The present invention discloses a target specific cross-linked heteroantibody and a method of producing the same. The cross-linked heteroantibodies of the present invention can cause normal autologous cells of the immune system to destroy any unwanted cell for which an antibody is available. Treatment or control of tumors, viral infected cells, fungi, bacteria, parasites and the like is now made possible through the use of the heteroantibody complex of the present invention.

BACKGROUND OF THE INVENTION 
1. Technical Field 
The present invention is related generally to the field of immunochemistry. 
More particularly, the present invention is related to antibody 
heteroaggregates which cause normal cytotoxic cells to attack specifically 
targeted cell types. 
2. State of the Art 
The most widely used methods for eliminating pathogenic or otherwise 
undesired cells is through the use of drugs. In most cases, however, drugs 
are not totally specific for the target, and serious side effects result. 
This is especially true for anti-cancer agents which are highly toxic for 
normal cells and produce severe adverse reactions. Moreover many types of 
neoplasms are unresponsive to chemotherapy. Similarly anti-viral or 
immunosuppresive drugs also often exhibit many side effects. 
One of the methods for obtaining increased specificity is through the use 
of target-specific antibodies, especially monoclonal antibodies. Such 
antibodies can, through their antibody binding sites, bind specifically to 
a designated target cell, for example a virally-infected cell, a tumor 
cell, a parasite, or a particular type of normal cell which expresses a 
distinctive cell surface antigen. However, the mere binding of antibodies 
to cells does not always lead to their destruction. Therefore, attempts 
have been made to render the antibodies cytotoxic by attaching a drug, 
toxin or radiolabelled isotope to them. Such "magic bullets", are under 
intense investigation. However, in many cases the antibody conjugates do 
not reach the target tissue because they are cleared rapidly from the 
circulation. Moreover, often large amount of antibodies must be injected 
and when they are taken up non-specifically by the wrong cells, serious 
side effects can result. Because of such limitations, a better method for 
destroying unwanted cells in vivo is needed. 
SUMMARY OF THE INVENTION 
It is, therefore, an object of the present invention to provide a means by 
which elements of the immune system can be targeted against specific types 
of unwanted or detrimental cells. 
It is a further object of the present invention to provide cross-linked 
antibodies capable of directing normal immune system to attack specific 
targets in vivo. 
It is yet another object of the present invention to provide a method of 
producing cross-linked heteroantibodies capable of causing normal 
cytotoxic cells to inactivate a specific cell type. 
It is an additional object of the present invention to provide antibody 
heteroaggregates which cause antibody-dependent cell-mediated cytolytic 
(ADCC) effector cells and cytotoxic T cells (CTL) to specifically lyse 
designated cell types. 
An advantage of the present procedure over other known procedures is that 
it utilizes an individual's own cellular immune mechanisms to render a 
variety of undesirable cells non-functional. Furthermore, unlike other 
antibody-dependent therapies, small amounts of cross linked antibody will 
provoke a relatively large cytotoxic response against pathogenic target 
cells because effector cells which have been treated with the cross-linked 
antibody can kill multiple target cells. Therefore, the possibilities of 
adverse side effects due to antibody cross reactivities, contaminants in 
the antibody preparations, and reactions against antibodies of the host 
are minimized. In the current invention, the specificity of lysis depends 
only upon the specificity of the anti-target antibody. Hence, as long as 
there is an anti-target antibody available, the efficacy and utility of 
the present system is unlimited. 
Additionally, the technique of the present invention does not introduce 
toxins, drugs, radioactive material and the like into the body. Rather, it 
activates the body's own immune system to attack the undesirable, harmful 
or pathogenic entity, thereby eliminating such deleterious entities by 
lysing or by rendering them functionally ineffective. 
Other objects and advantages will become evident as the detailed 
description of the present invention proceeds.

DETAILED DESCRIPTION OF INVENTION 
These and other objects and advantages of the present invention are 
achieved by target specific cross-linked heteroantibody and a method of 
producing the same. 
The various terms as used herein are defined as follows. 
The term "heteroantibody" or "antibody heteroaggregate" means two or more 
cross-linked, dissimilar antibodies one of which is directed against a 
specific receptor entity on a cytotoxic cell and the other is directed 
against a cell surface component on the target cell. These antibody 
heteroaggregates can also be designated as "antibody 1 x antibody 2", for 
example anti-T3.times.anti-K.sup.k. 
The term "target cell(s)" means those cells which are undesirable and need 
to be eliminated, attacked and/or destroyed functionally or otherwise. 
"Target specific" means directed against specific target cells. 
All other terms have the same meaning as is well established in the 
scientific literature or as is generally understood by those of ordinary 
skill in the art. 
Some of the essential features of this invention are: 
(1) That one of the antibodies be directed against the receptor (or 
receptor complex) on the cytotoxic effector cell which is responsible for 
triggering lysis. Antibodies against other cell surface components may not 
work. 
(2) That the second antibody be directed against a cell surface component 
on the target cell which needs to be destroyed. The target cell surface 
component must be in sufficiently high density to trigger lysis, and 
should not be present in secreted form in solution, as this may block the 
interaction of the effector cell with the target. 
(3) The two antibodies must be physically cross-linked to one another. 
Although any type of effector or cytotoxic cells could be used, the 
preferred effector cells particularly suitable for the present invention 
are those which belong to the two main classes of cytotoxic cells: (a) 
antibody-dependent cytolytic cells (ADCC effector cells); and (b) 
cytotoxic T cells (CTL). Cells belonging to ADCC and CTL categories 
normally bind the target cell by specific receptors and lyse the target 
cell. Without being bound to any particular theory, it is postulated that 
in accordance with the present invention, ADCC effectors bind and lyse 
antibody-coated target cells, while CTL bind to histocompatibility 
antigens on the target cells. What the heteroaggregates of the present 
invention do is to form an artificial bridge or a cross-linkage between 
the target cell on the one hand and the receptor on the cytotoxic cell on 
the other. Thus, by simply finding an antibody against the target cell or 
any part thereof, and incorporating this target-specific antibody as one 
of the components into the heteroaggregate (the other component of the 
heteroaggregate being a second antibody specific to the receptor complex 
or any part thereof on the cytotoxic effector cell), the specificity of 
lysis by the effector cell can be manipulated so as to be directed against 
any target cell having an antibody available thereto. 
For the purposes of the present invention, any cross linking agent can be 
utilized. However, it is preferable to use a reagent, such as SPDP vide 
infra, which only forms heteroaggregates, since homoaggregates will, of 
course, not retarget the effector cell, and could conceivably block 
retargeting. 
Although any similar or equivalent methods and material can be utilized in 
the testing and/or practice of the present invention, the preferred 
methods and materials are now described. All publications mentioned 
hereunder are incorporated herein by reference. Most of the hybridoma cell 
lines producing the antibodies used in the following descriptions are 
obtainable from the American Type Culture Collection (ATCC), Rockville, 
Md. They can also be obtained from individual investigators. Of course, 
the antibodies which can be employed in accordance with the present 
invention can either be monoclonal, polyclonal or a combination of the 
two. 
Materials and Methods 
Antibodies 
Both monoclonal and polyclonal antibodies are purified by standard 
laboratory procedures. For example, rabbit anti-2,4-dinitrophenyl (DNP) 
antibodies were isolated from immune serum by affinity chromatography 
followed by gel filtration as described by Segal, et al., Biochemistry 
15:5253 (1976). 
Monoclonal antibodies were grown as ascites in nude mice and isolated by 
gel filtration and ion-exchange chromatography as described by Unkeless, 
J. Exp. Med. 150:580 (1979). The precise methods of antibody production 
and purification are not important as long as pure, antibody is obtained. 
Polyclonal antibodies must be affinity purified in order for this method 
to work. 
In some instances it is preferable to use immunoglobulin fragments, e.g. 
Fab or F(ab').sub.2, rather than the intact immunoglobulin. 
Fab fragments were prepared by incubating purified antibodies for 4 h at 
37.degree. with a 1:100 (wt:wt)ratio of papain to immunoglobulin in 0.1 M 
sodium phosphate 0.1 M NaCl, lmM EDTA, 20 mM cystein, pH 8.0. The reaction 
was terminated by adding iodoacetamide to a final concentration of 30 mM. 
Analysis by SDS-PAGE indicated that the proteins had been completely 
digested to 50 Kd fragments. They were then dialyzed vs 0.1 M Tris.Cl, pH 
8.5 and passed over a protein A-Sepharose column to remove Fc fragments 
and trace contaminants of intact IgG. F(ab').sub.2 fragments from most 
monoclonal antibodies can be prepared by pepsin digestion in the pH range 
of 3.5-4.5, the exact pH required depending upon the antibody being 
digested (Dower et al, 1984, J. Immunol 132:751). 
Cross Linking of Antibodies 
Any procedure which forms active antibody heteroaggregates can be employed. 
Three preferred methods which have been used successfully are now 
described. 
(1) Heteroaggregates were prepared using SPDP 
(N-succinimidyl-3-(2-pyridyldithiol) propionate) according to the 
manufacturer's (Pharmacia Fine Chemicals, Pisctaway, N.J.) protocol. The 
detailed procedure for one prepration was as follows. Rabbit anti-DNP IgG 
antibodies (2ml, 12 mg/ml) and 2.4G2 (rat anti mouse Fc receptor 
(Fc.sub..gamma. R)) (4.6 ml, 5.2 mg/ml) were each dialyzed against 0.1 M 
potassium phosphate, 0.1 M NaCl, pH 7.5 (coupling buffer), and incubated 
separately for 2 h at room temperature with three fold molar excesses of 
SPDP (47 .mu.l of a 3.2-mg/ml solution of SPDP in ethanol was added to 
each sample). The 2.4G2 was redialyzed against coupling buffer and the 
anti-DNP was dialyzed against 0.1 M sodium acetate, 0.1 M NaCl, pH 4.5 
(reducing buffer). Dithiothreitol was then added to the anti-DNP to final 
concentration of 0.02 M. After 30 min at room temperature, the anti-DNP 
was passed through a Pharmacia PD10 column equilibrated with coupling 
buffer, and immediately added to the 2.4G2. After 4 h incubation at room 
temperature, 1 mg of iodoacetamide was added and the protein was eluted on 
a 2.6.times.90 cm Ultrogel AcA 22 column in borate buffered saline (BBS) 
plus 0.02% sodium azide. Polymerized material was collected in two 
fractions and concentrated using a Millipore CX-10 immersible membrane. 
This procedure produces a reducible heteroaggregate. 
(2) Non-reducible heteroaggregates are produced as follows: 10 mg of each 
protein was dialyzed against coupling buffer. To one protein was added a 3 
fold molar excess of SPDP, to the other a 3 fold molar excess of SMCC 
(Succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate). After 2 
hr at room temperature the SPDP-protein was dialyzed against reducing 
buffer and the SMCC protein against coupling buffer. The SPDP protein was 
then reduced with 0.02M dithiothreitol for 30 min at room temperature, and 
dialyzed 3 times, 2 hour each time, against 100 volumes of coupling buffer 
at 4.degree. C. It was then added to the SMCC-antibody and allowed to 
react 4 hr at room temperature (23.degree. C.-30.degree. C.). 
Approximately 50% of the starting material was cross linked. The material 
was active without fractionation. 
(3) The avidin-biotin interaction to produce cross linked antibody is as 
follows: 
(a) Biotin anti-target cell antibody: 
Dialyze 1 mg of antibody in 1 ml phosphate buffered saline (PBS) vs 0.1 M 
NaHCO.sub.3 pH 8.7. 
Dissolve 1 mg of N-hydroxysuccinimidobiotin (bio-NHS) in 1 ml 
dimethylsulfoxide. Add 7 ul of bio-NHS solution to protein (a 3 fold molar 
excess). Let sit 1 hr at room temperature. Dialyze vs PBS. 
(b) Avidin anti-receptor antibody 
Dialyze 12.5 mg of antibody, and 12.5 mg of avidin vs coupling buffer, each 
protein in 1 ml volume. Add a 3 fold molar excess of SPDP to the antibody 
(dissolve 3 mg SPDP in 0.5 ml ethanol, add 13 .mu.l ). Add 8 fold excess 
of SPDP (77 .mu.l ) to avidin. React 1 hr at room temperature. Dialyze 
avidin vs coupling buffer and antibody vs reducing buffer. Add 
dithiothreitol to antibody to a final concentration of 0.02 M. React 10 
min. at room temperature. Pass the antibody solution through a Pharmacia 
PD10 column equilibrated with coupling buffer. Add antibody to avidin. 
React 4 hr at room temperature. Pass cross linked protein on a 
1.6.times.90 cm Ultrogel AcA22 column. Collect and pool material of 
molecular weight 150,000 or greater. 
Bring pH of this material to 10.5 with 0.1 M lysine, pH 11.0. Apply to 
3.times.1 cm iminobiotin-Sepharose column. Wash with 0.1 M lysine, pH 
10.5. Elute bound protein with 1 M sodium acetate, pH 4.5. Dialyze eluted 
protein vs PBS. This is the purified avidin-antibody complex. 
To cross link biotin anti-target antibody to avidin anti-receptor, mix the 
two proteins together at a 2:1 (wt/wt) ratio of 
biotin-antibody:avidin-antibody, at a total protein concentration of about 
1 mg/ml. Add immediately to cells. 
Lytic Assays 
Effector Cells. Cells from the P388.sub.1 mouse macrophage line were grown 
in spinner culture as described by Koren, et al., J. Immunol. 114:894 
(1975), harvested, washed twice, and resuspended to a final concentration 
of 2-4.times.10.sup.7 cells/ml in culture medium containing the desired 
concentration (usually 10-20 .mu.g/ml) of cross-linked antibody. After 
incubation at 0.degree. C. for 30-45 min, cells were washed twice and 
resuspended to the desired concentration in culture medium. 
Human CTL clones 8.2, 8.4, 8.5 and 8.9 were carried in the laboratory of 
Dr. Steven Shaw and are specific for the HLA-DPw2 antigen (Biddison, et 
al., J. Exp. Med 159:783 (1984); Shaw, et al., J. Immunol. 134:3019 
(1985)). 
Mononuclear cells were isolated from freshly drawn, heparinized veinous 
blood from normal donors by density gradient separation over 
Ficoll-Hypaque (LSM, Bionetics, Kensington, MD). Monocytes were depleted 
by passage over Sephadex G10 as described (Hathcock, et al. (1980) Manual 
of Macrophage Methodology, Edited by H. B. Herscowitz et al., Marcel 
Dekker, NY, p.78) yielding a lymphocyte preparation. The leu 11.sup.+ 
cells (including most of the NK activity) were removed from the 
lymphocytes by incubating them with anti-Leu 11b (Becton-Dickinson, 
Mountain View CA) and rabbit complement (Pel Freeze, Rogers, AK) using 
Becton-Dickinson's suggested protocol. 
In some experiments, large numbers of cells were obtained from a normal 
donor by leukapheresis. In those experiments cells were cryopreserved and 
stored in liquid nitrogen (Holden, et al. (1977) In vitro Methods 
Cell-Mediated and Tumor Immunity. Ed. by B. R. Bloom and J. R. David. 
Academic Press New York p. 723) as either mononuclear cells, lymphocytes, 
or as Leu 11-depleted lymphocytes. 
Target Cells 
Target cells used in the assays are RDM4 and EL4 which are, respectively, 
H-2.sup.k and H-2.sup.b murine T-cell lymphomas, and M16 (Shaw et al, J. 
Exp. Med. 152:565 (1980)) is a human Epstein-Barr virus transformed 
lymphoblastoid cell line. Chicken red blood cells (CRBC) were also used as 
targets. In some experiments, target cells were modified with TNP by 
incubating them with 3 mM trinitrobenzene sulfonate for 10 min at 
37.degree. in PBS, pH 7.4. Cells were .sup.51 Cr-labeled as desribed 
(Jones et al. J. Immunol. 126:2457 (1981). Human tumor cells were obtained 
from various investigators. 
Cytotoxicity Assays 
Cytotoxicity was measured in culture medium using 96 well, U-bottom 
microtiter plates (Jones et al, 1981, J. Immunol. 126:2457). To each well 
was added varying numbers of effector cells in 100 .mu.l medium, followed 
by 20 ul of antibody or medium and either 1.times.10.sup.4 or 
5.times.10.sup.3 .sup.51 Cr-labeled target cells in 100 .mu.l medium. 
Plates were incubated for 4 hr. at 37.degree. in 5% CO.sub.2, 100% 
humidity. Superatants were harvested with a Titertek harvesting system 
(Skatron, Sterling, VA), and counted in a well-type gamma counter. 
Triplicate determinations were made for each sample. Maximum lysis was 
measured by incubating target cells in 5% Triton X-100, and spontaneous 
release was determined by incubating the target cells in medium alone. 
Percent lysis (P) was calculated from the formula: 
EQU P=100.times.(experimental release-spontaneous release)/(maximum 
release-spontaneous release). 
Results 
To demonstrate that anti-T3 containing heteroaggregates can cause human T 
cells to lyse any desired target cells, four different human anti-HLA-DPw2 
(SB2) cytotoxic T lymphocyte (CTL) clones 8.2, 8.4, 8.5 and 8.9, were 
tested. All four express the T3 and T4 antigens and target cell lysis 
mediated by three of the clones (8.4 being the exception) is inhibited by 
anti-T3 antibody (Biddison et al, J. Exp. Med. 159:783 (1984)). Clones 8.9 
and 8.5 were negative for FC.sub.65 R (as determined by flow cytometry) 
and therefore cannot mediate ADCC (clones 8.2 and 8.4 were not tested). 
Cross linked antibodies were prepared as described in the Methods Section. 
Two cross linked fractions were separated from monomeric IgG by gel 
filtration (FIG. 1). Tests showed that both were equally active and they 
were, therefore, used interchangeably. 
In Table I, the four clones were tested for the ability to lyse M16, a 
DPw2+human lymphoblastoid cell line, RDM4, an H-2.sup.k mouse tumor line, 
and EL-4, an H-2.sup.b mouse tumor line. The data indicate that these 
clones lyse the M16 target but not the murine tumor cells. Moreover, lysis 
of M16 by clones 8.9, 8.2 and 8.5 was blocked by 
anti-T3.times.anti-K.sup.k showing that anti-T3 remained active in the 
heteroaggregate. The main finding of Table I is that 
anti-T3.times.anti-K.sup.k causes each of the CTL clones to lyse the 
K.sup.k positive RDM4 targets, but not the K.sup.b -expressing EL-4 cells. 
RDM4 was also lysed if either effector or target cells were preincubated 
with the heteroaggregates and washed prior to experimentation. 
As expected, the antibody heteroaggregates cause a specific increase in 
conjugates between effector and target cells. Thus, when clone 8.9 and 
RDM4 were mixed, only 8% of the cells were in conjugates, whereas in the 
presence of anti-T3.times.anti-K.sup.k, 31% were in conjugates. 
TABLE I 
______________________________________ 
Anti-T3 .times. anti-K.sup.k heteroaggregates retarget CTL to 
K.sup.k -expressing cells 
% Specific lysis of target cell 
Clone anti-T3 .times. anti-K.sup.k 
M16 RDM4 EL-4 
______________________________________ 
8.9 - 47.0 6.6 .8 
8.9 + 11.5 53.3 .2 
8.2 - 44.9 9.1 3.3 
8.2 + 20.4 43.9 -.7 
8.4 - 49.8 7.2 4.3 
8.4 + 54.1 32.9 .8 
8.5 - 35.7 1.5 .6 
8.5 + 8.0 30.0 .4 
______________________________________ 
Lysis was measured in the presence or absence of 5 .mu.g/ml antiT3 .times 
antiK.sup.K at a 10:1 effector:target ratio in a 3 hr .sup.51 Cr release 
assay. Antibody was preincubated with effectors and targets for 30 min at 
0.degree., before warming to 37.degree.. Human CTL clones 8.2, 8.4, 8.5 
and 8.9 are specific for the HLADPw2 antigen. M16 is a human EpsteinBarr 
Virustransformed lymphoblastoid cell line which expresses HLADPw1 and DPw 
antigens. RDM4 and EL4 are H2.sup.k and H2.sup.b murine Tcell tumor lines 
Percent lysis was calculated as described in the methods section, and 
spontaneous release was 8.9, 5.2, and 6.8 percent for M16, RDM4, and EL4, 
respectively. 
In order to test the specificities required for cross linked antibodies to 
mediate lysis, anti-T3 was cross linked to anti-DNP, anti-T4 to 
anti-K.sup.k and anti-DNP to anti-K.sup.k. The data in Table II show that 
the anti-T3.times.anti-DNP confers anti-DNP specificity to T-cell clones; 
in the presence of this heteroaggregate, CTL lyse TNP-RDM4 and TNP-EL4 but 
not unmodified EL4 or RDM4. Moreover, DNP hapten in solution strongly 
inhibits lysis of the haptenated targets. It is noted that CTL even lyse 
hapten-modified chicken red blood cells-(CRBC) in the presence of 
anti-T3.times.anti-DNP. Thus, the antibody heteroaggregates seem to 
override the normal requirement for lysis that T-cell receptors bind to 
histocompatibility antigens on target cells, since it is unlikely that 
either CRBC or the murine T-cell tumor lines EL4 and RDM4 express 
histocompatibility antigens which could be recognized by the class II 
specific CTL clones used here. 
TABLE II 
__________________________________________________________________________ 
The specificity of CTL lysis in the presence of 
hetero-crosslinked antibodies 
% specific lysis of target cells by clone 8.9 
Antibody added 
anti-T3 .times. 
anti-T3 .times. 
anti-T3 .times. 
anti-K.sup.k + 
anti T3 .times. 
anti-DNP + 
anti-T4 .times. 
Target None 
anti-K.sup.k 
anti-T3 
anti DNP 
DNP anti-K.sup.k 
__________________________________________________________________________ 
M16 37.8 
-7.0 -29.7 22.3 -- 29.9 
RDM4 -8.7 
38.2 2.0 -8.6 -- 2.1 
EL-4 -4.9 
-4.2 -- -4.3 -- -- 
CRBC 1.1 -- -- 1.3 .0 -- 
M16-TNP 
55.1 
-- -- 32.3 43.8 -- 
RDM4-TNP 
-8.1 
-- -- 40.5 7.4 -- 
EL-4-TNP 
.4 -- -- 49.1 .3 -- 
CRBC-TNP 
0.8 -- -- 63.9 .0 -- 
__________________________________________________________________________ 
Experiments were performed as described in Table I using clone 8.9 cells 
as effectors. Clone 8.5 also lysed TNPCRBC in the presence of antiT3 
.times. antiDNP in a haptenspecific manner (data not shown). In all 
experiments shown in Table II, 10 .mu.g/ml cross linked antibody was used 
but 20 and 40 .mu.g/ml of antiT4 .times. antiK.sup.k were also tested wit 
results similar to those shown here. AntiT3 (50 .mu.g/ml, column 4) and 
DNPaminocaproate (0.1 mM, column 6) were used as inhibitors. 
DNPaminocaproate did not inhibit lysis of M16 by clone 8.9 (data not 
shown). Target cells were modified with TNP by incubating them for 10 min 
at 37.degree. in PBS, pH 7.4, containing 3 mM trinitrobenzene sulfonate. 
-- indicates experiment not done. Negative values for percent lysis 
indicate that chromium release was less than spontaneous release. In the 
test sample, spontaneous release values for the various targets were: M16 
28.6%; RDM4, 11.4%; EL4, 12.4%; CRBC, 2.9%; M16TNP, 7.4%; RDM4TNP, 15.4%; 
ELTNP, 4.5% AND CRBCTNP, 3.0%. 
It was important to establish if anti-T3 was a necessary component of the 
antibody heteroaggregate, or if mAb against any effector cell surface 
molecule would trigger lysis. Therefore, the effect of 
anti-T4.times.anti-K.sup.k upon lysis of M16 and RDM4 was tested. In the 
presence of anti-T4.times.anti-K.sup.k, 21% of effectors were conjugated 
while 9% were conjugated in its absence. However, as shown in Table II, 
this cross linked preparation neither promoted lysis of RDM4 nor 
significantly blocked lysis of M16 by clone 8.9. In a second test CTL 
clone 8.9 was treated with 1.5 mM trinitrobenzene sulfonate and tested for 
lysis against RDM4 in the presence of anti-DNP.times.anti-K.sup.k. Control 
experiments showed that anti-DNP.times.anti-K.sup.k promoted specific 
conjugate formation between TNP-8.9 and RDM4 and that the TNP-8.9 cells 
were able to lyse their natural target, M16. However, TNP-8.9 cells did 
not lyse RDM4 cells in the presence of anti-DNP.times.anti-K.sup.k (&lt;2% 
lysis over a concentration range of 5-40 .mu.g/ml heteroaggregate). 
Therefore, cross linking of target cells specifically to the T4 molecule 
and to a presumably large number of TNP-modified components on the 
effector cells did not lead to lysis. These data suggest that the cross 
linking of target cells directly to the T-cell receptor complex is a 
requirement for lysis. 
Lysis of Target Cells by Peripheral Blood T Cells 
In order to show that freshly drawn human peripheral blood T cells could 
also be rendered cytotoxic for specified targets with appropriate cross 
linked antibodies, mononuclear cells were incubated with .sup.51 
Cr-labeled RDM4 in the presence of anti-T3 x anti-K.sup.k or with .sup.51 
Cr-labeled TNP-RDM4 in the presence of anti-T3 x anti-DNP (Table III): In 
both cases, the mononuclear cells lysed the target cells when cross linked 
antibody was present, but did not mediate lysis in the absence of 
antibody. However, mononuclear cells also mediated antibody-dependent 
cellular cytotoxicity (ADCC), as shown by target cell lysis in the 
presence of anti-K.sup.k (which is an IgG2a antibody). Since the cross 
linked antibodies possessed Fc fragments, it was possible that tumor cell 
lysis in the presence of the antibody heteroaggregate represented ADCC and 
not heteroaggregate-dependent lysis mediated by peripheral blood T cells. 
To eliminate this possibility, mononuclear cells were first passed over 
Sephadex G10 to remove monocytes. This removed approximately 2/3 of the 
OKM1.sup.+ cells, and essentially all of the cells falling within the OKM1 
bright peak. As a result of monocyte depletion, ADCC activity was 
substantially reduced, while heteroaggregatedependent lysis increased 
(Table III). An aliquot of the G10-passed cells was then treated with 
anti-leu 11b and complement, to remove the K, NK subset of cells. (The Leu 
11 epitope is on the neutrophil and K cell Fc.sub..gamma. receptor, but 
not on monocyte Fc.gamma. receptors (Perussia et al., J. Immunol. 133:180 
(1984)). This treatment removed the reamining ADCC activity, (Table III) 
leaving a population of lymphocytes which was 87% T3.sup.+ and which 
required the anti-T3-containing heteroaggregate for mediating lysis. 
TABLE III 
__________________________________________________________________________ 
Lysis of RDM4 Cells by Franked Peripheral Blood T Cells 
Effectors 
Monocyte 
Leu 11 
depleted.sup.c 
depleted 
Mononuclear 
(lymphocytes) 
lymphocytes.sup.d 
IL-2.sup.e 
Exp 
Antibody Cells % lysis activated 
__________________________________________________________________________ 
1.sup.a 
none -0.6 1.8 -0.3 4.6 
anti-T3 .times. anti-K.sup.k 
10.1 16.2 26.0 43.2 
anti-K.sup.k 
18.5 8.4 -2.3 3.9 
2.sup.b 
none -1.8 -1.0 -1.4 3.1 
anti-T3 .times. anti-DNP 
6.2 7.5 8.4 38.2 
anti-K.sup.k 
5.7 2.1 0.0 2.6 
__________________________________________________________________________ 
.sup.a Tested against .sup.51 Crlabeled RDM4 at E:T = 20:1 in a 4 h assay 
AntiT3 .times. antiK.sup.k and antiK.sup.k were added to wells at 10 
.mu.g/ml and 5 .mu.g/ml final concentrations, respectively. AntiK.sup.k i 
367-5, a mouse IgG2.sub.a monoclonal antibody. 
.sup.b Same as in exp 1 except targets were TNPRDM4 and antiT3 .times. 
antiDNP was used at 5 .mu.g/ml. Effector cells were from a different dono 
than in exp 1. 
.sup.c By passage over Sephadex G10. 
.sup.d By treatment of the G10passed cells with antiLeu 11b and 
complement. 
.sup.e Leu 11 depleted lymphocytes were incubated overnight with 50 (exp 
1) or 30 (exp 2) units/ml of recombinant IL2. 
The data of Table III show that heteraggregate-dependent lysis mediated by 
human peripheral blood T-cells is markedly enhanced by overnight 
incubation with recombinant IL-2. Other data indicated that unlike IL-2, 
recombinant human interferon does not stimulate lysis. 
Retention of heteroaggregate-dependent Lytic activity by human T cells 
It was also important to determine for in vivo use of 
heteroaggregate-treated T cells, as to how long after treatment did the T 
cells retain activity when incubated at 37.degree., the physiological 
temperature. To test this, Leu 11-lymphocytes were activated with IL-2 and 
divided into two portions, one of which was incubated with 
anti-T3.times.anti-K.sup.k, the other with medium alone. The cells were 
then washed and incubated for 0, 4, 8, or 24 hr at 37.degree. and tested 
for lytic actvity against RDM4. As seen in Table IV, the treated cells 
retained considerable activity even after 24 hr incubation. 
Lysis of Human Tumor Cells by T-cells treated with Anti-T3.times.anti-tumor 
cell 
It has been deomonstrated above that cloned human CTL or human peripheral 
blood T cells can be induced, in vitro, to lyse target cells, which they 
normally would not lyse, by treating them with certain covalently 
crosslinked antibody heteroaggregates. In order to demonstrate that such 
effector cells can also be used to lyse tumor cells, several anti-tumor 
monoclonal antibodies were cross linked to anti-T3. The data presented 
below show that these heteroaggregate preparations cause human peripheral 
blood T cells to specifically lyse cultured tumor cells and fresh human 
tumor cells, but not normal cells from a variety of tissues. 
TABLE IV 
______________________________________ 
Effector Cells Retain Activity During Incubation at 37.degree. C. 
Treatment with 
Time of Incubation (hr) at 37.degree. C. 
Heteroaggregate.sup.b 
0 4 8 24 
Pre-coat.sup.c 
In medium.sup.d 
Percent Specific Lysis (lytic units).sup.a 
______________________________________ 
- - 2.2 3.7 4.5 6.5 
- + 33.7 (5.3) 
51.4 (7.4) 
42.4 (4.4) 
21.9 (1.3) 
+ - 33.0 (4.3) 
46.6 (4.3) 
48.5 (5.0) 
31.4 (2.4) 
+ + 32.0 (5.6) 
44.3 (3.9) 
49.2 (3.9) 
21.4 (1.4) 
______________________________________ 
.sup.a Percent lysis of RDM4 cells by IL2 activated, Leu 11.sup.- 
lymphocytes at E:T = 20:1. Lytic units, defined as the initial slope of a 
hyperbolic fit to the E:T dose response curve, are indicated in 
parentheses, where measurable. Effector cells were activated by overnight 
incubation with incubation with 100 units/ml recombinant IL2. 
.sup.b AntiT3 .times. antiK.sup.k 
.sup.c Effector cells were incubated 1 hr. at 0.degree. C. with (+) or 
without (-) 20 .mu.g/ml heteroaggregate and washed, prior to incubation a 
37.degree. C. 
.sup.d Heteroaggregate (5 .mu.g/ml) was either present (+) or absent (-) 
in the lytic assay medium. 
Human CTL clone 8.9 was treated with three different heteroaggregates and 
tested for lysis against various human tumor lines (Table V). Also shown 
in Table V are the results of fluorescence analyses of cells which were 
stained with the anti-tumor antibodies. Antibody 315F6, which was raised 
against a large cell lung carcinoma, binds to all of the tumor lines 
tested and, when crosslinked to T3, promotes lysis of all of the tumor 
lines by clone 8.9. Antibody 8H12 (anti-breast cancer) cross-reacts on 
small cell lung cancer and colon cancer lines and promotes their lysis, 
while HeFi-1 binds specifically to the L428 Reed-Sternberg Line, against 
which it was raised, and promotes lysis of that line when crosslinked to 
anti-T3. Thus, heteroaggregates containing anti-tumor antibodies can 
direct clone 8.9 to lyse human tumor targets, and the specificity of lysis 
parallels the specificity of the anti-tumor antibody. 
Having demonstrated that heteroaggregate-treated cloned CTL can lyse tumor 
lines, it was next determined that treated human peripheral blood T cells 
could lyse cells from either fresh tumor tissue or fresh normal tissue. 
Table VI shows that peripheral T cells treated with anti-T3 crosslinked to 
several anti-tumor antibodies specifically lyse fresh tumor cells but, 
with the exception of liver, do not lyse normal cells. Of the tumor cells 
that were tested, melanoma and colon cells were observed to be the most 
susceptible to lysis. It is not known with certainty why normal liver 
cells are lysed to a small extent by the treated T cells, but if some of 
the liver cells express Fc receptors, then they might bind to the T cells 
by the Fc portions of the heteroaggregates, rather than by the antibody 
combining sites. This could be prevented by using Fab fragments in the 
heteroaggregates. 
TABLE V 
__________________________________________________________________________ 
Heteroaggregate-dependent lysis of human tumor lines by cloned 
cytotoxic T cells.sup.a. 
Heteroaggregate.sup.b 
None Anti-T3 .times. 315F6 
Anti-T3 .times. 8H12 
Anti-T3 .times. HeFi-1 
Target cells.sup.c 
Percent specific lysis (mean fluorescent channel 
.times. 10.sup.-2).sup.d 
__________________________________________________________________________ 
BL-1 15 
(1.3) 
96 (6.9) 
5 (1.4) 
9 
L428 6 (1.4) 
90 (5.9) 
15 57 (5.0) 
N592 11 
(1.3) 
85 (5.5) 
67 (4.9) 
16 
N526 18 
(1.2) 
69 (6.5) 
70 (3.8) 
10 
H125 9 (1.3) 
42 (4.3) 
14 12 
LS174T 2 (1.3) 
69 (5.8) 
53 (4.6) 
12 
A549 1 (2.5) 
50 (6.1) 
2 (2.5) 
1 
__________________________________________________________________________ 
.sup.a The human antiDPW2 Tc clone, 8.9 was incubated with 20 ug/ml 
crosslinked antibody, washed, and tested for lysis against various target 
cells at an effector:target ratio of 5:1 in a 4hour .sup.51 Cr release 
assay. 
.sup.b AntiT3 was crosslinked to antitumor antibody as described in the 
methods. Preparations containing mainly dimers, trimers, and tetramers 
were used. 315F6 was a mouse monoclonal antibody (mAb) raised against a 
human large cell lung cancer line, the 8H12 mAb was generated against the 
MCF7 human breast carcinoma line, and mAb HeFi1 was raised against the 
L428 ReedSternberg line. 
.sup.c Target cells used in this table are: BL1, a Blymphoblastoid line; 
L428, a ReedSternberg line; N592 and N526, small cell lung cancer lines; 
H125, a large cell lung cancer line; LS174T, a colon line; and A549, a 
lung carcinoma line. 
.sup.d Tumor cells were incubated with biotinylated 315F6, 8H12, HeFi1 fo 
30 minutes on ice. Cells were washed, resuspended in 10 ul of 50 ug/ml 
fluorescein isothiocyanateavidin, and incubated for 15 minutes on ice. Th 
cells were washed and analyzed by flow cytometry. Mean log fluorescence i 
indicated with 100 channels corresponding to a factor of 2 increase in 
fluorescence (e.g., 315F6 stains BL1 with fluorescences about 50fold over 
background). 
TABLE VI 
__________________________________________________________________________ 
Lysis of fresh human normal and neoplastic tissue by heteroaggregate- 
treated peripheral blood T cells.sup.a. 
Target tissue.sup.b 
Lung 
Normal 
Colon 
Normal 
Normal 
Melanoma 
cancer 
lung tumor 
liver 
kidney 
Heteroaggregates.sup.c 
Percent specific lysis 
__________________________________________________________________________ 
none 1 9 1 1 -1 3 
anti-T3 .times. 96.5 
24 4 -3 -1 9 2 
anti-T3 .times. 315F6 
30 4 2 16 10 4 
anti-T3 .times. 8H12 
-1 7 3 41 8 1 
anti-T3 .times. 47D10 
2 5 -1 18 10 0 
__________________________________________________________________________ 
.sup.a Effector cells were prepared as described in the methods, incubate 
with 10 .mu.g/ml heteroaggregate, and tested for lysis against target 
cells at a 10:1 effector:target ratio in a 3hour .sup.51 Cr release assay 
.sup.b Single cell suspensions were prepared from fresh normal or 
neoplastic tissue. They were labeled with .sup.51 Cr by adding 0.2 .mu.l 
.sup.51 Cr stock to 0.5 ml of cells, incubating 4 hours at 37.degree. C. 
and washing. 
.sup.c 96.5 is an antimelanoma mAb, and 47D10 is an antilung carcinoma mA 
with crossreactivity against colon tumors. 
Lysis Mediated by Heteroaggregate-coated ADCC Effector Cells 
Initial studies (Karpovsky et al, 1984, J. Exp. Med. 160:1686) showed that 
mouse ADCC effector cells could be rendered cytotoxic with 
heteroaggregates containing antibodies against Fc receptors on the 
effector cells. Typical results from these studies are shown in Table VII. 
Here cells from a mouse macrophage line were coated with 
anti-Fc.sub..gamma. R.times.anti-DNP, and tested for lysis against .sup.51 
Cr-labeled chicken red blood cells as targets. Table VII shows that this 
heteroaggregate causes the effector cells to specifically lyse 
TNP-modified but not unmodified chicken red blood cells. Untreated 
effector cells will lyse antibody-coated target cells (Table VII, 
classical ADCC) but not TNP-CRBC. Unlike classical ADCC, lysis mediated by 
effectors coated with heteroaggregates is not blockable by immune 
complexes. For example, in one test, 10 .mu.g/ml of immune complexes 
inhibited classical ADCC by over 80% while inhibition of 
heteroaggregate-dependent lysis was undetectible (Karpovsky et al, 1984, 
J. Exp. Med. 160:1686). 
TABLE VII 
__________________________________________________________________________ 
Lysis of Target Cells by P388D.sub.1 Cells* 
Percent lysis.sup.++ of: 
Antibody- 
coated TNP 
TNP 
Treatment of effector cells 
Experiment 
CRBC CRBC CRBC 
__________________________________________________________________________ 
Incubated with medium only 
1 12.2 (0.5) 
-0.1 (4.1) 
-0.5 (0.4) 
2 34.5 (1.0) 
2.5 (0.5) 
0.4 (0.4) 
3 13.8 (1.1) 
3.0 (0.9) 
2.9 (0.6) 
4 11.1 (1.4) 
2.4 (0.1) 
-0.6 (0.7) 
5 31.4 (5.7) 
3.5 (0.6) 
1.2 (0.3) 
Franked with anti-Fc.gamma.R .times. anti DNP 
1 50.4 (2.4) 
44.5 (5.7) 
-0.8 (0.4) 
2 46.3 (3.8) 
34.8 (2.3) 
1.5 (0.5) 
3 107.5 (2.8) 
90.8 (1.6) 
-0.1 (0.5) 
4 40.5 (5.5) 
53.3 (2.8) 
-0.9 (0.5) 
5 62.7 (3.6) 
49.5 (2.1) 
0.9 (0.2) 
__________________________________________________________________________ 
*At effectorto-target ratios of 10:1. 
.sup.++ Means of triplicate samples followed by standard errors in 
parenthesis. 
P388D.sub.1 cells were treated with 10 ug/ml of antiFc.gamma.R .times. 
antiDNP. 
The data shown in Table VIII demonstrate that human peripheral blood 
lymphocytes contain a subset of cells which will mediate lysis of 
TNP-modified EL-4 tumor cells when coated with anti-Fc.sub..gamma. 
R.times.anti-DNP. These cells are clearly different from the cytotoxic T 
cells which mediate lysis when coated with anti-T3.times.anti-DNP, since 
removal of the T8+lymphocytes totally removes all of the T3-dependent 
activity but only partially removes the Fc.sub..gamma. R-dependent lysis, 
while removal of the Leu 11+ cells does the opposite. The data of Table 
VIII show that two different kinds of effector cells can be retargeted 
with appropriate antibody heteroaggregates and indicate that other types 
of cytotoxic effector cells could also be rendered lytic for designated 
target cells by using appropriate antibody heteroaggregates. The data in 
Table VIII also show that cross-linked Fab fragments are as effective as 
cross-linked intact antibodies in retargeting effector cells. This is 
important because it may be preferable to use the Fab-containing 
heteroaggregates for some in vivo applications. 
TABLE VIII 
__________________________________________________________________________ 
Heteroaggregate-dependent Lysis Mediated by Human 
Peripheral Blood T Cells and ADCC Effector Cells 
anti-T3(Fab) .times. 
anti-Fc.gamma.R(Fab) .times. 
Effector 
no anti-DNP(Fab) 
anti-DNP(Fab) 
Effector Cells 
Target Ratio 
antibody 
% Specific lysis of El-4-TNP 
__________________________________________________________________________ 
Lymphocytes 
20 3.5 39.8 81.6 
10 4.8 20.0 62.3 
5 3.9 13.7 39.5 
2.5 6.6 9.1 28.2 
Lymphocytes, 
20 9.4 6.2 43.5 
T8 depleted 
10 8.1 5.5 47.8 
5 6.2 4.6 35.0 
2.5 6.0 3.7 21.9 
Lymphocytes, 
20 3.7 36.7 7.8 
Leu 11 depleted 
10 3.0 19.5 4.5 
5 2.6 11.1 3.7 
2.5 4.6 6.9 2.3 
__________________________________________________________________________ 
Based on the present disclosure, it should be obvious to one of ordinary 
skill in the art that a multiplicity of cells from freshly drawn human 
peripheral blood such as granulocytes, monocytes, macrophages, K cells, T 
cells, and the like could be retargeted by this technique. In other words, 
many types of cytolytic cell can be targeted to destroy most types of 
cells using a suitable antibody heteroaggregate as described herein. Some 
of the examples of the target cells are tumor cells, virally infected 
cells, fungi, bacteria and parasites, unwanted normal cells such as 
autoreactive cells, and cells responsible for graft rejection. Tables 
I-VIII demonstrate the results obtained with cloned and peripheral blood T 
cells in accordance with the present invention. 
For in vivo use of the invention, many protocols could be adopted, 
depending upon the particular application. For example, normal peripheral 
blood leukocytes could be removed from a patient, incubated with 
appropriate antibody heteroaggregates, optionally with an activator such 
as IL-2, washed, and reintroduced into the circulation. If the treated 
cytotoxic cells eventually reached the target cells, they could initiate 
target cell destruction. This procedure renders an extremely efficient use 
of the antibody. For example 10.sup.9 human T cells, each with 10.sup.5 T3 
molecules on its surface would bind a maximum of 50 .mu.gm of 
heteroaggregate. Thus, only microgram amounts of antibody heteroaggregates 
would be required to retarget a large number of effector cells, and in 
addition, the heteroaggregates would not be free in the blood where they 
might be removed by elements of the reticuloendothelial system. Other 
techniques usually require much higher amounts of antibody, and the 
antibodies themselves are usually recognized as being foreign and are 
rapidly removed from the circulation. 
Further applications of target-specific heteroantibodies produced in 
accordance with the present invention for the treatment or control of 
abnormal conditions, the origin of which may be related, directly or 
indirectly to abnormal cellular function, are unlimited. As pointed out 
herein supra, the only requirements are that one of the antibodies be 
directed against the appropriate receptor on the cytotoxic effector cell 
and a second antibody be directed against a cell surface component on the 
target cell and the two antibodies be cross-linked. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and the 
scope of the appended claims.