2.0 cm removed. In the DHT assay, the test material(s) are recorded 
referencing their reaction to the controls (see Kadival, G. J. et al, J. 
Immunol., 139: 2447 (1987); and Keeney, R. T. et al, J. Immunol. Meth., 
101: 110 (1987)). 
SYNTHESIS EXAMPLE 6 
Diagnostic Reagent 
The following example describes the production of a T cell specific binding 
ligand which binds to cells which contain either MHC Class I and II 
molecules on the surface thereof which is useful as a diagnostic reagent. 
The following polypeptide, which contains about the first 10 amino acids in 
proper sequence of the M protein of 3 strains of streptococcal bacteria 
(see Beachley, E. H. et al, J. Exp. Med., 166: 647 (1987)), is treated 
with citraconic anhydride to block the amino group(s) of the lysine(s) as 
described above. After treatment with citraconic acid and subsequent 
purification, the molecules are reduced with 0.01M DTT to ensure that the 
cysteine is in an acceptable form: 
EQU TRVTRGTISVPRVFPRGTVENPVATRSQTDTSKc (MW=4209) 
The above polypeptide is then coupled to an equal molar amount of the 
following SPDP derivative of the IL-1.beta. polypeptide at positions 
163-171: 
EQU SPDP-AbuggVQGEESNDK (MW=1401) 
which is formed by reacting 2.0 mg (2.0 .mu.m) of the IL-1.beta. 
polypeptide with 0.05 ml of 24.8 mg/ml SPDP (4.0 .mu.m) in DMSO for 2 
hours at room temperature. The resulting heterofunctional cellular 
immunological reagent is shown below: 
##STR6## 
The following control polypeptides can be prepared in a similar manner: 
##STR7## 
SYNTHESIS EXAMPLE 7 
Diagnostic Reagent 
The following example describes the production of another T cell specific 
binding ligand which binds to cells which contain either MHC Class I and 
II molecules on the surface thereof which is useful as a diagnostic 
reagent. 
The streptococcal derived polypeptide of Synthesis Example 6 is coupled to 
the derivative of concanvalin A at positions 81-110: 
EQU gggLNDVLPEWVRVGLDSASTGLYKETNTILSWS (MW=4266) 
More specifically, 8.5 mg of the above concanvalin A polypeptide (2.0 
.mu.m) is reacted with 0.5 ml of the SPDP solution described in Synthesis 
Example 6 and then subsequently reacted with the streptococcal derived 
polypeptide of Synthesis Example 6 as described therein so as to obtain 
the following heterofunctional cellular immunological reagent: 
##STR8## 
The following control polypeptides can be prepared for the concanvalin A 
derivative in the same manner as described in Synthesis Example 6: 
##STR9## 
SYNTHESIS EXAMPLE 8 
Labelled Diagnostic Reagent 
The following example describes the labelling of a heterofunctional 
cellular immunological reagent for use in visualizing the binding of the 
heterofunctional cellular immunological reagent to the surface of T cells 
so as to diagnose the presence of T cells in a subject which are active 
against HIV. 
5.0 mg (1.0 .mu.mole) of the following heterofunctional cellular 
immunological reagent: 
##STR10## 
obtained as described above is dissolved in 2.0 ml of 0.05M sodium 
phosphate buffer (pH 7.0) containing 0.15M sodium chloride and reacted 
with 5.0 mg (10 .mu.mole) of DTAF (Eastman Kodak, Rochester, N.Y.) or 
NHS-Biotin (Pierce Chemical, Rockford, Ill.) at 15.degree. to 25.degree. 
C. so as to label the heterofunctional cellular immunological reagent. 
Since the heterofunctional cellular immunological reagent has a somewhat 
labile disulfide, the conditions are essentially as recommended by the 
manufacturer but the reaction is carried out for 2 hours and at a pH of 
7.0 and the reaction products are immediately separated by desalting and 
fractionating on a G-10 column using 0.05M potassium phosphate buffer (pH 
6.5) containing 0.15M sodium chloride, 0.001M EDTA, and 0.01% (w/v) 
PEG-6000. The resulting polypeptide is shown below: 
##STR11## 
The DTAF labelled heterofunctional cellular immunological reagent is used 
by incubating such with the T cells for 30-60 min at 2.degree. to 
8.degree. C. Then, the T cells are washed and examined under a 
fluorescence microscope for the presence or absence of binding the 
heterofunctional cellular immunological reagent to the T cell membrane. 
Next, appropriate quantitation of the percentage of T cells so labelled is 
carried out. 
If NHS-Biotin is used to label the heterofunctional cellular immunological 
reagent then such can be purified by use of an affinity column using 
avidin coupled to a solid support as described by the manufacturer, Pierce 
Chemical, Rockford, Ill. When NHS-biotin is used, avidin-FITC is used to 
visualize the binding of the heterofunctional cellular immunological 
reagent to T cells. 
If a trifunctional immunological reagent is under analysis for diagnostic 
uses then a total of eight heterofunctional cellular immunological 
reagents are possible if two options are available for each entity. Often 
by grouping and taking into account combinations which are not possible to 
synthesize, fewer heterofunctional cellular immunological reagents are 
required. 
SYNTHESIS EXAMPLE 9 USING SPDP AND MBS 
Trifunctional Reagent 
The following example describes the production of a trifunctional 
immunological reagent of the present invention. 
6.8 mg of the following LFA-3 T cell specific binding ligand: 
EQU AbuggSRHRYALIPIPLAVITTAbuIVLYNleNV (MW=3400) 
is activated with 4.0 .mu.mole of SPDP in 0.1M potassium phosphate buffer 
(pH 7.5) containing 0.15M sodium chloride and subsequently purified as 
described above. The purified product is then reacted with 4.0 mg (2.0 
.mu.mole) of the following plasmodium CSP-1 sequence which has been 
freshly reduced and purified: 
EQU y(QAQGDGANAGQP).sub.2 c (MW=1995) 
After purification, the resulting product is reacted with 4.0 .mu.mol of 
MSB to thiolyate the amino group of the tyrosine, after which the 
separated activated species is added to 2.0 .mu.mole of the following 
b-2-M T cell specific binding ligand: 
EQU KDWSFYLLYYTEFTPTEKDEYAC (MW=3396) 
to yield the trifunctional immunological reagent shown below: 
##STR12## 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.