Method for preparing a macromolecular monoclonal antibody composition

Macromolecular monoclonal antibody compositions are provided which are capable of selectively forming stable bonds to cells having a predetermined concentration of at least one surface antigen, such concentration being greater in such cells than in other cells in the cell population, wherein the composition comprises a substrate and a plurality of monoclonal antibodies specific to said surface antigen or antigens, which antibodies are covalently bonded to the substrate.

The present invention is directed to a method for selectively marking 
particular cells within a cell population by use of a macromolecular 
monoclonal antibody composition comprising a predetermined number and type 
of antibody molecules such that the composition is quantitatively specific 
to cells having a predetermined concentration of at least one target 
surface antigen. The predetermined concentration of the target surface 
antigen is such that the concentration of the antigen is greater on the 
surface of the cells to be selected than in other cells in the cell 
population. 
Among the uses for monoclonal antibodies, there are proposed methods of 
binding to specific antigen sites on selected cells. In particular, there 
are investigations in which monoclonal antibodies are used against 
malignant cells once the identity of cell surface antigens characteristic 
of the malignant cells are known. However, thus far the use of monoclonal 
antibodies in this way has not been generally satisfactory. Several 
reasons may be hypothesized to account for the unsatisfactory use of 
monoclonal antibodies in this respect. One reason may be that monoclonal 
antibodies may crossreact with unrelated proteins in other tissues having 
some structural portions in common with, or closely related to, the target 
antigen. Furthermore, some of the surface antigens thought to be 
characteristic only of malignant cells may also occur, to a lesser extent, 
in normal cells. Finally, normal cells may contain antigens which vary 
slightly in structure from those present on the malignant cells, but the 
structural differences may be so small so as to still enable a stable bond 
to be formed between the antibody and the normal cell surface antigen. For 
these reasons, the use of monoclonal antibodies against malignant cells 
may also be accompanied by attack of normal cells. Thus, the structural 
specificity of monoclonal antibodies is insufficient, in most cases, for 
obtaining the desired therapeutic effect without undesirable side effects. 
The present invention imparts quantitative specificity to a macromolecular 
monoclonal antibody composition, whereby the composition will be stably 
bound to a target cell only when a particular antigen, or antigens, are 
present at a concentration greater than a preselected threshold value. By 
antigen concentration is meant the concentration which is measured in a 
substantially two dimensional space, namely on the surface of a cell 
membrane. Since the antibody-antigen binding will occur at the cell 
surface the concentration of any antigenic material within the cell need 
not be considered. 
It is therefore an object of the present invention to provide a monoclonal 
antibody composition which achieves quantitative specificity thereby 
rendering said composition capable of selectively forming stable bonds 
with cells having at least one surface antigen in greater than a threshold 
concentration. 
Other objects of the invention will become apparent from the following 
specification and claims. 
In order to achieve quantitative specificity, the monoclonal antibody 
compositions of the present invention are prepared by forming a 
macromolecular unit. This macromolecular unit comprises a plurality of 
monoclonal antibody molecules, a substrate and one or more of the 
following: linkers, which are subunits by which the antibody molecule may 
be joined to the substrate; bridges, which are bifunctional cross-linking 
agents which may form bridges between antibody molecules; and toxins or 
other substances which may be incorporated into the macromolecular unit 
and which would be toxic to the cell to which the unit binds. 
The monoclonal antibodies utilized in the present invention may be 
generated by known techniques. The antibodies may be any of the antibody 
isotypes, allotypes or idiotypes, such as IgM, IgG, IgA, IgE, etc. The 
particular antibody which will be utilized will depend on the particular 
antigen which is found to be present in greater than normal concentration 
on the target cells. For example, it is known that human malignant 
melanomas express some normal surface antigens in greater than normal 
concentration. See Dippold et al., Proc. Natl. Acad. Sci. USA, 77, 6114 
(1980) and Yeh et al., Proc. Natl. Acad. Sci. USA, 76, 2927 (1979). In 
some cases it may be advantageous to utilize a plurality of different 
antibodies, each of which corresponds to a surface antigen present in the 
target cell in greater than normal concentration. For these cases, the 
macromolecular unit according to the present invention may comprise a 
mixture of monoclonal antibodies such that the corresponding 
concentrations of the various antigens on the cell surface to which the 
antibodies will bind will be reflected by the proportions of the different 
corresponding monoclonal antibodies in the macromolecular unit. The type 
and concentration of cell surface antigens in a particular cell population 
may be readily determined by conventional screening techniques by those of 
ordinary skill. See for example, Yeh et al., supra, and Herlyn et al., 
Proc. Natl. Acad. Sci. USA, 76, 1438 (1979). 
Since a target antigen may form a particularly strong antigen-antibody bond 
so that only a single antigen-antibody bond would be necessary in order 
for the macromolecular unit to be stably bound to the target cell, the 
quantitative selectivity would be lost because the macromolecular unit 
would be in such a case essentially acting as a single antibody molecule. 
Therefore, in many cases it may be necessary to modulate the binding site 
of the antibody by a chemical reaction such that it would require a 
plurality of modulated antibodies to form a stable bond between the 
macromolecular unit and the target cell surface. Such modulation may be 
accomplished, for example, by methylation of a tyrosine or serine hydroxyl 
group near, but not within, the binding site of the antibody. The aim of 
such chemical modifications is to decrease the affinity of the individual 
antibody bonding site for the antigen without appreciably affecting the 
antibody specificity. 
It is preferred that the macromolecular unit comprise a type of antibody 
such that it would require about three or four antibody-antigen bonds in 
order for the macromolecular unit to be bound to the target cell surface. 
It will be recognized that when antibodies such as divalent IgG are used 
only an even number of bonds may be formed. The number of antibody-antigen 
bonds, n, required to be formed in order to bind the macromolecular unit 
to the target cell surface will be dependent upon the affinity of the 
antigen for the particular antibody binding site. In practice, for a given 
monoclonal antibody, the lower limit of n necessary to distinguish the 
target cells from the remainder of the cell population may be readily 
determined by screening fractions of macromolecular units having different 
molecular weight for their activity against target cells. 
In order for n number of antibody-antigen bonds to be formed, it is 
necessary that all of the antibody molecules be arranged on the polymeric 
substrate so that they may simultaneously react with antigens on a cell 
surface. This requirement may be met by bonding the antibodies to a 
flexible substrate so that the antibodies are sufficiently flexible in 
their orientation to permit a plurality of antibody binding sites to 
simultaneously combine with antigens on a cell surface. The requirement 
may also be met by a polymeric substrate which is substantially planar so 
that all of the antibodies may be unidirectionally extending from the 
surface thereof. 
The substrates to be utilized according to the present invention which 
anchor the antibody molecules may be polymeric substances such as 
polysaccharides, or liposomes. 
The polysaccharides may be polydextrans, polyglucosides, etc. The polymeric 
substrate should be partially digestible by enzymes so that macromolecular 
units may be formed of a size which may be transported through the 
organism to the target cells. In the case of polysaccharides, many 
satisfactory enzymes are available such as dextranases, amylases, 
cellulases, etc. The polymeric substrate should be nonantigenic or not 
significantly antigenic in the host organism or culture. 
The macromolecular unit may also be formed by cross-linking antibody 
molecules with other organic molecules, such as non-antigenic proteins and 
polypeptides, with conventional bifunctional cross-linking agents. The 
macromolecular units may be formed by reacting antibody molecules and 
organic molecules in the presence of bifunctional reagents. In such cases, 
the polymer comprises the cross-linked antibodies and other molecules. 
Such organic molecules may be toxins or cytotoxic drugs which would cause 
the target cell to die. 
The antibodies may also be covalently bonded to liposome surfaces according 
to the method of Leserman et al., Nature, 293, 226 (1981), the disclosure 
of which is incorporated herein by reference. Monoclonal antibodies 
covalently bonded to liposomes carrying cytotoxic drugs, may be delivered 
into target cells, to eradicate the cells. While the mechanism of binding 
and ingesting the liposome bound antibodies is not clear, it is believed 
that the initial binding or non-binding mechanism which would determine 
whether the target cell is attached, is dependent upon the threshold 
concentration of the surface antigen or antigens which differentiate the 
target cells from the rest of the cell population. The use of liposomes is 
thus an alternative to the covalent linking of a toxin, such as the ricin 
A-chain, to an antibody molecule or polymeric substrate. 
In forming the macromolecular units according to the present invention, it 
is not necessary that the entire antibody molecule be utilized. Only the 
portion containing the antigen binding site is required and extraneous 
portions of the antibody molecule may be omitted. Various peptidases may 
be utilized to cleave the antibody protein and conventional techniques may 
be utilized to isolate the fraction containing the antigen binding site. 
The chemistry for either directly forming covalent bonds between the 
antibody or antibody fragment and substrate, or between the antibody, 
linker and substrate, respectively, is well known. See for example the 
reagents and procedures described in A. N. Glazer, The Proteins, Vol. IIA, 
3rd Ed., and Neurath and R. L. Hill, Editors, Academic Press, pp. 1-103 
(1976); and A. N. Glazer et al., "Chemical Modification of Proteins", 
Laboratory Techniques in Biochemistry and Molecular Biology, Vol. IV, Part 
I, T. S. Work and E. Work, Editors, North-Holland Publishing Company 
(1975); and K. Peters et al., Ann. Rev. Biochem., 46, 423-51 (1977), the 
descriptions of which are incorporated herein by reference. Further 
examples of commercially available linking agents are disclosed in the 
Pierce 1981-82 Handbook and General Catalogue, pp. 161-166, Pierce 
Chemical Company, Rockford, Ill. Known linking procedures as described in 
the above publications may be employed. For example, the monoclonal 
antibody peptide may be reacted with iminothiolane, thereby placing an 
accessible sulfhydryl group thereon. The substrate or other linker protein 
may be activated by a reaction with succinimidylpyridylthiopropionate. The 
mixture of these two prepared components would result in joining thereof 
through dsulfide bonds. 
The macromolecular units according to the present invention may be utilized 
according to known methods for introducing monoclonal antibodies to cell 
culture or for administering monoclonal antibodies in mammals. See, for 
example, Levy et al., New Eng. J. Med., 306, 517 (1982). If administered 
to mammals, such as mice, rats, rabbits or guinea pigs, the monoclonal 
antibody composition may be administered subcutaneously, intramuscularly, 
intravenously, intraperitoncally, or otherwise, depending upon the most 
efficacious way of reaching the target cells. The dose of the 
macromolecular unit which is administered or introduced will depend on the 
mass, location and distribution of the target cells within one mammal or 
culture. Exemplary techniques utilized for use of monoclonal antibodies in 
humans, animals, and cell cultures are described by Levy et al., supra, 
Young et al., Science, 211, 487 (1981), and Yeh et al., supra, 
respectively, the disclosures of which are incorporated by reference 
herein. 
Another approach in utilization of the quantitative specificity according 
to the present invention is to form a collection of various monoclonal 
antibodies to be generated against various antigens which occur on the 
surfaces of fetal cells. From this collection those antibodies which will 
react with non-target adult cells are to be eliminated. The residual 
monoclonal antibodies are to be utilized to react with target adult cells. 
Antibodies identified in this second reaction are retained and utilized to 
form the macromolecular units according to the present invention. The 
approach of this method is based on the theory that fetal cells may 
contain antigens which are most generally present in malignant target 
cells from the adult and which are most generally absent from non-target 
adult cells. 
The macromolecular units prepared according to the present invention for 
achieving quantitative specificity of antibodies may be utilized for 
marking, for purposes of isolation, various types of cells which may be 
finely divided in terms of their state of differentiation of physiological 
state when these states may be characterized by absolute quantities of a 
given antigen or mixture of antigens on the cell surface. The cell 
isolation technique may be utilized, for example, in the following way. It 
is known that in certain types of cells the phenomenon of capping occurs, 
i.e., the congregation in one area of the cell surface of most of the 
antigen molecules. Thus if capped cells are to be isolated the 
macromolecular unit according to the present invention will react with 
that capped area of the cell surface, thereby targeting the cell for 
destruction or isolation. 
The macromolecular units may be utilized in the area of curative medicine. 
For example, they may be used for the control of allergy or for 
eradicating malignant cells. For example allergy causing antibodies are 
produced by IgE secreting cells of the B lymphocyte type. These IgE 
secreting cells may not have on their surfaces any antigens that 
distinguish them sufficiently from cells which produce other antibody 
isotypes, therefore ordinary monoclonal antibodies made against IgE or 
against other antigens present on the surface of IgE producing cells may 
crossreact with other B cells as well as with other different types of 
cells. If, however, a macromolecular unit exhibiting quantitative 
specificity is formed against one or several antigens present on the 
surface of IgE producing B cells, these cells may be singled out with a 
degree of specificity not achievable heretofore. Destruction of these 
cells may reduce the IgE secretion and eliminate the allergic effects. 
The quantitatively specific macromolecular units may also be useful in 
diagnostic medicine. In particular, diagnosis in the central nervous 
system has proven to be heretofore difficult. The central nervous system 
contains cells with fine, yet important differences from one another. It 
is believed that there has been heretofore unavailable an effective means 
for sorting such fine differences especially, in situ, in living 
organisms. However, the quantitatively specific macromolecular units may 
be used for selective treatment of cells in the central nervous system, 
and in other anatomical locations, which may be identified with a high 
degree of specificity. 
There are other areas in which isolation and/or identification of specific 
cell types are important. For example in the field of diagnosis, the 
macromolecular units may be used as imaging vectors. Imaging may be 
attained by binding the macromolecular units to an appropriate molecular 
payload. 
Chemical microsurgery may be accomplished by attaching a cytotoxic payload 
to the macromolecular unit and allowing the unit to seek and selectively 
kill stringently defined subtypes of cells in the central nervous system 
or elsewhere in the organism. Finally, the use of quantitative selectivity 
has apparent uses in biological and molecular biological research, 
particularly in the investigation of cell development, differentiation and 
activation. For example, it is known that glycoprotein 70 is expressed in 
activated mouse lymphocytes, but may be found in lesser amounts on other 
cells. Klenner et al., Proc. Natl. Acad. Sci. USA, 79, 1250 (1982). This 
observation therefore may be a basis for identifying activated cells by 
macromolecular units comprising monoclonal antibodies which complement 
glycoprotein 70. Also differences in antigenic specificity among cell 
types and subtypes in the central nervous system have been reported. See 
Sternberger et al., Proc. Natl. Acad. Sci. USA, 79, 1326 (1982). The 
macromolecular units may thus be utilized to isolate and/or kill 
selectively finely defined subtypes which are present during development 
and differentiation stages of the organism and which would be beneficial 
to gaining new insights into such cell mechanisms.

The following examples are presented to illustrate the present invention, 
however the invention is not deemed to be limited to the particular 
embodiments set forth. 
EXAMPLE 1 
Spleen lymphocytes obtained from mice immunized against human melanoma cell 
line and melanoma-mouse hybrid cells are fused with the P3x63 Ag8 mouse 
myeloma according to procedures described by Koprowski et al., Proc. Natl. 
Acad. Sci. USA, 75, 3405 (1978), to produce hybrids secreting monoclonal 
antibodies against a human melanoma. Among the twenty-nine hybrid cultures 
obtained, nine show the presence of antimelanoma antibody. Among these 
nine cultures, two (691-2, 691-12) react with all (six total) melanomas 
used by screening activity, four out of the five colorectal carcinomas and 
all three of the normal human cultures. 
Antibodies from the cultures 691-2 and 691-12 are respectively isolated 
from the culture medium (see Jensenius et al., Eur. J. Immunol, 4, 91 
(1974)), treated with iminothiolane, and covalently linked to dextran 
activated with succininidylpyridylthiopropionate. The substrate bound 
antibodies are then treated with dimethyl sulfate to modulate the binding 
potential of the antigenic binding sites. The dextran substrate is then 
partially degraded by digestion with dextranase and the fractions 
according to molecular weight. At least one fraction of the substrate 
bound 691-2 and 691-12 monoclonal antibody, respectively, will react with 
one or more of the melanomas or colorectal carcinomas used previously, and 
will not react with any of the three normal human cultures. 
EXAMPLE 2 
Monoclonal antibody IgG is linked to dextran. The substrate is then 
degraded enzymatically by dextranase according to known techniques to 
block progressive degradation. Soluble fragments will be obtained carrying 
different numbers of antibody molecules and fractions may be separated by 
molecular weight according to known techniques such as electrophoresis, 
chromatography, etc. The procedure may be illustrated as follows, wherein 
Ab indicates a monoclonal antibody molecule: 
##STR1## 
EXAMPLE 3 
Monoclonal antibody molecules are cross linked with the bifunctional linker 
dipyridylsulfide. The fraction containing the desired number of n antibody 
molecules and physical properties may be separated according to 
conventional techniques. This scheme is illustrated below: 
##STR2## 
A mixture of monoclonal antibodies and the protein toxin ricin A-chain may 
be reacted with a bifunctional cross-linker such as dialdehyde or 
diimidoether according to known techniques. See Krolick et al., Proc. 
Natl. Acad. Sci. USA, 77, 5419 (1980). This produces a macromolecular 
structure comprising cross-linked antibody and toxin molecules. This 
macromolecular structure is then separated into fractions to isolate the 
macromolecular unit containing the desired number of n antibody units. 
This scheme is illustrated below: 
##STR3## 
EXAMPLE 4 
The procedure described above in Example 2 is modified by directly bonding 
the antibody and/or toxin protein to a low molecular weight water-soluble 
polymer carrying functional groups capable of reacting with functional 
groups present on the antibody and toxin protein. Such polymers may be, 
for example, polyacrylates, polyanhydrides, polyaldehydes, 
polysaccharides, etc. The resultant macromolecular units will contain 
antibody molecules and toxin molecules directly bonded to the substrate 
and may be fractionated according to conventional methods to isolate the 
particular desired structure having the required number of antibody units. 
These methods are illustrated by the following scheme: 
##STR4## 
EXAMPLE 5 
The procedures given above in Examples 2, 3 and 4 may be modified for use 
of a mixture of monoclonal antibodies. For example, in a case where the 
macromolecular unit is required to have two different antibodies, antibody 
1 and antibody 2, linking agents will be chosen having functional groups 
F.sub.1 and F.sub.2, or low molecular weight polymeric substrates will be 
chose having functional groups F.sub.1 F.sub.2, such that one of the 
F.sub.1 or F.sub.2 groups will react only with antibody 1. The other 
F.sub.1 or F.sub.2 group will react with any common functional group 
present in a protein, such as HN.sub.2. Therefore, by sequential reactions 
with antibody 1 and antibody 2 separated by a fractionation step, the 
desired macromolecular unit having the desired antibody 1 and antibody 2 
ratios, (antibody.sub.1).sub.m (antibody.sub.2).sub.n may be isolated. 
These schemes are illustrated below: 
##STR5## 
EXAMPLE 6 
In some cases the antibody or antibodies which are utilized may be partly 
inactivated by utilizing the conventional chemical procedures described 
above in Examples 1 through 4. In such a case if partial inactivation is 
not desired the respective antibody binding sites may be protected by 
first reacting the antibody with its corresponding antigen, thereby 
protecting the antibody binding site. The bound antibody may then be 
functionized with a linker group in a heterogeneous phase containing the 
bound antibody, followed by release of the antibody from the antigen to 
yield an antibody functionalized with a linker group. These methods are 
illustrated below, wherein Ag is an antigen molecule: 
##STR6## 
The cross-linked antibody moiety, Ab.sub.2 Ab.sub.1, may then be treated 
according to any of the procedures described in the previous examples.