Methods and compositions concerning homogenous immunotoxin preparations

Immunotoxin preparations are described in which the preparations are enriched for a single species of immunotoxin. Also described are methods for the preparation of the substantially purified immunotoxins (ITs). Also disclosed are methods for determining the most effective species of immunotoxin conjugates for treated diseases and pharmaceutical preparations for such treatments.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to the field of immunotoxins, i.e., 
toxins conjugated to targeting molecules, and to methods for their 
preparation and use. The invention particularly concerns novel methods of 
purifying immunotoxins and determining the most effective species of 
immunotoxin conjugates for treatment of specific diseases. 
2. Description of the Related Art 
Ricin A chain (RTA) is the catalytic subunit of the plant toxin ricin and 
is frequently used for the construction of immunotoxins (ITs) (Lord et 
al., 1988). To be pharmacologically active, RTA-Its must have a disulfide 
bond between the antibody and the RTA (Masuho et al., 1982). The chemical 
linkage of RTA to antibody involves the introduction of activated thiol 
groups into the molecule of antibody and the reaction of the thiolated 
antibody with reduced RTA. The free thiol groups of the RTA molecule 
displace the pyridyl disulfide from the activated thiol groups of the 
antibody to form disulfide linkages. The resulting IT is then purified to 
remove the free RTA and antibody by two successive chromatographic steps 
on Blue-SEPHAROSE SEPHACRYL S-200 (Ghetie et al., 1988a; 1991). 
Unfortunately, the IT that results from such methods is not a homogeneous 
product, but contains mixtures of conjugates comprising several molecular 
species with one, two or more RTAs linked to each molecule of antibody. 
These mixtures are not easily separated, particularly during scale-up, 
because their MWs and electrical charges are similar. 
This formation of mixtures of conjugates that contain different proportions 
of deglycosylated A chain (dgA) or other toxin is the principal problem in 
the preparation of ITs by chemical methods. The mixture usually comprises 
a major component containing equimolar amounts of antibody and toxin and 
several other conjugates with two or more toxin molecules per molecule of 
antibody. This heterogeneity results from the introduction of various 
numbers of thiol groups per molecule of antibody. Due to the nature of the 
chemical reaction between the N-succinimidyl group of the crosslinker and 
the primary amino groups of the antibody, populations of antibody 
molecules substituted with none, one, two, three and even more than three 
thiol groups are obtained. 
For example, it was reported that when the crosslinker 
N-succinimidyl-oxycarbonyl-.alpha.-methyl-(2-pyridyl-dithio)toluene (SMPT) 
was added to the molecule of antibody (mouse IgG.sub.1) in a 5-fold molar 
excess, the average number of thiol (MPT groups) introduced was 2 (Ghetie 
et al., 1991). However, when the IgG.sub.1 -MPT was chromatographed 
through an activated thiol-AGAROSE column in the inventors' laboratory, 
25% of the protein did not bind and after treatment with dithiothreitol, 
it did not release pyridine-2-thione, demonstrating that 25% of the 
IgG.sub.1 molecules were not substituted. This indicated that the average 
number of MPT groups introduced was 2.7 and not 2. Further, when this 
thiolated antibody was reacted with dgA, the main component (50%) 
contained one molecule of dgA per molecule of antibody. This suggested 
that not all MPT groups introduced into the IgG.sub.1 molecule are able to 
bind dgA and therefore, that the IT may contain some molecules of antibody 
with unreduced MPT groups. 
To decrease the number of MPT groups introduced into the molecule of 
antibody, a lower molar ratio between SMPT and IgG.sub.1 was used (2-3). 
In this case, the degree of substitution averaged 1 MPT group per molecule 
of IgG.sub.1 and a 180 kDa IT was formed after reacting with dgA. However, 
the yield of IT obtained (10-15%) was considerably lower than the yield 
obtained when an IgG.sub.1 -MPT with 2 MPTs per molecule of IgG.sub.1 was 
used (50-60%) (Ghetie et al., 1991). Therefore, merely adjusting the molar 
ratio between SMPT and IgG.sub.1 is not an efficient way to produce ITs of 
a particular toxin to antibody molar ratio. 
This inability to produce pure antibodies of a particular toxin/antibody 
ratio has also made it difficult to assess which immunotoxin preparation 
would be the most beneficial in a pharmacological preparation. Although a 
comparison of two ITs constructed with either one or two molecules of 
intact ricin (A+B chains) has been reported (Marsh and Neville, 1986), 
this comparison has not been done with the ricin A chain conjugated to an 
antibody. Therefore, there exists an immediate need for a procedure for 
large scale, efficient preparation of immunotoxins which can be separated 
according to the toxin/antibody molar ratio. 
SUMMARY OF THE INVENTION 
The present invention seeks to overcome these and other drawbacks inherent 
in the prior art by providing efficient procedures for the large scale 
preparation of immunotoxins for use in various embodiments, such as, for 
example, in the treatment of cancer and retroviral diseases. The invention 
also provides novel immunotoxin preparations which are enriched for 
specific immunotoxin species comprising an antibody molecule conjugated to 
one, two or three toxin chains, respectively. 
As used herein, the term "species" of immunotoxin refers to an immunotoxin 
of a particular, defined toxin/antibody ratio. For example, an immunotoxin 
of a single toxin chain conjugated to a single antibody molecule is a 
separate species from an immunotoxin of two toxin chains conjugated to a 
single antibody molecule. The terms "enriched" and "substantially 
purified" are used herein interchangeably. These terms are used to refer 
to immunotoxin preparations in which a single immunotoxin species is 
present as a larger proportion of the total immunotoxin in a preparation 
than in the heterologous conjugation reaction mix. 
In certain embodiments, an immunotoxin which has a toxin to antibody ratio 
of one toxin chain to one antibody molecule (dgA.sub.1 /IgG), is enriched 
for that species such that from about 50% to more than about 95% by weight 
of the total immunotoxin present in the preparation is dgA.sub.1 /IgG. 
While it is understood that any preparation containing more than about 50% 
of that species is within the scope of the present claimed invention, 
preparations of more than 60% or preparations of more than 80% and even 
preparations of about 95% of the total immunotoxin being the dgA.sub.1 
/IgG species are also encompassed within the present claimed invention. In 
other embodiments, an immunotoxin which has a toxin to antibody ratio of 
two toxin chains to one antibody molecule (dgA.sub.2 /IgG), is enriched 
for that species such that from about 30% to about 92% by weight of the 
total immunotoxin present in the preparation is dgA.sub.2 /IgG. While it 
is understood that any preparation containing more than about 30% of that 
species is within the scope of the present claimed invention, preparations 
of more than 50% or preparations of more than 70% or even preparations of 
about 92% by weight of the total immunotoxin is the dgA.sub.2 /IgG species 
are also encompassed within the scope of the present claimed invention. In 
other embodiments, an immunotoxin which has a toxin to antibody ratio of 
three toxin chains to one antibody molecule (dgA.sub.3 /IgG), is enriched 
for that species such that from about 19% to about 81% by weight of the 
total immunotoxin present in the preparation is dgA.sub.3 /IgG. While it 
is understood that any preparation containing more than about 19% of that 
species is within the scope of the present claimed invention, preparations 
in which more than 30%, or preparations in which more than 60% or even 
preparations in which about 81% by weight of the total immunotoxin is the 
dgA.sub.2 /IgG species are also encompassed within the scope of the 
present claimed invention. 
As used herein, the term "immunotoxin (IT)" is used to refer to a cytotoxic 
agent that comprises a cell-binding moiety otherwise described as a 
"targeting agent" and a toxin moiety linked via a chemical cross-linker, 
natural peptide, disulfide bond or any other suitable means. The targeting 
agent or moiety can be an antibody or a fragment thereof, such as a Fab' 
or Fab fragment, a growth factor, or a hormone that binds selectively to 
certain cell types. Most often, the targeting agent will be an antibody or 
a fragment thereof, and preferably will be a monoclonal antibody such as a 
mouse or human monoclonal antibody or so-called humanized antibody 
constructs which are known in the art. In certain embodiments, the 
invention is exemplified by the use of mouse IgG.sub.1 as the targeting 
agent. 
The targeting agent is also conjugated to a toxin moiety. The toxin moiety 
of the immunotoxin may be any one of a variety of toxins that are commonly 
employed in the art. It may be an intact toxin, a toxin A chain, or a 
naturally occurring single-chain ribosome-inactivating protein (RIP). 
Toxins which are encompassed by the invention include, for example, 
diphtheria toxin (DT) and DT(CRM-45); pseudomonas endotoxin (PE) and PE40; 
ricin and abrin and blocked forms of both of these; gelonin and saporin. 
Preferred toxins are contemplated to be those involving ricin, such as 
ricin A chain, and most preferably, deglycosylated ricin A chain. 
The heterologous mixture of immunotoxins is obtained by crosslinking the 
toxin and antibody molecules by any appropriate method. A preferred method 
of crosslinking is by using SMPT 
(N-succinimidyl-oxycarbonyl-.alpha.-methyl-(2-pyridyldithio) toluene). The 
unreacted toxin chains, antibody molecules and small particles are then 
removed by chromatography and filtration, for example, or by a combination 
of methods. These methods separate molecules based on the differences in 
molecular size or in molecular charge at certain pH and ionic strength 
conditions. These methods are well known in the art and preferred methods 
include affinity chromatography over activated dye/agarose beds and gel 
filtration through acrylamide beads, for example. The heterologous mixture 
is then purified or enriched for the individual immunotoxin species by any 
of the available separation methods which include, but are not limited to 
adsorption, partition, ion-exchange or molecular sieve chromatography, 
electrophoresis, or a combination of such methods. Preferred methods are 
affinity chromatography over an agarose/dye column and gel filtration. 
A certain embodiment of the present invention is a method of purifying 
immunotoxin preparations. In particular, the purification methods of the 
invention include obtaining and purifying a heterologous mixture of ITs 
with different molar ratios of toxin/antibody and then enriching the 
immunotoxin preparations for a single immunotoxin species. In preferred 
embodiments, the immunotoxin species is an antibody crosslinked to one, 
two or three toxin molecules. The antibody is preferably a monoclonal 
antibody and most preferred is a mouse monoclonal antibody. The preferred 
toxin is ricin A and more preferred is a deglycolsylated ricin A chain. 
Another embodiment of the present invention is a method of obtaining 
enriched immunotoxin preparations comprising the steps of chemically 
crosslinking toxin molecules to antibodies to obtain immunotoxin 
conjugates, removing free toxins and antibodies and separating the 
individual immunotoxin species. The immunotoxin species may be separated 
by affinity chromatography and exclusion chromatography, or gel 
filtration. In certain embodiments, the immunotoxin species may be 
separated by electrophoresis, preferably SDS-polyacrylamide gel 
electrophoresis. 
Another embodiment of the present invention concerns compositions of 
immunotoxins of known toxin/antibody molar ratio obtainable by the methods 
disclosed herein, i.e., immunotoxin preparations and pharmaceutical 
formulations obtainable by subjecting a heterologous mixture of 
immunotoxin conjugates to separation methods such as affinity 
chromatography and gel filtration. Most preferred immunotoxins are those 
with toxin/Ab ratios of one, two or three. 
In certain embodiments the invention concerns pharmaceutical preparations 
comprising a substantially purified immunotoxin in combination with a 
pharmacologically acceptable carrier. For certain clinical uses, ITs with 
two molecules of toxin conjugated to one antibody molecule may be 
preferred. 
The invention also provides methods for introducing immunotoxins into 
animals, including human subjects, and methods for treating various 
conditions and diseases, including cancers and lymphomas. These methods 
generally comprise administering to an animal an effective amount of a 
pharmaceutically acceptable immunotoxin composition comprising a 
substantially purified immunotoxin prepared as disclosed herein, in 
combination with a pharmacologically acceptable carrier. The formulations 
may be administered parenterally, such as via intravenous, intramuscular 
or sub-cutaneous injection and the like and are preferably administered 
parenterally and most preferably are administered intravenously. 
It is understood that the preparations of the present invention will have 
wide utility as compositions for use against a variety of diseases such as 
cancer and retroviral infections including human immunodeficiency virus 
(HIV). Also, for the first time, the toxicity, immunogenicity and 
effectiveness of particular dgA/IgG immunotoxins can be assessed to 
determine the best molar ratio to be used in pharmacological preparations. 
It is also understood that in addition to the clinical applications of the 
preparations of the present invention, the methods of preparation present 
a new and efficient method of large scale preparation and purification of 
immunotoxins for clinical, pharmaceutical and various other scientific 
applications.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Previous attempts to purify immunotoxins have resulted in a heterologous 
mixture of immunotoxins comprising one antibody molecule conjugated to 
one, two, three or possibly more toxin chains. In addition, larger, 
undefined species of immunotoxins which might comprise a single antibody 
molecule conjugated to more than three toxin chains, or which might 
comprise more than one antibody molecule were present in the mixture. 
These species of immunotoxins were difficult to separate because of their 
similarities in size and charge. 
The present inventors made the surprising discovery that the heterologous 
mixture of immunotoxins, when rechromatographed, can be enriched for the 
individual species of immunotoxins. For example, immunotoxins of one, two 
or three toxin chains per molecule of antibody respectively, can be 
separated from each other by simple procedures. These new, homogenous 
immunotoxin preparations could then be evaluated to determine the most 
effective species to be used in therapeutic applications. This is an 
important step in immunotoxin therapy because the practitioner must 
balance the tumoricidal benefits with the side effects caused by 
nonspecific cytotoxicity and the patient's immune response to the 
immunotoxin. Having the homogenous immunotoxin preparations of the present 
invention available will aid greatly in the determination and 
administration of the most effective therapies. 
Immunotoxins 
Immunotoxins combine into a single molecule, the exquisite specificity of a 
ligand and the extraordinary toxicity of a toxin. Despite their conceptual 
simplicity, IT's are large and complex molecules that are continually 
undergoing improvements for optimal in vivo activity since each of their 
components--the binding moiety, the cross-linker, and the toxin, 
introduces a different set of problems that must be addressed for the IT 
to function optimally in vivo. 
The targeting ligand of an IT is most often an antibody (Ab). The origin or 
derivation of the antibody or antibody fragment for use in the invention 
(e.g., Fab', Fab or F(ab').sub.2) is not crucial to the practice of the 
invention, so long as the antibody or fragment that is employed has the 
desired properties for the ultimately intended use of the IT. Thus, where 
monoclonal antibodies are employed, they may be of human, murine, monkey, 
rat, hamster, chicken or even rabbit origin. The invention also 
contemplates the use of human antibodies, "humanized" or chimeric 
antibodies from mouse, rat, or other species, bearing human constant 
and/or variable region domains, single chain antibodies, Fv domains, as 
well as recombinant antibodies and fragments thereof. Of course, due to 
the ease of preparation and ready availability of reagents, murine 
monoclonal antibodies will typically be preferred. 
In certain therapeutic embodiments, one may use known antibodies, such as 
those having high selectivity for solid tumors, such as B72.3, PRBC5 or 
PR4D2 for colorectal tumors; HMFG-2, TAG 72, SM-3, or anti-p 185.sup.Her2 
for breast tumors; anti-p 185.sup.Her2 for lung tumors; 9.2.27 for 
melanomas; and MO v18 and OV-TL3 for ovarian tumors. Anti-CD2 and anti-CD4 
immunotoxins may be purified according to the invention and used to kill 
malignant T cells or HIV-infected cells. Also, CD3-specific immunotoxins 
may be purified and used to prevent acute graft-host disease after bone 
marrow transplantation. 
In other embodiments, one may use another immunogen and prepare a new 
monoclonal antibody. The technique for preparing monoclonal antibodies is 
quite straightforward, and may be readily carried out using techniques 
well known to those of skill in the art, as exemplified by the technique 
of Kohler & Milstein (1975). Generally, immunogens are injected 
intraperitoneally into mice. This process is repeated three times at 
two-weekly intervals, the final immunization being by the intravenous 
route. Three days later the spleen cells are harvested and fused with 
SP2/0 myeloma cells by standard protocols (Kohler & Milstein, 1975): 
Hybridomas producing antibodies with the appropriate reactivity are then 
cloned by limiting dilution. 
The toxins that have been used to form ITs are derived from bacteria or 
plants and are inhibitors of protein synthesis. They are among the most 
powerful cell poisons known. Fewer than ten molecules will kill a cell if 
they enter the cytosol (although many times that number must bind to the 
cell surface because the entry process is inefficient). This extraordinary 
potency initially led to the concern that such poisons were too powerful 
to control. However, the toxins can be rendered innocuous (except when 
directed to the target cells) simply by removing or modifying their 
cell-binding domain or subunit. The remaining portion of the toxin 
(lacking a cell-binding domain) is then coupled to a ligand (e.g., an 
antibody) that targets the toxic portion to the target cell. By selecting 
an antibody lacking unwanted cross-reactivity, ITs are safer and have 
fewer non-specific cytotoxic effects than most conventional anticancer 
drugs. The other main attraction of toxins is that because they are 
inhibitors of protein synthesis, they kill resting cells as efficiently as 
dividing cells. Hence, tumor or infected cells that are not in cycle at 
the time of treatment do not escape the cytotoxic effect of an IT. 
"Toxin" is employed herein to mean any anticellular agent, and includes 
chemotherapeutic agents, radioisotopes as well as cytotoxins. In the case 
of chemotherapeutic agents, agents such as a hormone, asteroid for 
example; an antimetabolite such as cytosine arabinoside, fluorouracil, 
methotrexate or aminopterin; an anthracycline; mitomycin C; a vinca 
alkaloid; demecolcine; etoposide; mithramycin; or an antitumor alkylating 
agent such as chlorambucil or melphalan, may be used. 
However, preferred toxins will be plant-, fungus- or bacteria-derived 
toxins, which, by way of example, include various A chain toxins, 
particularly ricin A chain, ribosome inactivating proteins such as saporin 
or gelonin, .alpha.-sarcin, aspergillin, restrictocin, ribonucleases such 
as placental ribonuclease, angiogenin, diphtheria toxin, and Pseudomonas 
exotoxin, to name just a few. 
Plant toxins often contain two disulfide-bonded chains, the A and B chains. 
The B chain carries both a cell-binding region (whose receptor is 
uncharacterized) and a translocation region, which facilitates the 
insertion of the A chain through the membrane of a acid intracellular 
compartment into the cytosol. The A chain then kills the cell after 
incorporation. For their use in vivo, the ligand and toxin must be coupled 
in such a way as to remain stable while passing through the bloodstream 
and the tissues and yet be labile within the target cell so that the toxic 
portion can be released into the cytosol. 
The most preferred toxin moiety for use in connection with the invention is 
ricin A chain, and particularly toxin A chain which has been treated to 
modify or remove carbohydrate residues, so-called deglycosylated A chain. 
Deglycosylated ricin A chain (dgA) is available commercially from Inland 
Laboratories, Austin, Tex. 
However, it may be desirable from a pharmacologic standpoint to employ the 
smallest molecule possible that nevertheless provides an appropriate 
biological response. One may thus desire to employ smaller A chain 
peptides which will provide an adequate anti-cellular response. To this 
end, it has been discovered by others that ricin A chain may be 
"truncated" by the removal of 30 N-terminal amino acids by Nagarase 
(Sigma), and still retain an adequate toxin activity. It is proposed that 
where desired, this truncated A chain may be employed in conjugates in 
accordance with the invention. 
Alternatively, one may find that the application of recombinant DNA 
technology to the toxin A chain moiety will provide additional significant 
benefits in accordance the invention. In that the cloning and expression 
of biologically active ricin A chain has now been enabled through the 
publications of others (O'Hare et al., 1987; Lamb et al., 1985; Halling et 
al., 1985), it is now possible to identify and prepare smaller or 
otherwise variant peptides which nevertheless exhibit an appropriate toxin 
activity. Moreover, the fact that ricin A chain has now been cloned allows 
the application of site-directed mutagenesis, through which one can 
readily prepare and screen for A chain derived peptides and obtain 
additional useful moieties for use in connection with the present 
invention. 
The cross-linking of the toxin A chain region of the conjugate with the 
binding agent region is an important aspect of the invention. Where one 
desires a conjugate having biological activity, it is believed that a 
cross-linker which presents a disulfide function is required. The reason 
for this is unclear, but is likely due to a need for the toxin moiety to 
be readily releasable from the binding agent once the agent has 
"delivered" the toxin to the targeted cells. Each type of cross-linker, as 
well as how the cross-linking is performed, will tend to vary the 
pharmacodynamics of the resultant conjugate. Ultimately, one desires to 
have a conjugate that will remain intact under conditions found everywhere 
in the body except the intended site of action, at which point it is 
desirable that the conjugate have good "release" characteristics. 
Therefore, the particular cross-linking scheme, including in particular 
the particular cross-linking reagent used and the structures that are 
cross-linked, will be of some significance. 
Cross-linking reagents are used to form molecular bridges that tie together 
functional groups of two different proteins (e.g., a toxin and a binding 
agent). To link two different proteins in a step-wise manner, 
heterobifunctional cross-linkers can be used which eliminate the unwanted 
homopolymer formation. An exemplary heterobifunctional cross-linker 
contains two reactive groups: one reacting with primary amine group (e.g., 
N-hydroxy succinimide) and the other reacting with a thiol group (e.g., 
pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine 
reactive group, the cross-linker may react with the lysine residue(s) of 
one protein (e.g., the selected antibody or fragment) and through the 
thiol reactive group, the crosslinker, already tied up to the first 
protein, reacts with the cysteine residue (free sulfhydryl group) of the 
other protein (e.g., dgA). 
The spacer arm between these two reactive groups of any cross-linkers may 
have various length and chemical composition. A longer spacer arm allows a 
better flexibility of the conjugate components while some particular 
components in the bridge (e.g., benzene group) may lend extra stability to 
the reactive group or an increased resistance of the chemical link to the 
action of various aspects (e.g., disulfide bond resistant to reducing 
agents). 
The most preferred cross-linking reagent is SMPT, which is a bifunctional 
cross-linker containing a disulfide bond that is "sterically hindered" by 
an adjacent benzene ring and methyl groups. It is believed that steric 
hindrance of the disulfide bond serves a function of protecting the bond 
from attack by thiolate anions such as glutathione which can be present in 
tissues and blood, and thereby help in preventing decoupling of the 
conjugate prior to its delivery to the site of action by the binding 
agent. The SMPT cross-linking reagent, as with many other known 
cross-linking reagents, lends the ability to crosslink functional groups 
such as the SH of cysteine or primary amines (e.g., the epsilon amino 
group of lysine). Another possible type of cross-linker includes the 
heterobifunctional photoreactive phenylazides containing a cleavable 
disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) 
ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with 
primary amino groups and the phenylazide (upon photolysis) reacts 
non-selectively with any amino acid residue. 
Although the "hindered" cross-linkers will generally be preferred in the 
practice of the invention, non-hindered linkers can be employed and 
advantages in accordance herewith nevertheless realized. Other useful 
cross-linkers, not considered to contain or generate a protected 
disulfide, include SATA, SPDP and 2-iminothiolane. The use of such 
cross-linkers is well understood in the art. 
New Methods for Immunotoxin Preparation 
Immunotoxins comprising monoclonal antibodies covalently bound to 
deglycosylated ricin A by hindered disulfide linkers have recently entered 
clinical trials for the treatment of non-Hodgkin's (B cell) lymphoma. 
These "second generation" immunotoxins are stable, long lived and display 
potent cytotoxicity to target cells. Standardized procedures for rapid 
preparation of high yields of these immunotoxins have been developed 
(Ghetie et al., 1991). 
The procedure for preparation of the immunotoxins comprises the 
derivitization of antibodies with SMPT and reduction of deglycosylated 
ricin A (dgA) with dithiothreitol (DTT), followed by the reaction of the 
two components to establish a hindered interchain disulfide bond. The 
chemical crosslinking reaction results in a mixture of antibody, toxin and 
immunotoxins which are then purified, initially to remove the free 
antibody and free toxin molecules and subsequently to separate the 
different immunotoxin species which comprise one antibody molecule 
conjugated with one, two, three or more than three toxin molecules, 
respectively. The unreacted components of the crosslinking reaction may be 
removed by successive chromatographies on an affinity chromatography 
column such as activated dye/agarose to remove free antibody followed by 
gel filtration to remove high molecular weight material and free toxin. 
The result of this procedure is a mixture of conjugates of various 
toxin/antibody ratios. An important embodiment of the present invention is 
the further purification of this mixture to obtain preparations 
essentially comprising immunotoxins of a single toxin/antibody ratio 
separated from immunotoxins of different toxin/antibody ratios. This 
purification is accomplished by further chromatographic separation which 
may be accomplished by affinity chromatography for example, using a salt 
gradient to elute the various species of immunotoxins and gel filtration 
to separate the immunotoxins from larger molecules. 
Another important embodiment of the present invention is the ability to 
determine which of the dgA/IgG ratios is the most effective cytotoxic 
agent to be used in pharmacological preparations. For example, it was 
found that the unpurified mixture is more cytotoxic than the dgA.sub.1 
/IgG preparation, and that the dgA.sub.2 /IgG is the most effective 
cytotoxic component of the preparation. The isolation and characterization 
of each of the single species of immunotoxin made possible by the present 
invention is of particular advantage in clinical applications as it allows 
the practitioner to exercise more precise control over the effective 
amount of immunotoxin to be administered in a particular situation. In 
addition, the purified immunotoxins, dgA.sub.1 /IgG and dgA.sub.2 /IgG are 
shown to be less toxic and less immunogenic than the nonpurified mixture 
and would therefore be expected to have less severe side effects in a 
patient. 
Gel Filtration 
A gel to be used in the procedures of the present invention is a three 
dimensional network which has a random structure. Molecular sieve gels 
comprise cross-linked polymers that do not bind or react with the material 
being analyzed or separated. For gel filtration purposes, the gel material 
is generally uncharged. The space within the gel is filled with liquid and 
the liquid phase constitutes the majority of the gel volume. Materials 
commonly used in gel filtration columns include dextran, agarose and 
polyacrylamide. 
Dextran is a polysaccharide composed of glucose residues and is 
commercially available under the name SEPHADEX (Phamacia Fine Chemicals, 
Inc.). The beads are prepared with various degrees of cross-linking in 
order to separate different sized molecules by providing various pore 
sizes. Alkyl dextran is cross-linked with N,N'-methylenebisacrylamide to 
from SEPHACRYL-S300 which allows strong beads to be made that fractionate 
in larger ranges than SEPHADEX can achieve. 
Polyacrylamide may also be used as a gel filtration medium. Polyacrylamide 
is a polymer of cross-linked acrylamide prepared with 
N,N'-methylenebisacrylamide as the cross-linking agent. polyacrylamide is 
available in a variety of pore sizes from Bio-Rad Laboratories (USA) to be 
used for separation of different size particles. 
The gel material swell in water and in a few organic solvents. Swelling is 
the process by which the pores become filled with liquid to be used as 
eluant. As the smaller molecules enter the pores, their progress through 
the gel is retarded relative to the larger molecules which do not enter 
the pores. This is the basis of the separation. The beads are available in 
various degrees of fineness to be used in different applications. The 
coarser the bead, the faster the flow and the poorer the resolution. 
Superfine is to be used for maximum resolution, but the flow is very slow. 
Fine is used for preparative work in large columns which require a faster 
flow rate. The coarser grades are for large preparations in which 
resolution is less important than time, or for separation of molecules 
with a large difference in molecular weights. For a discussion of gel 
chromatography, see Freifelder, Physical Biochemistry, Second Edition, 
pages 238-246, incorporated herein by reference. 
The most preferred methods of gel filtration for use in the present 
invention are those using dextran gels, such as SEPHADEX, and those using 
dextran-polyacrylamide gels such as SEPHACRYL which are able to separate 
molecules in the 180 to 240 kilodalton range. 
Affinity Chromatography 
Affinity chromatography is generally based on the recognition of a protein 
by a substance such as a ligand or an antibody. The column material may be 
synthesized by covalently coupling a binding molecule, such as an 
activated dye, for example to an insoluble matrix. The column material is 
then allowed to adsorb the desired substance from solution. Next, the 
conditions are changed to those under which binding does not occur and the 
substrate is eluted. The requirements for successful affinity 
chromatography are that the matrix must adsorb molecules, the ligand must 
be coupled without altering its binding activity, a ligand must be chosen 
whose binding is sufficiently tight, and it must be possible to elute the 
substance without destroying it. 
A preferred embodiment of the present invention is an affinity 
chromatography method wherein the matrix is a reactive dye-agarose matrix. 
Blue-SEPHAROSE, a column matrix composed of Cibacron Blue 3GA and agarose 
or SEPHAROSE may be used as the affinity chromatography matrix. The most 
preferred matrix is SEPHAROSE CL-6B available as Reactive Blue 2 from 
Sigma Chemical Company, catalogue #R 8752. This matrix binds the 
immunotoxins of the present invention directly and allows their separation 
by elution with a salt gradient. 
Pharmaceutical Preparations 
Pharmaceutical aqueous compositions of the present invention comprise an 
effective amount of the IT dissolved or dispersed in a pharmaceutically 
acceptable carrier or aqueous medium. The phrases "pharmaceutically or 
pharmacologically acceptable" refers to molecular entities and 
compositions that do not produce an adverse, allergic or other untoward 
reaction when administered to a human. As used herein, "pharmaceutically 
acceptable carrier" includes any and all solvents, dispersion media, 
antibacterial and antifungal agents, isotonic and absorption delaying 
agents and the like. The use of such media and agents for pharmaceutical 
active substances is well known in the art. Except insofar as any 
conventional media or agent is incompatible with the active ingredient, 
its use in the therapeutic compositions is contemplated. Supplementary 
active ingredients can also be incorporated into the compositions. 
The following buffers and reagents are particularly contemplated for use in 
the preparation of pharmaceutical preparations of the present invention: 
dgA, deglycosylated ricin A chain; DMF, dimethylformamide (Pierce, 
Rockford, Ill.); DTT (Pierce); PBE, 0.05M sodium phosphate, pH 7.5 with 1 
mM EDTA; PBES, 0.05M sodium phosphate, pH 7.5 with various concentrations 
of NaCl (such as 0.1M, 0.2M, 0.3M, 0.4M and 0.5M NaCl) and 1 mM EDTA; 
PBSE, 0.01M sodium phosphate, pH 7.5 with 0.17M NaCl and 1 mM EDTA; SMPT, 
N-succinimidyl-oxycarbonyl-.alpha.-methyl-.alpha.(2-pyridyldithio)toluene 
(Pierce). All buffers may be prepared with endotoxin-free distilled water 
using enzyme grade salts (Fisher Biotec, Springfield, N.J.). 
The ITs may be formulated for parenteral administration, e.g., formulated 
for injection via the intravenous, intramuscular or sub-cutaneous routes. 
The preparation of an aqueous composition that contains an IT as an active 
ingredient will be known to those of skill in the art in light of the 
present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 
16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. 
Moreover, for human administration, it will be understood that 
preparations should meet sterility, pyrogenicity, general safety and 
purity standards as required by FDA Office of Biological Standards. 
Typically, such compositions can be prepared as injectables, either as 
liquid solutions or suspensions; solid forms suitable for using to prepare 
solutions or suspensions upon the addition of a liquid prior to injection 
can also be prepared; and the preparations can also be emulsified. The 
compositions will be sterile, be fluid to the extent that easy 
syringability exists, stable under the conditions of manufacture and 
storage, and preserved against the contaminating action of microorganisms, 
such as bacteria and fungi. It will be appreciated that endotoxin 
contamination should be kept minimally at a safe level, for example, less 
that 0.5 ng/mg protein. 
Although it is most preferred that solutions of ITs be prepared in sterile 
water containing other non-active ingredients, made suitable for 
injection, solutions of ITs can also be prepared in water suitably mixed 
with a surfactant, such as hydroxypropylcellulose, if desired. Dispersions 
can also be prepared in liquid polyethylene glycols, and mixtures thereof 
and in oils. The carrier can also be a solvent or dispersion medium 
containing, for example, water, ethanol, polyol (for example, propylene 
glycol, and liquid polyethylene glycol, and the like), suitable mixtures 
thereof, and vegetable oils. The proper fluidity can be maintained, for 
example, by the use of a coating, such as lecithin, by the maintenance of 
the required particle size in the case of dispersion and by the use of 
surfactants. 
The prevention of the action of microorganisms can be brought about by 
various antibacterial ad antifungal agents, for example, parabens, 
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many 
cases, it will be preferable to include isotonic agents, for example, 
sugars or sodium chloride. Prolonged absorption of the injectable 
compositions can be brought about by the use in the compositions of agents 
delaying absorption, for example, aluminum monostearate and gelatin. 
Upon formulation, solutions will be administered in a manner compatible 
with the dosage formulation and in such amount as is therapeutically 
effective. For parenteral administration in an aqueous solution, for 
example, the solution should be suitably buffered if necessary and the 
liquid diluent first rendered isotonic with sufficient saline or glucose. 
These particular aqueous solutions are especially suitable for 
intravenous, intramuscular, subcutaneous and intraperitoneal 
administration. In this connection, sterile aqueous media which can be 
employed will be known to those of skill in the art in light of the 
present disclosure. Some variation in dosage will necessarily occur 
depending on the condition of the subject being treated. The person 
responsible for administration will, in any event, determine the 
appropriate dose for the individual subject. 
It is particularly contemplated that suitable pharmaceutical IT 
compositions will generally comprise from about 10 to about 100 mg of the 
desired IT conjugate admixed with an acceptable pharmaceutical diluent or 
excipient, such as a sterile aqueous solution, to give a final 
concentration of about 0.25 to about 2.5 mg/ml with respect to the 
conjugate, in, for example, 0.15M NaCl aqueous solution at pH 7.5 to 9.0. 
The preparations may be stored frozen at -10.degree. C. to -70.degree. C. 
for at least 1 year. 
ELISA 
ELISAs may be used in conjunction with the invention. In an ELISA assay, 
proteins or peptides incorporating dgA antigen sequences are immobilized 
onto a selected surface, preferably a surface exhibiting a protein 
affinity such as the wells of a polystyrene microtiter plate. After 
washing to remove incompletely adsorbed material, it is desirable to bind 
or coat the assay plate wells with a nonspecific protein that is known to 
be antigenically neutral with regard to the test antisera such as bovine 
serum albumin (BSA), casein or solutions of milk powder. This allows for 
blocking of nonspecific adsorption sites on the immobilizing surface and 
thus reduces the background caused by nonspecific binding of antisera onto 
the surface. 
After binding of antigenic material to the well, coating with a 
non-reactive material to reduce background, and washing to remove unbound 
material, the immobilizing surface is contacted with the antisera or 
clinical or biological extract to be tested in a manner conducive to 
immune complex (antigen/antibody) formation. Such conditions preferably 
include diluting the antisera with diluents such as BSA, bovine gamma 
globulin (BGG) and phosphate buffered saline (PBS)/TWEEN. These added 
agents also tend to assist in the reduction of nonspecific background. The 
layered antisera is then allowed to incubate for from 2 to 4 hours, at 
temperatures preferably on the order of 25.degree. to 27.degree. C. 
Following incubation, the antisera-contacted surface is washed so as to 
remove non-immunocomplexed material. A preferred washing procedure 
includes washing with a solution such as PBS/TWEEN, or borate buffer. 
Following formation of specific immunocomplexes between the test sample and 
the bound antigen, and subsequent washing, the occurrence and even amount 
of immunocomplex formation may be determined by subjecting same to a 
second antibody having specificity for the first. To provide a detecting 
means, the second antibody will preferably have an associated enzyme that 
will generate a color development upon incubating with an appropriate 
chromogenic substrate. Thus, for example, one will desire to contact and 
incubate the antisera-bound surface with a urease or peroxidase-conjugated 
anti-human IgG for a period of time and under conditions which favor the 
development of immunocomplex formation (e.g., incubation for 2 hours at 
room temperature in a PBS-containing solution such as PBS-Tween). 
After incubation with the second enzyme-tagged antibody, and subsequent to 
washing to remove unbound material, the amount of label is quantified by 
incubation with a chromogenic substrate such as urea and bromocresol 
purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and 
H.sub.2 O.sub.2, in the case of peroxidase as the enzyme label. 
Quantification is then achieved by measuring the degree of color 
generation, e.g., using a visible spectra spectrophotometer. 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventor to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. 
EXAMPLE I 
Preparation of Immunotoxins 
The IT containing deglycosylated RTA (dgA) and a murine IgG.sub.1 
monoclonal directed against the human CD22 molecule (RFB4) was prepared 
(RFB4-SMPT-dgA) and purified as reported below (Ghetie et al., 1991). The 
IT was stored at -70.degree. C. 
Buffers and reagents 
The following buffers and reagents were used: dgA, deglycosylated ricin A 
chain; DMF, dimethylformamide (Pierce, Rockford, Ill.); DTT (Pierce); PBE, 
0.05M sodium phosphate, pH 7.5 with 1 mM EDTA; PBES, 0.05M sodium 
phosphate, pH 7.5 with various concentrations of NaCl (such as 0.1M, 0.2M, 
0.3M, 0.4M and 0.5M NaCl) and 1 mM EDTA; PBSE, 0.01M sodium phosphate, pH 
7.5 with 0.17M NaCl and 1 mM EDTA; SMPT, 
N-succinimidyl-oxycarbonyl-.alpha.-methyl-.alpha.(2-pyridyldithio)toluene 
(Pierce). All buffers were prepared with endotoxin-free distilled water 
using enzyme grade salts (Fisher Biotec, Springfield, N.J.). 
Antibodies 
Hybridoma cells secreting mouse IgG1 anti-CD22 (RFB4) were obtained from 
Dr. G. Janossy (London, U.K.). Hybridoma cells secreting mouse IgG1 
antiCD19 (HD37) can be obtained from Dr. G. Moldenhauer and Dr. B. Dorken 
(Heidelberg, F. R. G.), if desired. 10-20 g batches of purified antibodies 
from these hybridoma cells were produced by Abbott Laboratories (Needham 
Heights, Mass.) and contained less than 5% impurities as determined by 
sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 
low levels of endotoxin (2 EU/ml). The proteins were supplied in 0.01M 
phosphate buffered saline, pH 7.2 and were stored at -70.degree. C. 
Protein concentrations were calculated from the optical density of the 
solution using an absorption coefficient of 1.4 (1 mg/ml, 1 cm, 280 nm). 
Deglycosylated ricin A chain (dgA) 
The dgA was purchased from Inland Laboratories, Austin, Tex., and was 
prepared and characterized as described (Fulton et al., 1986). The protein 
was dissolved in phosphate buffered saline, pH 7.2 containing 50% glycerol 
and was stored at -20.degree. C. The protein contained less than 0.1 U of 
endotoxin per 3 mg/ml. Protein concentrations were calculated from the 
optical density of the solution using an absorption coefficient of 0.77 (1 
mg/ml, 1 cm, 280 nm). 
Preparation of immunotoxins 
All procedures were performed in good laboratory practice (GLP) laboratory 
using a chromatographic system described in (Ghetie, et al., 1988a; 1991). 
The buffers were maintained in sterile square barrels at 4.degree. C. and 
pumped directly into the chromatographic system. All columns and gels were 
purchased from Pharmacia (Piscataway, N.J.). The preparation required 2-3 
days and the purification was performed in 2 days. Provided that the 
system did not require cleaning with 0.1M NaOH (e.g. to remove endotoxin 
or adsorbed protein) four batches of immunotoxin could be prepared per 
month, each yielding 1.5 g from 6.0 g of antibody and 3.0 g of dgA. This 
is enough to treat approximately 85-90 patients at the MTD. 
RFB4-SMPT-dgA; Preferred Method of Preparation 
Six grams of RFB4 antibody dissolved in 800 ml of 0.01M phosphate buffered 
saline, pH 7.2 were adjusted to pH 7.5 with 10N NaOH. The protein solution 
was then mixed with 1/50 of its volume of SMPT (5 mg/ml DMF) giving a 
final concentration of 0.1 mg SMPT/ml which is equivalent to 5.0-fold 
molar excess over the antibody. The mixture was then stirred gently under 
N.sub.2 for 1 h at room temperature and applied to a Sephadex G-25M column 
(25.times.60 cm) equilibrated with N.sub.2 -flushed PBE. The protein 
eluate containing RFB4-MPT was collected and concentrated to 1.5 liter 
(approximately 4 mg/ml) using the Amicon CH2 ultrafiltration unit at 
2.degree.-8.degree. C. Samples were removed for the determination of the 
average number of MPT groups per molecule of IgG using the method of 
Carlson et al. (1978). The pH of the dgA solution (3 mg/ml) was adjusted 
to 7.5 with 10N NaOH and 3 g of this solution was then mixed with 1/10 its 
volume of DTT (7.7 mg/ml) in PBE and stirred in the dark for 1 h at room 
temperature. The mixture (approximately 1 liter) was then applied to a 
SEPHADEX G-25M column (25.times.60 cm) in N.sub.2 -flushed PBE. The 
reduced dgA was collected and concentrated to 750 ml (approximately 4 
mg/ml) and immediately mixed with SMPT-derivatized antibody solution (1500 
ml at 4 mg/ml). The reaction mixture was sterilized by filtration through 
a 0.22 .mu.m membrane and was maintained at room temperature for 2 days. 
Samples were removed for SDS-PAGE and LAL testing. The reaction mixture 
was applied to a 11.6.times.30 cm column of Blue-SEPHAROSE CL-6B 
equilibrated with PBE. After washing to remove unbound protein (nonreacted 
antibody) the immunotoxin and free dgA were collected by elution with 
PBES. The protein solution was concentrated to 1.5 liter and filtrated 
through a SEPHACRYL S-200HR column (25.times.90 cm) equilibrated with 
0.15M NaCl, containing 5 mM lysine. The first major peak containing the 
immunotoxin was concentrated to 0.5 mg/ml, sterilized through a 0.22 .mu.m 
filter and distributed into vials of appropriate capacity. 
RFB4-SMPT-dgA; Alternative Method of Preparation 
Six grams of RFB4 antibody dissolved in 500 ml of 0.01M phosphate buffered 
saline, pH 7.2 were applied to a SEPHACRYL S200-HR column (25 cm 
diameter.times.90 cm length) equilibrated in BSE at 2.degree.-8.degree. C. 
The 150 kDa (monomer) peak was collected and concentrated to 7.5 mg/ml in 
an Amicon CH2 ultrafiltration unit at 2.degree.-8.degree. C. The protein 
solution was then mixed with 1/50 of its volume of SMPT (Pierce) (4.5 
mg/ml DMF) giving a final SMPT concentration of 0.09 mg/ml which is 
equivalent to a 4.7-fold molar excess over the antibody. The mixture 
(containing 5.5 g of IgG in 700 ml) was then stirred gently under N.sub.2 
for 1 h at room temperature and applied to a SEPHADEX G-25M column 
(25.times.60 cm) equilibrated in N.sub.2 -flushed PBSE. The protein eluate 
(containing MPT antibody free of unreacted SMPT) was collected and 
concentrated as indicated above to approximately 2 liters (2.5 mg/ml). 
Samples were removed for SDS-PAGE and LAL testing and for the 
determination of the average number of MPT groups per molecule of IgG 
using the method of Carlson et al. (1978). The pH of the dgA solution was 
adjusted to 7.5 with 10N NaOH. Three grams of dgA were then mixed with 0.1 
vol. of DTT (7.7 mg/ml PBSE) and stirred in the dark for 1 h at room 
temperature. The mixture (approximately 1 liter) was then applied to a 
SEPHADEX G25M column (25.times.60 cm) in N.sub.2 -flushed PBSE. The 
DTT-free, reduced dgA was collected and samples were removed for SDS-PAGE 
and LAL testing. The dgA solution (approximately 3 liters) was mixed with 
the SMPT-derivatized antibody (2 liters) and the mixture was concentrated 
to 5 mg/ml (total protein) at 2.degree.-8.degree. C. (approximately 1.8 
liter, final volume). The reaction mixture was sterilized by filtration 
through a 0.22 .mu.m membrane (Nalgene disposable filterware, Nalge 
Company, Rochester, N.Y.) and maintained at room temperature under N.sub.2 
for 3 days. Samples were removed for SDS-PAGE and LAL testing. 
The reaction mixture was mixed with 1/100 of its volume of cysteine (free 
base) (2.5 mg/ml) in PBSE. After stirring for 6 h at room temperature, the 
mixture was filtered through a 0.8 .mu.m filter to remove any particulate 
matter and the solution was applied to a SEPHACRYL S-200HR column 
(25.times.90 cm) in BE at 28.degree.-8.degree. C. The peaks with molecular 
masses between 150 and 210 kDa were collected. This semi-purified 
immunotoxin (containing free IgG) was concentrated to approximately 1.5 
mg/ml (total protein) and samples were removed for SDS-PAGE and LAL 
testing. Unreacted RFB4 antibody was removed by affinity chromatography on 
a 11.6.times.30 cm Blue -SEPHAROSE CL-6B column (capacity 3 g dgA) 
equilibrated with BE. The column was washed with this buffer until all 
unbound antibodies were removed and then the purified RFB4-SMPT-dgA was 
displaced from the column with BES. The protein solution was dialyzed by 
diafiltration in an Amicon CH2 unit into 0.15M NaCl containing 5 mM lysine 
at 2.degree.-8.degree. C. using approximately 10-15 liters of 
lysine-containing saline. The protein concentration was adjusted to 0.5 
mg/ml using an absorption coefficient of 1.3 (for 1 mg/ml, 1 cm, 280 nm). 
Finally, the immunotoxin was sterilized through a 0.22 .mu.m filter and 
distributed into vials. 
Analysis of the immunotoxins 
All assays except the measurement of antibody activity have been described 
by Ghetie et al., (1988a), incorporated herein by reference. To measure 
antibody activity, samples of Daudi cell suspension (10.sup.6 cells/0.1 
ml) were treated with various concentrations of immunotoxin or 
unconjugated antibody (from 0.05 to 5 nM) and after a 15 min incubation at 
4.degree. C. the cells were washed twice with phosphate buffered saline 
containing 0.02% sodium azide. Cells were then treated with 3 .mu.l of 
fluorescent goat antimouse Ig (Kirkegaard and Perry Labs, Gaithersburg, 
Md.) per 50 .mu.l cells suspension containing 10.sup.6 cells. After 
another 15 minutes of incubation at 4.degree. C. the cells were washed 
once and suspended in 0.5 ml 1% paraformaldehyde in phosphate buffered 
saline with sodium azide and the percentage of fluorescent cells 
determined by FACS. The percentage of fluorescent cells vs. protein 
concentration in nM was plotted and the protein concentration staining 50% 
of treated cells was determined. The relative antibody activity of the 
immunotoxin was calculated as a percentage of the initial antibody 
activity with the equation: 
##EQU1## 
where [Ab] and [immunotoxin] are the concentrations of antibody and 
immunotoxin staining half of the treated cells. 
Cleaning the chromatographic system 
When endotoxin contamination of the chromatographic system occurred, 
columns were `cleaned` before use by incubation with 0.1M NaOH for 48-72 h 
at 2.degree.-8.degree. C. as described (Ghetie et al., 1988a). 
EXAMPLE II 
Purification of Immunotoxins 
Methods 
The cell free rabbit reticulocyte assay and the Daudi cell killing assay 
have been described (Press et al., 1988; Ghetie et al., 1988b; each 
incorporated herein by reference). SDS-PAGE was carried out using the 
Pharmacia Phast System with a 4-15% gel gradient and low and high MW 
standards. The LD.sub.50 was determined in groups of four BALB/c mice 
injected intraperitoneally with different doses of the IT. The primary 
antibody response against dgA was determined in groups of 3 mice injected 
i.p. with 5 .mu.g/g animal of ITs. The animals were bled two weeks after 
injection. The antibody titer was estimated using an ELISA (Amlot et al., 
1993). The half-life was determined in mice injected with .sup.125 
I-labeled ITs as previously described (Fulton et al., 1988). 
High performance liquid chromatography (HPLC) was performed on 
7.5.times.600 mm SEC-250 analytical columns (Bio-Rad, Hercules, Calif.). 
The retention times of various peaks were compared to those of standard 
proteins of known MWs. Gel filtration on SEPHACRYL S-300 was performed on 
15.times.600 mm columns equilibrated with phosphate buffered saline. 
Chromatography on Blue-SEPHAROSE was performed in 30 ml packed gel 
columns, equilibrated in 0.05M phosphate buffer with 0.003M Na.sub.2 EDTA, 
pH 7.5 (PBE). A stepwise gradient comprising 0.1M, 0.2M, 0.3M and 0.5M 
NaCl dissolved in PBE was applied to elute the bound protein. 
Results 
RFB4-SMPT-dgA has been described previously and is currently being used in 
clinical trials (Amlot et al., 1993). This IT was submitted to SDS-PAGE 
and HPLC. The results presented in FIGS. 1 and 2 demonstrate that the 
preparation is heterologous and comprises several molecular species with 
molecular weights (MWs) ranging between 180 KDa and&gt;250 kDa. The major 
chromatographic peak (76%) has a MW ranging between 180-210 kDa and 
includes a mixture of two ITs, containing one and two molecules of dgA 
linked to one molecule of IgG.sub.1. The second peak (19%) has a MW of 240 
kDa and is thought to comprise three molecules of dgA linked to one 
molecule of antibody. The first peak (5%) has a MW&gt;250 kDa and may contain 
a mixture of conjugates with dgA/IgG.sub.1 molar ratios of 4 or greater. 
When this material was analyzed on SDS-PAGE five bands were observed. The 
first three had MWs corresponding to molecules of dgA/IgG.sub.1 with molar 
ratios of 1, 2 and 3. The two faint bands had MWs in a range that could 
not be determined on this gel. The apparent discrepancy between the 
results of HPLC and SDS-PAGE is due to the fact that the SEC-250 column 
does not allow separation of the 180 kDa and 210 kDa ITs while these two 
ITs are completely separated by SDS-PAGE. 
Pure immunotoxin preparations were obtained by an additional chromatography 
step. The heterologous mixture was chromatographed on Blue-SEPHAROSE. A 
NaCl gradient was used to separate the conjugates comprising different 
molar ratios of dgA/IgG.sub.1 (FIG. 3). Unexpectedly, 50% of the protein 
applied to the Blue-SEPHAROSE column did not bind, despite the fact that 
the same buffer (PBE) had been used to purify the original IT (Ghetie et 
al., 1991). Hence, 50% of the IT had lost its ability to interact a second 
time with Blue-SEPHAROSE. When this material was analyzed by SDS-PAGE and 
HPLC, it contained only the first band (or the third peak) (MW-180 kDa) 
and a small amount (10%) of the fourth and fifth bands (or the first peak) 
(MW&gt;250 kDa) (FIGS. 4 and 6D). The difference in the MW of these species 
facilitated complete separation of the 180 kDa IT from the heavier MW ITs 
by gel filtration on SEPHACRYL S-300 (FIG. 5). The second major peak 
contained only the 180 kDa IT as shown in FIG. 6C. 
After collecting the non-bound fraction, a stepwise gradient was used to 
elute material from the Blue-SEPHAROSE column. Four fractions were 
collected by eluting the column with 0.1M, 0.2M, 0.3M and 0.5M NaCl. The 
0.1M fraction contained a small percentage of 180 kDa IT and the bulk of 
the 210 kDa IT, while the 0.2M fraction contained the 210 kDa IT almost 
exclusively. The 0.3M and 0.5M fractions contained equal proportions of 
210 kDa IT and&gt;250 kDa IT (FIG. 4; Lanes C, D, E and F). 
The unpurified RFB4-SMPT-dgA IT also contains a small amount of higher MW 
components. The exact MW of these two heavy MW ITs (bands 4 and 5 in 
SDS-PAGE and peak 1 in HPLC) cannot be determined accurately by these 
procedures, but they are&gt;250 kDa. It is, therefore, possible that these 
ITs contain 4 or more dgA molecules per molecule of antibody, but it is 
also possible that antibody dimerization occurred and these conjugates 
contain two molecules of dimerized antibody linked to an undetermined 
number of dgA molecules. This latter possibility is favored by the fact 
that the heavy MW ITs are not bound to Blue-SEPHAROSE in 0.05M PBE pH 7.5, 
a behavior quite unexpected for a conjugate having 4 or more dgA molecules 
bound to one molecule of antibody. 
It is unclear why half of the initial IT eluted from Blue-SEPHAROSE during 
the first purification did not bind to the Blue-SEPHAROSE during the 
second passage. The lack of binding is not due to freezing or to the high 
salt concentration since similar results were obtained using freshly 
prepared IT and an IT obtained by elution in 0.15M NaCl. The dgA in the 
unbound fraction (containing mainly dgA.sub.1 /IgG) had the same enzymatic 
activity as the dgA in the fraction eluted at 0.2M (containing the 
dgA.sub.2 /IgG conjugate) as shown in Table 1. Therefore, the lower 
activity of the dgA.sub.1 /IgG vs dgA.sub.2 /IgG was not due to partial 
inactivation of the dgA moiety in the IT containing one dgA molecule. The 
fact that the initial IT preparation is more active than that with one dgA 
molecule (1.3 vs 2.6.times.10.sup.-12 M) is not a consequence of a partial 
inhibition of the dgA activity in the dgA.sub.1 /IgG conjugate, but 
results from the fact that the initial IT contained 30% dgA.sub.2 /IgG 
(which is 7 times more active than dgA.sub.1 /IgG). 
EXAMPLE III 
Biological Activity of Purified Immunotoxins 
The biological activity of the purified 180 kDa IT was tested and compared 
with that of the initial preparation of IT (containing all 5 
electrophoretic bands) and that of the fraction eluted with 0.2M NaCl, 
containing mainly the 210 kDa IT (two molecules of dgA linked to one 
molecule of antibody, dgA.sub.2 /IgG). The results are presented in Table 
1. 
The results indicate that the ability of the different species of IT to 
inhibit protein synthesis in a cell free system is not dependent on the 
dgA/IgG.sub.1 molar ratio since all three preparations had comparable 
activity. In contrast, when the different ITs were tested for their 
ability to specifically kill Daudi cells, in vitro, the IT containing two 
molecules of dgA per molecule of antibody was 7 times more cytotoxic than 
the IT with one dgA molecule per molecule of antibody. The latter was 
2-fold less active than the unpurified IT preparation. Preliminary studies 
presented in Table 1 also indicate that the unpurified IT is more toxic 
than the purified 180 kDa and 210 kDa components and that the primary 
antibody response against the unpurified IT is higher than for the 
purified 180 kDa and 210 kDa preparations. This suggests that it may be 
possible to decrease both toxicity and immunogenicity of the ITs by 
further purification. The halflife of the IT containing two molecules of 
dgA was significantly shorter than that of the IT with one dgA molecule, 
indicating that the latter will persist longer in the circulation of 
tumor-bearing individuals. 
The present results indicate that target cell killing is increased about 7 
fold when a conjugate containing two molecules of dgA is used compared to 
a conjugate containing one molecule of dgA. This finding suggests that the 
former IT may have an increased therapeutic index in vivo. The 
immunogenicity of the two appear similar. Since the half-life of the two 
ITs are not the same, it will be of considerable importance to determine 
if there is a significant advantage of one molecular species over another 
in tumor therapy studies in mice. 
TABLE 1 
__________________________________________________________________________ 
The biological activity of purified and unpurified RFB4- 
SMPT-dgA 
Biological 
Starting Purified 180 
Purified 210 
Activity 
Material kDa component 
kDa component 
__________________________________________________________________________ 
Inhibition of 
4.0 .times. 10.sup.-11 
3.0 .times. 10.sup.-11 
4.1 .times. 10.sup.-11 
cell free 
protein 
synthesis 
(IC50, M) 
Cytotoxicity 
1.3 .+-. 0.2 .times. 10.sup.-12 
2.6 .+-. 0.35 .times. 10.sup.-12 
0.4 .+-. 0.04 .times. 10.sup.-12 
to Daudi cells 
(IC50, M).sup.a 
Half-life in 
27.5 .+-. 3.0 
32.0 .+-. 3.5 
19.2 .+-. 0.6 
mice (hrs).sup.b 
Toxicity to 
6.2 10.0 9.2 
mice.sup.c 
(LD50, ug/g 
animal) 
Anti-dgA titer 
3.9 2.5 2.6 
in mice 
(ug/ml).sup.d 
__________________________________________________________________________ 
.sup.a P &lt; 0.01 for the difference in activity between the 180 kDa and 21 
kDa components (mean of 5 experiments) 
.sup.b 3 mice/group 
.sup.c 4 mice/group 
.sup.d 3 mice/group 
EXAMPLE IV 
Clinical Use of Immunotoxins 
The present example is provided to outline the use of ITs for the treatment 
of lymphoma, and is just one example of the many uses in which ITs 
prepared in accordance with the invention may be employed. 
Patients 
To be eligible for treatment, patients should preferably be over 18 years 
of age with histologically confirmed, relapsing non-Hodgkin's lymphoma 
(NHL); a Karnofsky performance status of .+-.30% and a life expectancy of 
.+-.2 months; .+-.15% of their lymphoma cells should express CD22 antigen 
at .+-.10% of its density on a Daudi B cell line; and they would have 
clinically or radiologically measurable disease. Patients would have 
generally relapsed following at least one course of conventional therapy 
and most have been intensively treated with combination chemotherapy 
(100%), radiotherapy (56%) and autologous bone marrow transplantation 
(19%). Conventional therapy should be stopped at least 2 weeks before 
treatment except for corticosteriods which may be maintained at their 
previous dosage throughout. 
Patients would be excluded from the study if they had CNS disease, severe 
infection, autoimmune vasculitis, inflammatory arthritis, cardiac, renal 
(creatinine .+-.170 .mu.M) or hepatic dysfunction (bilirubin .+-.25 
.mu.M), allergy or antibodies to mouse immunoglobulin (M1 g) or dgA. 
Immunophenotyping of the lymphoma 
Blood, bone marrow (BM) and/or lymph node (LN) biopsies would be obtained 
from each patient. Lymphoid cells would be separated from blood and BM 
using Lymphoprep and mechanically teased from lymph nodes. The lymphoid 
cells would be stained for a variety of T and B cell antigens (CD2, CD3, 
CD4, CD5, CD6, CD10, CD19, CD20, CD21, CD22 [RFB4], CD37 and 
immunoglobulin [.kappa., .lambda., .mu., .delta., .gamma., and .alpha. 
chains], and were assessed for the intensity of CD22 (RFB4) antigen 
expression (compared to Daudi cells) by flow cytometry. Lymphoma cells and 
Daudi cells would be stained in parallel with an irrelevant IgGlk 
(MOPC-21) and RFB4, followed by FITC-goat anti-mouse Ig. 
Immunotoxin administration 
RFB4-SMPT-dgA would be prepared as described herein. For intravenous 
access, an indwelling subclavian catheter would be inserted before 
starting treatment. The immunotoxin would be filtered through a 0.22 um 
filter and infused over 4 hours in 100 ml of saline; one would preferably 
administer 4 infusions of immunotoxin 48 hours apart. The rationale of 
this approach is to give all the immunotoxin before any host antibody 
response against the immunotoxin (usually by 10-14 days following 
xenogenic immunoglobulin) could occur. 
Two patients could be treated at each total dose level and dosages could 
start at 5% of the mouse LD.sub.50 rising to 10%, 15% and so on until 
grade III or IV toxicity was encountered in any patient. The total dose 
could be achieved by intra-patient escalation of RFB4-SMPT-dgA doubling 
with each successive dose. Thus at 10%LD50 successive doses of 0.67%, 
1.33%, 2.67% and 5.33% LD50 would be given representing 1/15, 2/15, 4/15 
and 8/15 of the planned total dose. Patients could also receive the 
planned total dose as 4 equally divided doses. Patients could receive 
between 2 and 12 infusions. The number of infusions would, of course, be 
dependent upon the grade of any toxicity produced by previous infusions. 
The LD50 would be taken as the standard for biological activity of 
different Lots of immunotoxin and if the LD50 does not differ between the 
Lots, dosage would be expressed as mg/m2. 
Patient examination 
Before entry into the study patients would undergo a physical examination, 
measurement of vital signs, respiratory function tests, EKG, chest X-rays, 
MUGA scans, CT scans of chest, abdomen and pelvis. Laboratory measurements 
would include full blood counts, lymphoma phenotyping, examination of CSF, 
PT, PTT, fibrinogen levels, serum creatinine, creatine kinase, 
electrolytes, urea, AST, ALT, bilirubin, albumin, total serum protein and 
immunoglobulins. Physical examination, EKG and laboratory tests would be 
performed daily during immunotoxin administration until 2 days after the 
last dose and then weekly thereafter. Serum samples would be taken weekly 
after the immunotoxin treatment for evaluation of human anti-mouse (HAMA) 
and anti-ricin (HARA) responses until patients came off study. 
Grading of toxicity 
Adverse effects would be graded as Grade I (mild), II (moderate), III 
(severe) or IV (life threatening) based on WHO guidelines with 
modifications as appropriate. If patients experience Grade I or lower 
toxicity, they would complete a scheduled four doses of immunotoxin and 
could have a further four doses thereafter if (i) the tumor burden was 
reduced by at least 50% and (ii) there was no HAMA or HARA. After Grade II 
toxicity further doses could be delayed for 24 hours to allow improvement. 
At Grade III toxicity administration of immunotoxin would be stopped until 
improvement had occurred and doses could then be continued at half the 
previous level. Grade 4 toxicity would be an absolute contraindication to 
further immunotoxin therapy. 
Pharmacokinetics 
The assays used to determine immunotoxin levels in the blood are well 
known. Serum samples would be obtained at 0, 1, 3, 4, 8, 12, 24 and 48 
hours after each infusion. Half-lives and other pharmacokinetic parameters 
would be analyzed using the PKCALC program (1987) developed by Dr. R. C. 
Shumaker, Merrel Dow Research Institute, Cincinnati, Ohio. Pharmacokinetic 
analysis would be performed on every infusion with a sufficient series of 
detectable immunotoxin levels. 
Evaluation of tumor responses 
Clinical responses would be scored using WHO criteria to define partial 
(PR) and complete responses (CR) by linear measurement of masses 
detectable clinically and by CT scanning. Evaluation of linearly, 
unmeasurable disease such as BM infiltration, leukemic cells and 
ill-defined tissue infiltration would not be undertaken unless evaluating 
a possible CR in which case all previously involved sites would have to be 
proven free of disease by biopsy. Physical examination would be performed 
weekly after treatment and CT scans would be carried out between 2-7 days 
and again at one month after treatment. Patients would come off study when 
their disease progressed and/or they were treated with chemotherapy, 
radiotherapy or an increased dose of corticosteroids. Patients with large 
tumors (L: 10 cm in maximum diameter or 100 cm total measurable area) 
would be distinguished from those with smaller masses (S: 10 cm and 100 cm 
). 
Statistical analysis 
Statistical analysis may be performed using the SPSS statistical package, 
for example. SPSS Inc. Illinois, USA and Student's T-test. ANOVA. 
Mann-Whitney U-test and Pearson product moment correlation techniques may 
be used depending on whether continuous, categorical, multiple or skewed 
distribution variables are being analyzed. 
While the compositions and methods of this invention have been described in 
terms of preferred embodiments, it will be apparent to those of skill in 
the art that variations may be applied to the composition, methods and in 
the steps or in the sequence of steps of the method described herein 
without departing from the concept, spirit and scope of the invention. 
More specifically, it will be apparent that certain agents which are both 
chemically and physiologically related may be substituted for the agents 
described herein while the same or similar results would be achieved. All 
such similar substitutes and modifications apparent to those skilled in 
the art are deemed to be within the spirit, scope and concept of the 
invention as defined by the appended claims. 
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