Methods and compositions for the treatment of Hodgkin's disease

Disclosed are methods and compositions for the treatment of Hodgkin's disease and processes involving Hodgkin's disease cells or Reed-Sternberg cells, through specific elimination of Hodgkin's disease cells through the application of immunotoxin technology. The compositions of the invention include toxin conjugates composed of a Hodgkin's disease cell binding ligand conjugated to a toxin A chain moiety such as ricin A chain or deglycosylated ricin A chain, by means of a cross-linker or other conjugation which includes a disulfide bond. In preferred aspects of the invention, therapeutic amounts of conjugates composed of a CD-30 or IRac antibody or fragment thereof conjugated to deglycosylated A chain by means of an SMPT linker is administered to a Hodgkin's disease patient so as to specifically eliminate Hodgkin's disease cells without exerting significant toxicity against non-tumor cells. Also disclosed are particular hybridomas and monoclonal antibodies, and associated methodology, which may be employed, e.g., in the preparation of these immunotoxins as well as other uses such as diagnostic applications.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to methods and compositions useful in the treatment 
of patients suffering from Hodgkin's disease and other conditions such as 
large cell anaplastic lymphoma and graft-versus-host disease. In 
particular, this invention relates to antibody-toxin conjugates 
(immunotoxins) capable of selectively killing Hodgkin's and Reed-Sternberg 
cells and other cells such as activated lymphoid cells and monocytoid 
cells, and, in further embodiments, to hybridomas and antibodies useful in 
the preparation of such immunotoxins. 
2. Description of the Related Art 
Chemotherapy of Hodgkin's disease is undoubte of the major breakthroughs in 
clinical oncology over the last 25 years. The introduction of the 
multi-agent chemotherapy regimens such as MOPP (1) and ABVD (2) and the 
optimized use of radiation in early stages of the disease has improved the 
probability of curing these patients from less than 5% in 1963 to about 
70% at the present time (3-5). 
Despite the high proportion of cures in patients with Hodgkin's disease who 
respond to first line treatment, the outlook for those who relapse or fail 
to achieve complete remission is bleak. Second line combination 
chemotherapy can produce good remission rates although cures are uncommon 
(6-8). High dose chemotherapy with autologous bone marrow transplantation 
has been reported to be effective in relapsed or resistant cases of 
Hodgkin's disease (HD) but is associated with major toxicity, resulting in 
up to 26% treatment-related deaths (9,10). Of those patients achieving 
complete remission, 15-20% will develop a second malignancy as a 
chemotherapy-related side effect (11). There is therefore a need for new 
modes of treatment for this disease. In particular, there is a need for 
new agents for the management of or treatment of Hodgkin's disease that 
are free from mutagenic side effects. 
An approach proposed by the present invention to preparing new, 
non-mutagenic reagents for the therapy of Hodgkin's disease would be to 
couple the ribosome-damaging A-chain of ricin or other toxins to 
antibodies directed against Hodgkin cell-associated antigens. In several 
laboratories, ricin A-chain has been linked to antibodies against 
tumour-associated antigens to form immunotoxin reagents that are 
selectively toxic to malignant cells in vitro (reviewed in 12,13). 
However, in vivo studies in rodents, and more recently, in man have given 
variable results. In rodents, good antitumor effects have generally been 
observed in leukemia and lymphoma models, whereas solid tumors appear to 
be less responsive (12,13). In humans, the antitumor effects obtained in 
melanoma (14) and leukemia (15) patients have so far been disappointing, 
whereas in patients with steroid-resistant graft-versus-host disease, 
remarkable benefit has been obtained (16). 
Thus, although there have been many reports of the high cytotoxic potency 
and specificity of immunotoxins in vitro, relatively few workers have 
reported good antitumor effects of immunotoxins on solid tumors in vivo: 
Bernhard et al. (17) and Hwang et al. (18) reported that a single i.v. or 
s.c. injection of an abrin A-chain immuno-toxin reduced or completely 
abolished the growth of solid L10 hepatocarcinoma cell line tumors in 
guinea pigs. Leonhard et al. (19) described 11/46 complete remissions of 
solid human T cell line tumors in mice intravenously treated with CD5 
ricin A-chain immunotoxins. Roth et al. (20) demonstrated a reduction in 
the number of pulmonary metastases after systemic administration of ricin 
A-chain immunotoxins to mice bearing TRF (transformed rat fibro-blasts) 
tumors. Others have needed to give multiple injections with dosages up to 
125% of the LD.sub.50 (21) or intratumoral injections to show antitumor 
effects of their immunotoxins (22). Accordingly, the use of immunotoxin in 
the treatment of tumour, particularly solid tumors, has heretofore been 
unpredictable at best. 
For the foregoing and other reasons, it can be appreciated that there is 
currently a need for novel approaches to the treatment and control of 
patients suffering from Hodgkin's disease. In particular, there is a need 
for improved treatment modalities which exhibit one or more advantages 
over existing approaches to treatment. Furthermore, there is a need for 
treatments which may be employed in those cases where more traditional 
approaches have not proven effective. The present invention addresses one 
or more shortcomings in the art through application of immunotoxin 
technology. 
SUMMARY OF THE INVENTION 
The present invention addreses one or more deficiencies in the prior art by 
providing improved methods and compositions for the treatment of Hodgkin's 
disease, as well as other diseases such as those involving large cell 
anaplastic lymphoma or graft-versus-host disease. It is proposed that the 
methodology and compositions disclosed herein will provide a means for 
treating Hodgkin's disease and other diseases which are caused by 
activated lymphoid cells, monocytoid cells or CD30- or IRac-positive 
cells, through the application of immuno-toxin technology, wherein a 
specific cell surface binding ligand is conjugated to a toxin moiety, with 
the binding ligand serving as a means for directing the toxin moiety to 
the cells to be treated, in this case, Hodgkin, Reed-Sternberg, activated 
lymphoid cells or monocytoid cells. 
Thus, the invention provides, in a general sense, an immunotoxin conjugate 
which includes a cell-surface binding ligand comprised of an antibody or 
antibody fragment having binding affinity for Hodgkin or Reed-Sternberg 
cells, and a toxin moiety conjugated to the binding ligand by means of a 
disulfide linkage. As used herein, the term disulfide linkage is meant to 
refer to any means of connecting the toxin moiety to the binding ligand 
wherein the connecting means includes a disulfide bond. A disulfide or 
similar biologically releasable bond is important to the realization of a 
clinically active immunotoxin in that the toxin moiety must be capable of 
being released from the binding ligand once the binding ligand has entered 
the target cell. Numerous types of linking constructs are known, including 
simply direct disulfide bond formation between sulfhydryl groups contained 
on amino acids such as cysteine, or otherwise introduced into respective 
protein structures. However, the term "disulfide linkage" in also meant to 
include the use of a linker moiety which includes a disulfide bond, as 
discussed further herein below. 
In certain preferred embodiments of the invention the conjugate will 
include a binding ligand which, prior to conjugation with a toxin moiety, 
will exhibit a binding affinity (i.e., Kd) of less than about 200 nM for 
the targeted antigen. The inventors have found that a binding affinity of 
much higher than 200 nM will generally not have a high enough binding 
affinity to be of particular usefulness in connection with the treatment 
of clinical disease. For the purposes of the present invention, the 
binding affinities will typically be measured with reference to a 
Hodgkin's disease-derived cell line such as the L540 cell line. Moreover, 
where a binding ligand is said to exhibit a Kd of less than about 200 nM 
for L540 Hodgkin's cells, this phrase is meant to imply the binding 
affinity of such a ligand for such cells when carried out in accordance 
with the procedures set forth hereinbelow in the examples. 
Although it is believed that useful immunotoxins may be prepared where the 
binding ligand exhibits a Kd of less than about 200 nM for, for example, 
L540 Hodgkin cells, in more preferred embodiments the binding ligand will 
exhibit Kds that are significantly less than 200 nM. For example, binding 
affinity, in terms of Kd, of less than about 40 nM and even those less 
than about 20 nM will be particularly preferred for uses in accordance 
herewith. The present inventors have discovered that these high binding 
affinities may be achieved against certain particular antigens, including 
the 70 Kd antigen recognized by the IRac antibody or the CD-30 antigen, as 
discussed in more detail hereinbelow. Of course, it will nevertheless be 
necessary to screen hybridoma banks to identify monoclonal antibodies 
which exhibit the desired binding specificity and affinity. In any event, 
the inventors have been able to obtain binding ligands having extremely 
high binding affinities, for example, on the order of less than about 7 to 
about 27 nM for L540 Hodgkin cells for the most preferred binding ligands 
provided in accordance herewith. 
An important aspect of the present invention is the ability to prepare 
immunotoxins which include not only a binding ligand with a high binding 
affinity, but more importantly, immunotoxins which themselves exhibit very 
high anti-cellular cytotoxicity. Cytotoxicity is not always directly 
related to binding affinity, and therefore the identification of 
monoclonal antibodies which can be employed in the preparation of highly 
cytotoxic immuno-toxins will not necessarily be evident from a high 
binding affinity. Therefore, in addition to identifying anti-bodies or 
binding ligands having high binding affinity, one will also desire to 
screen such antibodies for their ability to provide highly cytotoxic 
immunotoxins, as well as immunotoxins with little or no cross-reactivity 
with non-tumor cells. The present invention provides in certain 
embodiments immunotoxins having exceedingly high cytotoxicities, for 
example, as measured in terms of the concentration at which they will 
inhibit by 50% the proliferation, protein synthesis or some other vital 
function of the cells (i.e., the "IC.sub.50 "). Accordingly, in certain 
embodiments, the invention is directed to conjugates which exhibit an 
IC.sub.50 of less than or equal to about 10.sup.-9 M on the targeted 
Hodgkin's disease cells. However, as a useful reference, the inventors 
disclose the use of the L540 Hodgkin's cell line in an assay for measuring 
a relative IC.sub.50 of the respective conjugate embodiments. The 
preparation of immunotoxin conjugates which exhibit an IC.sub.50 of less 
than or equal to about 10.sup.-9 M on L540 Hodgkin's disease cells is 
believed to be predictive of conjugates that will exhibit certain clinical 
advantages in accordance with the present invention. That is, those 
conjugates having an IC.sub.50 of less than about 10.sup.-9 M will be 
expected to exhibit a preferred degree of usefulness in treating Hodgkin's 
disease. However, in even more preferred embodiments, the invention is 
concerned with the preparation of conjugates which exhibit an IC.sub.50 of 
less than or equal to about 10.sup.-10 M on L540 Hodgkin's cells. It is 
believed that Hodgkin's directed immunotoxins having this very high degree 
of cytotoxicity will be of particular usefulness in the treatment of this 
disease. 
In more particular embodiments, the inventors disclose herein the 
preparation of various immunotoxin conjugates which exhibit an IC.sub.50 
of between about 7.times.10.sup.-10 and about 1.times.10.sup.-11 M when 
measured on L540 Hodgkin's cells. This range is exemplary of the range of 
IC.sub.50 observed for the most preferred and clinically useful 
immunotoxin conjugates in accordance herewith. 
While the present invention is not limited to the targeting of any one 
particular Hodgkin's disease cell antigen, it has been found that certain 
antigens are to be preferred over others, both in terms of the ability to 
generate high affinity antibodies and high specific activity immunotoxins 
for such antigens, but further in that these antigens are found to define 
highly selective immunotoxins. One such antigen is known as the CD-30 
antigen complex. The CD-30 antigen (also known as the Ki-1 antigen) is 
composed of two non reducible subunits of 105 and 120 kDa molecular 
weight. 
While it was at one time thought that the Ki-1 or CD-30 antigen was 
specific for Hodgkin or Reed-Sternberg cells, it has now been found on a 
variety of other cells, including large cell anaplastic lymphomas, 
peripheral T-cell lymphomas, cutaneous lymphoid infiltrates and tumor 
cells of embryonal carcinoma. Furthermore, the CD-30 antigen can be 
induced on B and T cells by phytohemagglu-tinin (PHA), human T-cell 
leukemia viruses 1 and 2 (HTLV 1 and 2) as well as Epstein-Barr virus. In 
any event, however, it has been found that the CD-30 antigen can be 
employed for the generation of highly useful immunotoxins in accordance 
herewith. 
A second antigen which the inventors have found to be particularly useful 
for the targeting of Hodgkin's disease-directed immunotoxins is the 70 kDa 
antigen which has been characterized by Hsu et al. (47, 48) through the 
use of the IRac antibody. The IRac antibody was developed through the use 
of Hodgkin's disease cells from tumor samples which were purified and 
stimulated with phorbol acetate (TPA). In any event, the inventors have 
found that the IRac antibody is particularly useful both in the 
preparation of immunotoxins and in the identification of cross-blocking 
immunotoxins which might be similarly useful. 
Of course, it is not intended that this invention be limited to antibodies 
against these particular anti-Hodgkin's disease cell antigens. It is 
proposed that a number of target antigens are known which can suitably be 
employed in the practice of the present invention. Other possible targets 
include, for example, the interleukin 2 (IL2) receptor alpha or beta chain 
(CD25 antigen), the CD15 antigen, HLA-DR, the transferrin receptor, the 
leukocyte common antigen (CD45), and the like (see, e.g., references 
73-74). Through the application or use of the techniques or materials set 
forth or referred to herein, it is proposed that one will be able to 
prepare binding ligands having a suitable degree of binding affinity and 
capable of providing immunotoxins having a suitable degree of specific 
cytotoxicity. 
Furthermore, culture deposits have been made of hybridomas which secrete 
particular preferred antibodies directed against either the CD-30 antigen 
(i.e., the HRS-3 antibody, has been deposited with the PHLS Centre for 
Applied Microbiology & Research, European Collection of Animal Cell 
Cultures, Division of Biologics, Porton Down, Salisbury, Wiltshire, 
England, on Nov. 16, 1989, as accession No. 89111607) as well as 
hybridomas which secrete the IRac antibody also deposited with the PHLS 
Centre on Nov. 16, 1989, as accession No. 8911608. As discussed in more 
detail hereinbelow, these monoclonal antibodies may be employed either 
directly or in the initial screening and identification of antibodies 
having specificity for these two respective antigens. 
Thus, in certain embodiments, the present disclosure is further directed to 
the preparation of hybridomas which may be employed in the preparation of 
antibodies useful in the practice of the invention, including in 
particular, CD-30 or IRac antibodies which exhibit binding characteristics 
similar to HRS-3 or IRac. Techniques are disclosed herein which may be 
employed both in the preparation of hybridoma libraries which secrete 
anti-Hodgkin's disease cell antibodies, and in screening these libraries 
to identify and select hybridomas secreting antibodies having many, if not 
most, of the desirable attributes of HRS-3 or IRac. 
In general, the hybridoma libraries are prepared through the application of 
known monoclonal antibody techniques, with some important modifications. 
Typically one will desire to employ as the initial immunogen in hybridoma 
development, a Hodgkin's disease or Reed-Sternberg cell, cell line or cell 
derived protein or fraction. Advantageously, one may simply desire to 
employ whole tumor cells for this purpose, preferably tumor cells from a 
Hodgkin's disease cell line such as the L428 or L540. Since one of the 
preferred antibody species, HRS-3, was prepared using the L540 cell line, 
one may typically desire to use this cell line as the starting immunogen. 
However, it should be pointed out that the other preferred species, IRac, 
was prepared using tissue from diseased patients. Thus, the use of tissue 
biopsy samples, for example, should not be excluded. In any event, 
following immunization and fusion of lymphoid cells with an appropriate 
fusion partner (e.g., X63Aj8.635 or P3-NS1/1-Ag4-1), one will desire to 
screen the resultant hybridomas in various manners to identify an 
appropriate colony. 
One such screening will typically be to exclude hybridomas which secrete 
antibodies not directed against cell surface antigens of the Hodgkin's 
disease cells. These techniques are well known in the art. One might also 
wish to screen putative positives against other Hodgkin disease cell lines 
or tissues to ensure that the secreted monoclonal is more or less 
pan-reactive with Hodgkin's disease cells. Another screening might be 
performed to exclude antibodies that are reactive with normal tissues such 
as tonsils or other normal tissues, particularly "life-sustaining tissues" 
as discussed hereinbelow. 
Further, to assist in identifying CD-30 monoclonal antibodies (or IRac 
related antibodies) having the parti-cular desirable attributes of HRS-3 
(or IRac), one may find advantages in the use of the cross-blocking assay 
disclosed herein, e.g., using the HRS-3 (or IRac) antibody itself as the 
competing antibody. Using the foregoing techniques, one will be enabled to 
identify anti-Hodgkin cell antibodies that will "compete" with the HRS-3 
or IRac antibody for its particular CD-30 or 70 Kd antigen epitope, 
respectively. Antibodies identified in this manner should therefore be 
reactive with the same, or associated, CD-30 epitope as HRS-3, or 70 Kd 
antigen epitope as IRac, as the case may be. 
However, in addition to the identification of anti-bodies reactive with the 
same or similar CD-30 epitope, it will further be preferred to select 
monoclonal antibodies having the additional desirable characteristics of 
HRS-3 or IRac, including their high binding affinity (i.e., low Kd), their 
ability to form highly cytotoxic immunotoxins against Hodgkin's disease 
cells, the exhibition of little or no binding to life-sustaining "normal" 
tissues, and even the ability of the stability of the underlying hybridoma 
and its ability to secrete useful quantities of the antibody. 
In addition to their usefulness in the preparation of immunotoxins, it is 
proposed that the anti-Hodgkin's disease cell antibodies disclosed or 
otherwise enabled herein will find utility in other respects. For example, 
it is contemplated that these antibodies may be useful in therapeutic 
modalities directly without toxin conjugation, in that these antibodies 
apparently exhibit direct anti-cellular activities. Furthermore, it is 
contemplated that due to the highly selective nature of their binding to 
Hodgkin's disease antigens, it is proposed that these antibodies will find 
utility in diagnostic embodiments, such as in the performance of RIAs or 
ELISAs for disease diagnosis or for following the course of the disease. 
In any event, it is specifically pointed out that this aspect of the 
invention is not limited to the preparation of immunotoxins. 
In certain embodiments, the invention is directed to binding ligands, such 
as antibodies on antibody fragments capable of at least about 70% 
cross-blocking of the HRS-3 or IRac binding to L540 Hodgkin's cells, when 
said binding ligand is present at about 100-fold excess with respect to 
said HRS-3 or IRac antibody. It is proposed that where a desired antibody 
is capable of cross-blocking the binding of HRS-3 or IRac to the extent of 
about 70% when the cross-blocking is carried out as disclosed hereinbelow, 
one will thereby identify a binding ligand having a particular utility in 
the preparation of immunotoxins in accordance herewith. Even more 
preferably, through the use of cross-blocking techniques one will be 
enabled to select binding ligands that will cross-block either the HRS-3 
or IRac antibodies to the extent of at least about 90% cross-blocking. 
In still further embodiments, it will be desirable to identify binding 
ligands that are essentially, effectively or pharmacologically free of 
binding affinity for normal tissues. As used herein, the term "essentially 
free of binding affinity for normal tissues" is intended to refer to 
binding ligands which exhibit little or no binding affinity for 
life-sustaining tissues, such as one or more tissues selected from bone 
marrow, colon, kidney, brain, breast, prostate, thyroid, gall bladder, 
liver, lung, adrenals, heart, muscle, nerve fibers, pancreas, skin, or 
other life-sustaining organ or tissue in the human body. The 
"life-sustaining" tissues that are the most important for the purposes of 
the present invention, from the standpoint of low cross-reactivity, 
include heart, kidney, central and peripheral nervous system tissues, 
liver and lung. By the term "little or no" binding is meant an antibody or 
antibody fragment, which, when applied to the particular tissue under 
conditions suitable for immuno-histochemistry, will elicit either no 
staining or a mixed staining pattern with only a few positive cells of 
large lymphoid or monocytoid morphology scattered among a field of mostly 
negative cells. 
The origin or derivation of the antibody or antibody fragment (e.g., Fab', 
Fab or F(ab').sub.2) is not believed to be particularly crucial to the 
practice of the invention, so long as the antibody or fragment that is 
actually employed for immunotoxin preparation otherwise exhibits the 
desired properties. Thus, where monoclonal antibodies are employed, they 
may be of human, murine, monkey, rat, hamster, chicken or even rabbit 
origin. The invention therefore contemplates the use of human antibodies, 
"humanized" or chimaeric antibodies from mouse, rat, or other species, 
bearing human constant and/or variable region domains, single domain 
antibodies (e.g., DAbs), 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 anti-bodies will typically be 
preferred. 
The ligand-toxin conjugate composition of the invention will typically 
comprise a Hodgkin cell binding ligand conjugated to a toxin moiety 
through a disulfide linkage. This is because it has been found that the 
disulfide linkage is important where one desires to employ a toxin moiety 
such as ricin A chain in connection with anti-cellular therapy. While the 
mechanism is not entirely clear, it appears as though a disulfide linkage 
allows decoupling of a toxin moiety such as a ricin A chain moiety 
delivered to target cells by the binding ligand, thereby freeing the A 
chain moiety to exert its anti-cellular effect in the cytosol. 
It is proposed that the configuration of cross-linking between, e.g., ricin 
A chain and the binding function is an important consideration in that 
this configuration appears to play an important role pharma-ceutically. 
This is likely a function of a somewhat complex set of variables, 
including the vulnerability of the disulfide bond to "decoupling" as well 
as its ability to release the toxin upon binding on the surface of target 
cells. 
The general construction of conjugates by means that will provide a 
disulfide bond between the ligand and the toxin moiety is generally known 
in the art, as reviewed in references such as 53 and 54. Disulfide 
coupling may be achieved directly between cysteine residues of the 
respective proteins, e.g., by means of disulfide exchange reactions 
wherein the protein is reduced and derivatized with Ellman's reagent. 
However, direct disulfide bond formation between many binding ligands and 
toxin will generally not be preferred, since a cysteine in the ligand is 
not accessible for coupling. Reduction of cysteine bridges in the ligand, 
to provide reactive SH groups, may damage the functional integrity of the 
ligand. 
Accordingly, one will generally find it preferable, in the case of ligands 
lacking free cysteine residues, to employ a cross-linking group which will 
provide suitable release characteristics and resultant therapeutic 
parameters. A variety of cross-linkers having disulfide groups are known 
in the art, as exemplified by SPDP, SATA, 2-IT and SMPT (34, 54). 
Generally speaking, suitable cross-linkers will include structures 1) 
having the ability to covalently couple to amino groups of lysine, or the 
like; and 2) incorporating a disulfide or other desired releasable 
functionality. Useful groups of cross-linkers include the 
heterobifunctional cross-linkers described above. 
Particular useful cross-linkers found to have desirable characteristics in 
terms of stability, yields and long in vivo half-lives of resulting 
conjugates include SATA (N-succinimidyl-S-acetylthioacetate), SPDP 
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB 
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT 
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). 
SPDB and SMPT generate linkages containing a hindered disulfide bond and 
are particularly preferred. A variety of additional reagents for the 
purposes of cross-linking conjugates in accordance with the present 
invention are known in the art and can be substituted for those referred 
to herein. 
As used herein, the phrase "hindered disulfide bond" is intended to refer 
to a disulfide bond having groups near or adjacent to the disulfide bond 
that reduce its susceptibility to reduction, e.g., by thiols, by 3-fold or 
more, preferably greater than 5-fold, relative to the disulfide bonds 
generated by SPDP or 2-iminothiolane hydrochloride reagents. The rate of 
reduction of immunotoxin into the ligand and the toxin component may be 
measured, e.g., by treating a known concentration of immunotoxin with a 
known concentration of a thiol such as dithiothreitol or 2-mercaptoethanol 
and measuring the extent of dissociation of the immunotoxin at various 
time intervals. This measurement may be performed in a number of ways, 
e.g., by SDS-polyacrylamide electrophoresis and densitometric scanning of 
gels. By this approach, the ratio of the sum of the areas under the free 
antibody and free toxin peaks to the total area under all the peaks gives 
the fraction of immunotoxin that has dissociated. 
The toxin molecule of the present invention will typically comprise a toxin 
A chain or toxic derivative thereof. Numerous A chains believed to have 
suitable anti-cellular properties in the practice of the invention are 
known in the art. Exemplary "A chains" which may be employed in connection 
with the invention, as this term is used herein, include the A chain of 
ricin, diphtheria toxin, volkensin, modeccin, Shigella toxin, abrin or the 
like; or the "free A chains", known as ribosome-inactivat-ing proteins, 
e.g., gelonin, trichosanthin, saporin, bryodin, momordin, alpha-sarcin, 
dianthins, pokeweed antiviral protein, barley toxin, or the like; or other 
known toxin moieties such as Pseudomonas exotoxin A, diptheria toxin, 
genetically engineered versions or derivatives of any of the foregoing 
toxins (see, e.g., ref. 73,74), intact ricin that has been "blocked" to 
prevent nonspecific B-chain binding (see, e.g., refs. 75-76), or fragments 
of any of the foregoing. Of these, the ricin A chain molecule is the most 
preferred due to its high intrinsic anti-cellular activity and the 
clinical experience in humans indicating only modest side effects. 
In addition to the whole A chain molecule, one may desire to simply employ 
that portion of the A chain that is necessary for exerting anti-cellular 
effects. For example, it has been found that the ricin A chain molecule 
can be truncated by removal of the first 30 amino acids and nevertheless 
obtain a toxin molecule that exerts sufficient anti-cellular activity to 
be of use in connection herewith. Such termination is achieved by either 
genetic engineering or proteolytic degradation, e.g., with Nagarase (55), 
the product being referred to herein as "truncated" A chain. 
In the more preferred embodiments of the present invention, a 
deglycosylated A chain such as deglycosylated ricin A chain (dgA) or 
variants thereof is employed. Deglycosylated A chain is A chain that has 
been treated so as to remove or destroy carbohydrate moieties (e.g., 
mannose, fucose) which are incorporated into naturally produced A chain 
molecules. It has been found that the presence of mannose/fucose on the 
oligosaccharide side chains of the A chain promote rapid clearance by the 
liver and reduced therapeutic effect of the toxin or A chain by cells such 
as the reticuloendothelial cells of the liver and spleen which have 
receptors that recognize these structures. The inventors have found that, 
through the use of deglycosylated A chains, one may achieve particular 
advantages in terms of both increased potency and increased half life of 
the conjugate in the circulation and reduced hepatotoxicity, by reducing 
the clearance of the conjugate by the liver. 
While deglycosylated ricin A chain is preferred, there is no reason that 
other nonglycosylated toxin A chains or ribosome-inactivating proteins 
could not be employed in connection with the invention. In any event, the 
preparation and use of deglycosylated A chain is known in the art as 
illustrated by references such as Thorpe et al. (30, 56). Moreover, 
deglycosylated A chain is now available commercially from Inland 
Laboratories, Austin, Tex. 
Additionally, the preparation of ricin A chain by recombinant means is now 
known, as exemplified by O'Hare et al. (57). Thus, it is now possible to 
alter the amino acid structure through the application of in vitro 
muta-genesis technology. Through the judicious selection of amino acid 
sequence alterations or modifications based on knowledge of interactive 
forces between amino acids, one can readily modify or alter the A chain 
sequence and provide a means for selecting variant proteins having 
improved toxicity, pharmacologic or release properties. 
In still further embodiments of the invention, it is contemplated that 
several binding ligands may be conjugated to a single toxin A chain 
moiety. It is proposed that such constructs, containing up to, for 
example, 5 or so binding ligands per toxin moiety, may find particular 
therapeutic benefits. It is, for example, believed that such constructs 
may have a particular high binding affinity for target cells, thereby 
providing enhanced ability to deliver toxin to the targeted cells and 
thereby kill them. 
It has been found that ricin B-chains alone, or coupled to antibody, can 
serve to greatly enhance the specific cytotoxicity of immunotoxins 
containing ricin. B chains are the "lectin" binding regions of the toxin 
complex that are responsible for the native toxin's broad ranging 
cell-binding capability. It has been proposed by others that not only do B 
chains stimulate immunotoxin action, but that one can "separate" 
pharmacologically this action from the cell-binding function by chemical 
or heat modification of the B chain (58). It is thus proposed that the 
application of toxin B chains in combination with the A chain conjugates 
may provide advantages in terms of even further specific cytotoxicity 
against targeted cells. 
An important aspect of the invention is the prepara-tion of pharmaceutical 
compositions which incorporate the binding ligand-toxin conjugates in 
therapeutically effective amounts. Of course, where pharmaceutical 
compositions are prepared, one will desire to employ conjugates that are 
essentially free of unconjugated material and, further, do not contain any 
undesired impurities. Therefore, one will generally find it necessary to 
purify conjugates prepared in accordance with the invention through the 
application of purification technology. Techniques are known for isolating 
and purifying conjugates to a very high degree. 
In certain aspects, the present invention is thus concerned with techniques 
for purifying immunoconjugates, including conjugates such as the 
anti-Hodgkin's cell ligand-toxin conjugates of the present invention. 
Parti-cular techniques which have been found useful in the purification of 
conjugates in accordance herewith include affinity chromatography 
techniques employing Blue- (or Red) Sepharose, molecular exclusion 
chromatography on Sephacryl or even gel permeation chromatography by HPLC. 
(See, e.g., U.S. Ser. No. 07/150,190, filed 1/29/88, incorporated herein 
by reference) 
Pharmaceutical compositions comprising conjugates of the present invention 
are typically prepared by combining the purified conjugate with a 
pharmaceutically acceptable diluent or excipient for parenteral 
administration. A variety of suitable carrier vehicles and their 
formulation are described, for example, in reference 29. Suitable carriers 
include sterile aqueous solutions including stabilizing agents, e.g., 
buffers and other protein and pH-stabilizing agents, salts and the like. 
Typically, sterile aqueous compositions of the desired conjugate will 
include a dose concentration of between about 0.25 and about 2.5 mg/ml, to 
allow for administration of convenient amounts. 
In certain embodiments, the appropriate dose of conjugate to be 
administered will be somewhat dependent upon the particular patient. Those 
of skill in the art of immunotoxin administration will appreciate that 
variations in optimal doses will exist from patient to patient, depending 
on a variety of variables. Typically, one will desire to administer on the 
order of 10 to 250 mg (for an average 70 kg human), depending upon the 
type of conjugate employed and the appearance of untoward side effects 
such as vascular leak syndrome (VLS), myalgia, fatigue and/or fever. Other 
considerations include the administration of the conjugates in 2-20 
fractional doses. 
In still further embodiments, the present invention is directed to the 
preparation of "cocktails" which incorporate more than one immunotoxin 
species. Prefer-ably, such cocktails employ immunotoxins having 
speci-ficity for different antigens on the Hodgkin's disease or 
Reed-Sternberg cells. For example, in particular preferred embodiments, 
one will desire to prepare a "cocktail" pharmaceutical composition which 
comprises one immunotoxin directed to the CD-30 antigen, and a second 
immunotoxin directed to the 70 kDa/IRac antigen. In these embodiments, one 
will typically desire to employ the respective immunotoxins in an overall 
amount such that their total toxicity will not be high enough to cause 
undesirable side effects. It is proposed that by the use of immunotoxins 
having differing specifities, one will decrease the opportunity for the 
development of resistant tumors, such as tumors wherein a single targeted 
antigen such as the CD-30 antigen is no longer being expressed. Where two 
or more independent epitopes on one or more antigens are targeted, such as 
through the use of such cocktails, it is believed that a much improved 
overall antitumor therapeutic effect will be realized.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Introduction 
The present invention relates to the preparation and use of immunotoxins 
which recognize antigens located on the cell surface of Hodgkin and 
Reed-Sternberg cells. Immunotoxin conjugates of the invention are composed 
of a cell surface binding ligand, typically an antibody or antibody 
fragment such as an Fab' fragment, having binding affinity for cell 
surface antigens located on Hodgkin or Reed-Sternberg cells, with the 
binding ligand being conjugated to a toxin moiety by means of a disulfide 
linkage between the toxin and the binding ligand. The disulfide linkage 
may be formed by direct conjugation of the binding ligand to the toxin 
moiety, or may involve the use of a chemical linker which incorporates a 
disulfide group. The use and nature of the disulfide linker is thought to 
be important to the pharmacologic properties of the immunotoxin in that, 
preferably, the conjugate should remain intact while circulating through 
the blood stream, but, once the conjugate attaches itself to a target 
cell, the toxin moiety should be able to dissociate from the ligand and 
enter the cell to work its toxic effect upon the target. 
The cell surface binding ligand employed in connection with the practice of 
the invention will typically be an antibody or antibody fragment having a 
degree of specificity or affinity for Hodgkin or Reed-Sternberg cells. 
Hodgkin's disease is one of the major cancerous diseases of the lymphoid 
system. It is characterized by an infiltration of the lymphoid organs by 
Hodgkin and Reed-Sternberg cells which results in a progressive 
enlargement of lymph nodes, spleen, liver and eventually an infiltration 
of bone marrow, lung and other organs, depending on the stage of the 
disease. Hodgkin's disease is considered to be a malignant neoplasm of 
transformed early lymphoid progenitor cells or of dendritic cells. The 
characteristic tumor cells in this disease are the mononucleated Hodgkin 
cells and the bi-nucleated Reed-Sternberg cell, which are found in 
infiltrated tissue in association with lymphocytes and eosinophilic 
leukocytes and fibrosis. It has been proposed that the Reed-Sternberg cell 
may in fact be the clonal cell responsible for the malignant aspect of 
Hodgkin's disease, thus making such cells a principal target in any 
therapeutic regimen. 
The exact nature of Hodgkin's disease has been disputed over the years. The 
suggested origin of Reed-Sternberg cells in Hodgkin's disease includes 
histiocytes, macrophages and B or T lymphocytes. It has been proposed that 
Hodgkin or Reed-Sternberg cells are probably related to histiocytes or 
interdigitating reticulum cells, in that it has been possible to identify 
phenotypic markers that are shared by Hodgkin or Reed-Sternberg cells, 
interdigitating reticulum cells and histiocytes. 
The present invention takes advantage of the existence of antigenic 
cell-surface molecules that are found on Hodgkin or Reed-Sternberg cells, 
but are not found, or found in only limited amounts or degrees, on 
non-cancerous tissues. However, due to the presence of many such Hodgkin 
or Reed-Sternberg cell surface antigen on certain cell types such as 
interdigitating reticulum cells or activated T or B cells, immunotoxins of 
the present invention which employ antibodies which recognize these 
antigens may also be useful in treating conditions of these cells as well, 
such as graft-versus-host disease, organ or tissue allograft rejection 
episodes, autoimmune disorders, histiocytomatosis, histiocytosis X, large 
cell anaplastic lymphomas, or even lymphomatoid papulosis. 
In any event, it will typically be the case that anti-Hodgkin or 
Reed-Sternberg antibodies which recognize antigens found on activated T or 
B cells, or on interdigitating reticulum cells, will nevertheless prove 
useful therapeutically and such a reactivity will not in itself present a 
problem of untoward reaction due to reactivity with these cells. Of 
course, it will not be desirable to employ antibodies which react with 
cells other than activated lymphoid cells or monocytoid cells in major 
normal or "life-sustaining" tissues. Reactivity with normal tissues such 
as these will tend to provide an immunotoxin that will not be of 
particular use clinically. 
In a general sense, the preparation of antibodies having specificity for 
Hodgkin or Reed-Sternberg antigens can be accomplished using more or less 
standard hybridoma technology, but with some important modifications. For 
example, one will desire to employ Hodgkin or Reed-Sternberg cells or cell 
derived antigen(s) for the initial immunization of animals that are being 
employed to generate the hybridomas. In the preparation of antibodies, one 
may find particular advantage in the use of a Hodgkin disease cell line, 
such as the L-428, L540, DEV, L591, KM-H2 or even the HDLM cell line, 
which have been developed as permanent cell lines (see, e.g., references 
66-68, 71 and 72) and can be employed as the initial immunogen. 
For example, in the preparation of useful monoclonal antibodies in 
accordance herewith, one may desire to immunize a mammal such as a BALB/c 
mouse with whole L428 cells, or cells from another Hodgkin's disease cell 
line such as L540, obtain spleen cells from the immunized mouse and fuse 
the spleen cells with cells of, preferably, a non-secreting myeloma line 
that is compatible with the particular mammalian source that is being 
employed, such as X63-Ag8.653 (59) or Sp2/O -Ag14, in t he case where 
murine hybridomas are being employed. Although murine hybridomas are to be 
preferred due to their ease of preparation, as well as their general 
acceptability for use in connection with human administration, there is no 
reason why other mammalian sources of programmed spleen cells or 
lymphocytes cannot be employed where desired, including lymphocytes from 
humans, monkeys, rats, hamsters, chickens or even rabbits. 
Additional advantages may be realized where, in addition to the use of 
Hodgkin cells for the initial immunization, one employs a means of 
stimulation of the Hodgkin cells in a manner which induces or enhances the 
appearance of Hodgkin related antigens. For example, one may employ 
substances such as phorbol esters (e.g., 12-O-tetra-decanoyl 
phorbol-13-acetate; TPA) which stimulate cellular differentiation, to 
enhance the appearance of desirable target epitopes on the cell surface of 
cells being employed for initial immunization in the preparation of 
anti-Hodgkin disease antibodies. Other agents which might be similarly 
useful include interleukin-1 (IL-1), phytohemagglutinin (PHA), 
Epstein-Barr virus, or even human T-cell leukemia virus 1 (HTLV-1). 
Regardless of the type or origin of cells being employed for the initial 
generation of hybridoma banks, one will desire to screen the bank to 
identify those hybridomas which secrete antibodies having the desired 
binding capabilities. In general, for uses in accordance with the present 
invention, one will preferably desire to select those hybridomas which 1) 
secrete antibodies having a high affinity for the target cells, 2) are 
capable of forming immunotoxins which exhibit a low IC.sub.50 (i.e., 50% 
inhibitory concentration), and 3) exhibit minimal binding to all or most 
normal tissues. These are the attributes that are believed to be most 
important in the formation of immunotoxins in accordance with the present 
invention. 
The inventors have identified certain cell surface antigens that are 
associated with Hodgkin's disease or Reed-Sternberg cells which appear to 
provide particularly useful monoclonal antibodies in the foregoing 
regards. For example, antigens which have found particular utility in 
providing antibodies having particularly desirable attributes are the CD30 
antigen complex and the 70 kd Hodgkin and/or Reed-Sternberg associated 
antigen. It should be appreciated, though, that the present invention 
contemplates that other Hodgkin or Reed-Sternberg associated antigens or 
epitopes can be employed in connection with the practice of the invention 
so long as antibodies against such or other antigens or epitopes exhibit 
the desired binding capacity, tissue selectivity and killing capability 
when formed into an immunotoxin. 
The CD30 Antigen Complex 
An important finding of the inventors is that CD30 immunotoxins directed 
against the Ki-1 antigen on Hodgkin cells may be identified which have 
high potency and specificity of cytotoxic effect and sufficiently 
restricted binding to normal human tissues that they are candidates for 
the treatment of Hodgkin's disease in man. 
The Ki-1 antigen (CD30) was first described by Schwab et al., 1982 (23). It 
is composed of two nonreducible subunits of 105 and 120 kDa molecular 
weight (24). The antibody, which was raised against the Hodgkin cell line 
L428 (25), was originally thought to be specific for Hodgkin and 
Reed-Sternberg cells. It has since been demonstrated to be present on 
anaplastic large cell lymphomas (26), peripheral T cell lymphomas (26), 
cutaneous lymphoid infiltrates (27) and tumor cells of embryonal carcinoma 
(28). The Ki-1 antigen is not expressed on resting mature or precursor B 
or T cells, but it can be induced on these cells by PHA, HTLVl and IL-1 or 
Epstein-Barr virus (EBV). 
Since such induction is accompanied by the expression of other activation 
markers such as HLA-DR, transferrin receptor and Il-2 receptor it was 
concluded that Ki-1 identifies both activated normal T- and B-lymphocytes 
and lymphomas derived from such cells (26). Because the Ki-1 antigen is 
expressed on all cases of Hodgkin's disease apart from the 
lymphocyte-predominant subtype (27), and has very limited expression on 
normal tissue (26), it appears to be a good target for immunotherapy. One 
of the five CD30 antibodies tested in this study (HRS-4) exhibited a 
strong crossactivity with a vital organ (pancreas) that would reduce its 
clinical usefulness as an immunotoxin. Thus, it is now clear that certain 
CD30 immunotoxins are to be preferred over others. 
The preparation of monoclonal antibodies against the CD30 complex can be 
achieved in a number of fashions. Typically and most readily, one employs 
a Hodgkin cell line such as the L428 or L540 line, and immunizes a 
selected mammal with the Hodgkin disease cells until an adequate immune 
response to the cells is obtained. Spleen cells from the immunized mammals 
are then employed in the preparation of hybridomas. Once a hybridoma bank 
has been obtained, one will desire to screen the bank to identify colonies 
which secrete antibodies having the desired pharmacokinetics, including 
binding strength (Kd), ability to form highly toxic immunotoxins 
IC.sub.50), as well as tumor cell selectivity. For most purposes in 
accordance with the present invention, the inventors propose that useful 
monoclonal antibodies will be characterized by Kd of at least about 200 nM 
for L540 or L428 cells, and even more preferably less than about 40 or 
even 20 nM. The most preferred antibodies will have a binding affinity of 
between about 7 and about 27 nM for L540 cells, or even lower. 
As discussed in more detail in the Examples herein-below, the inventors 
have identified certain monoclonal antibodies which have particularly 
desirable pharmacologic characteristics in terms of the criteria discussed 
above. The most preferred anti-CD30 antibodies identified exhibit very low 
Kds, very low IC.sub.50 s when incorporated into toxin A chain 
immunotoxins, and exhibit very low binding to non-tumor tissues. The most 
preferred of the anti-CD30 monoclonal antibody is known as HRS-3, and was 
originally developed by Dr. Michael Pfreundshuh. Hybridomas secreting 
HRS-3 have been deposited with the PHLS Centre for Applied Microbiology 
and Research and accorded accession No. 89111607. 
While one may find particular benefit through the use of hybridomas which 
secrete HRS-3, other antibodies against the CD30 complex can be employed 
and nevertheless obtain useful results in accordance herewith. Moreover, 
one may desire to use the HRS-3 antibody itself as a means of identifying 
other useful antibodies. For example, a cross-blocking technique is set 
forth hereinbelow which will allow the identification and selection of 
antibodies which have the same or similar binding specificity as HRS-3. 
Thus, one may find some benefit in employing such a cross-blocking assay 
in the initial screening of hybri-doma clones to identify those which 
secrete antibodies capable of competing with the HRS-3 or other antibodies 
found to be useful in accordance herewith. Such other antibodies may then 
be further screened to identify those having additional useful and 
desirable attributes such as high binding capability and selectivity for 
Hodgkin cells, ability to form highly toxic immunotoxins, hybridoma 
stability, ability to secrete large amounts of antibody, stability of the 
antibody and resultant immunotoxin, ability to give Fab' fragments in good 
yield, and the like. 
The 70 kDa Antigen 
Another Hodgkin cell determinant has been identified which, in addition to 
the CD30 complex, has been found by the inventors to be particularly 
useful in the preparation of anti-Hodgkin or Reed-Sternberg cell 
immunotoxins. This antigen has been identified as a 60 or 70 kd Hodgkin 
cell surface antigen by Drs. P-L and S-M Hsu, who first charac-terized the 
antigen through the preparation of monoclonal antibodies having binding 
specificity for Hodgkin mono-nuclear cells and Reed-Sternberg cells (47, 
48). One antibody developed by these individuals has been designated as 
"IRac" based on its ability to recognize the 70 kd antigen, which has been 
found to also be associated with interdigitating reticulum ("IR") cells. 
In contrast to the HRS series antibodies which were developed through the 
use of Hodgkin disease cell lines, the IRac antibody was developed through 
the use of Hodgkin disease tissue samples from Hodgkin disease patients. 
In particular, the IRac antibody was developed by immunizing mice with 
TPA-induced Hodgkin/Reed-Sternberg cells. The cells were obtained from 
surgical specimens from either lymph node or spleen of Hodgkin's disease 
patients which had been diagnosed based on established criteria. The 
sterile tumors were minced and filtered through a nylon mesh and the cells 
collected by Ficoll-Hypaque gradient centrifugation. The cells collected 
from the gradient were suspended in an appropriate medium (e.g., RPMI 1640 
medium containing 10% fetal calf serum) and enriched by 
complement-mediated cytolysis of contaminating T cells, B cells and 
monocytes (60), followed by another round of centrifugation on 
Ficoll-Hypaque. Cells enriched in this manner were then cultured at about 
4.times.10.sup.5 to about 2.times.10.sup.6 cells/ml in RPMI 1640 medium 
(Gibco) supplemented with 10% fetal calf serum, 2 mM glutamine, 50 uM 
2-mercaptoethanol, and 50 ug/ml gentamycin at 37.degree. C. in a 
humidified, 5% CO2 atmosphere. The developers of IRac have indicated that 
these cultures could be maintained for up to 7 days, with cell viability 
ranging from 70 to 80%. 
For antigen induction, TPA was dissolved in DMSO at about 14 ug/ml and 
added to the above cell cultures at a final concentration of about 2 
ng/ml, with fresh TPA containing medium being added about every second 
day. The induction was carried out for 3 days, and its effect monitored by 
immunocytochemical staining with anti-CD30 and anti-2H9 on cytospin 
smears. Successful induction was judged by the loss of CD30 and 2H9 from 
cell membranes, as well as cytologic changes, as evidenced by a decrease 
in the nuclear/cytoplasmic rations, increased size and number of 
cytoplasmic projections, as well as decreased cell proliferation. For 
generation of the IRac monoclonal, the procedure described for the 
generation of HeFi-1 and anti-2H9 was followed (61, 62). For intra-splenic 
immunization, a total of about 1.times.10.sup.7 Hodgkin's disease-derived 
cells in 0.5 ml of RPMI medium was injected directly into the spleen of 
BALB/c mice, with a booster injection being given 21 days later, about 3-4 
days prior to hybridization. 
To screen for reactivities of monoclonal antibodies produced in the 
foregoing manner, the avidin-biotin-peroxidase (ABC)-immunoperoxidase 
technique was used on frozen sections of normal lymphoid tissue on 
cytospin smears of TPA-induced Hodgkin/Reed-Sternberg cells. Briefly, 
sections (or smears) were fixed in acetone at room temperature for 5 
minutes. After being washed in Tris-buffered saline (TBS), 0.01M, pH 7.6, 
the sections or smears were incubated with hybridoma culture supernatant, 
and then with biotin-labeled horse anti-mouse Ig (1:200) and ABC. Each 
incubation lasted about 30-60 minutes, with an interval of about 5 minutes 
for washing with TBS. The slides were developed in a DAB-H.sub.2 O.sub.2 
-NiCl.sub.2 solution (63). Antibodies which had selective reactivity with 
the Hodgkin/Reed-Sternberg cells, but not with normal lymphoid cells, were 
subcloned and selected for further analysis. 
Thus, the foregoing technique presents an alternative to the use of Hodgkin 
disease cell lines in the preparation of useful Hodgkin's disease cell 
directed monoclonal antibodies. The IRac antibody prepared through the 
application of the foregoing techniques has been deposited with PHLS 
Centre and accorded accession number 89111608. Thus, as with the anti-CD30 
antibodies, and the HRS-3 antibody in particular, one might find 
particular benefit in the application of screening steps directed to the 
identification of antibodies which are capable of cross-blocking the 
binding of IRac for its epitope upon the 70 kd antigen species identified 
by Hsu et al. 
Thus, for such studies, one might, e.g., wish to test hybridoma fluids for 
the ability of antibodies therein that effectively block the binding of 
IRac (or HRS-3, etc.) antibody for its epitope on target cells. A useful 
technique for measuring the cross-blocking ability is set forth 
hereinbelow in Examples I and II. Those hybridomas secreting antibodies 
capable of cross-blocking either HRS-3 or IRac to at least about 70%, or 
more preferably 80 or even greater than 90%, will be particularly 
preferred for uses in accordance herewith, assuming that such antibodies 
meet the additional preferred criteria of non-reactivity with most normal 
tissues, high binding affinities (i.e., low Kd) and high cytotoxicity when 
conjugated with toxin moieties. 
Preparation of Immunotoxins 
While the preparation of immunotoxins per se is, in general, well known in 
the art (see, e.g., U.S. Pat. Nos. 4,340,535, and EP 44167, both 
incorporated herein by reference), the inventors are aware that certain 
advantages may be achieved through the application of certain preferred 
technology, both in the preparation of the immunotoxins and in their 
purification for subsequent clinical administration. For example, while 
IgG based immunotoxins will typically exhibit better binding capability 
and slower blood clearance than their Fab' counterparts, Fab' 
fragment-based immunotoxins will generally exhibit better tissue 
penetrating capability as compared to IgG based immunotoxins. 
Additionally, while numerous types of disulfide-bond containing linkers are 
known which can successfully be employed to conjugate the toxin moiety 
with the binding ligand, certain linkers will generally be preferred over 
other linkers, based on differing pharmacologic characteristics and 
capabilities. For example, the inventors have discovered that linkers 
which contain a disulfide bond that is sterically "hindered" are to be 
preferred, due to their greater stability in vivo, thus preventing release 
of the toxin moiety prior to binding at the site of action. Furthermore, 
while certain advantages in accordance with the invention will be realized 
through the use of any of a number of toxin moieties, the inventors have 
found that the use of ricin A chain, and even more preferably 
deglycosylated A chain, will provide particular benefits. 
The most preferred toxin moiety for use in connection with the invention is 
toxin A chain which has been treated to modify or remove carbohydrate 
residues, so called deglycosylated A chain. The inventors have had the 
best success through the use of deglycosylated ricin A chain (dgA) which 
is now 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 (64), 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 ligand region is an important aspect of the invention. As 
discussed in the Summary section, 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 ligand once the ligand 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 
ligand). 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 cross-linker, 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 
ligand. The SMPT cross-linking reagent, as with many other known 
cross-linking reagents, lends the ability to cross-link 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 (53). The use of such 
cross-linkers is well understood in the art. 
Once conjugated, it will be important to purify the conjugate so as to 
remove contaminants such as unconjugated A chain or binding ligand. It is 
important to remove unconjugated A chain because of the possibility of 
increased toxicity. Moreover, it is important to remove unconjugated 
binding ligand to avoid the possibility of competition for the antigen 
between conjugated and unconjugated species. In any event, a number of 
purification techniques are disclosed in the Examples below which have 
been found to provide conjugates to a sufficient degree of purity to 
render them clinically useful. In general, the most preferred technique 
will incorporate the use of Blue-Sepharose with a gel filtration or gel 
permeation step. Blue-Sepharose is a column matrix composed of Cibacron 
Blue 3GA and agarose, which has been found to be useful in the 
purification of immunoconjugates (69). The use of Blue-Sepharose combines 
the properties of ion exchange with A chain binding to provide good 
separation of conjugated from unconjugated binding. 
The Blue-Sepharose allows the elimination of the free (non conjugated) 
binding ligand (e.g., the antibody or fragment) from the conjugate 
preparation. To eliminate the free (unconjugated) toxin (e.g., dgA) a 
molecular exclusion chromatography step is preferred using either 
conventional gel filtration procedure or high performance liquid 
chromatography. 
After a sufficiently purified conjugate has been prepared, one will desire 
to prepare it into a pharmaceutical composition that may be administered 
parenterally. This is done by using for the last purification step a 
medium with a suitable pharmaceutical composition. 
Suitable pharmaceutical compositions in accordance with the invention will 
generally comprise from about 10 to about 100 mg of the desired 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. Such formulations will 
typically include buffers such as phosphate buffered saline (PBS), or 
additional additives such as pharmaceutical excipients, stabilizing agents 
such as BSA or HSA, or salts such as sodium chloride. For parenteral 
administration it is generally desirable to further render such 
compositions pharmaceutically acceptable by insuring their sterility, 
non-immunogenicity and non-pyrogenicity. Such techniques are generally 
well known in the art as exemplified by reference 70. It should be 
appreciated that endotoxin contamination should be kept minimally at a 
safe level, for example, less that 0.5 ng/mg protein. Moreover, for human 
administration, preparations should meet sterility, pyrogenicity, general 
safety and purity standards as required by FDA Office of Biologics 
standards. 
A preferred parenteral formulation of the HRS-3.dgA or IRac.dgA is 0.25 to 
0.5 mg conjugate/ml in 0.15M NaCl aqueous solution at pH 7.5 to 9.0. A 
preferred parenteral formulation of the HRS-3 Fab'.dgA is 1.0 to 2.5 mg/ml 
in 0.15M NaCl aqueous solution at pH 7.5. The preparations may be stored 
frozen at -10.degree. C. to -70.degree. C. for at least 1 year. 
The following examples are representative of techniques employed by the 
inventors in the preparation and use of exemplary and preferred 
immunotoxins for use in the treatment of Hodgkin's disease and other 
conditions involving activated lymphocytes or neoplastic histiocytes or 
lymphoid cells. It should be appreciated that while these techniques are 
exemplary of preferred embodiments for the practice of the invention, 
those of skill in the art, in light of the present disclosure, will 
recognize that numerous modifications can be made without departing from 
the spirit and intended scope of the invention. 
EXAMPLE I 
Evaluation of Ricin A-chain Containing Immunotoxins Directed Against CD30 
I. Materials and Methods 
A. Materials 
Tissue culture medium RPMI 1640 and fetal calf serum were purchased from 
Gibco-Biocult Ltd. (Paisley, Scotland). Blue Sepharose CL-6B, Sepharose 
G25 (fine grade), Staphylococcal protein A-Sepharose, DEAE Sepharose and 
Sephacryl S-200 HR were obtained from Pharmacia Ltd. (Milton Keynes, 
England). Carrier free [.sup.125 I] Iodine and L-[4,5-.sup.3 H] leucine 
(TRK 170) were purchased from Amersham International (Amersham, England). 
IODO-GEN was from Pierce Ltd. (Chester, England). Pepsin was from Sigma 
(Poole, England). Silicone fluids Dow Corning 200/1CS, 5CS and 550 were 
purchased from Dow Corning Corp. (Midland, USA). 
B. Cells 
The cell line L540, which derived from a patient with Hodgkin's disease 
(25) was maintained in RPMI 1640 supplemented with 20% (v/v) fetal calf 
serum, 4 mM L-glutamine, 200 U/ml penicillin and 100 ug/ml streptomycin. 
C. Preparation of dgA 
The ricin A-chain was purified by the method of Fulton et al. (29). 
Deglycosylated ricin A was prepared as described by Thorpe et al. (30). 
For conjugation with antibodies or Fab' fragments, the A-chain was reduced 
with 5 mM DTT and subsequently separated from DTT by gel filtration on a 
column of Sephadex G25 in PBS, pH 7.5. 
D. Antibodies 
Five monoclonal antibodies which recognize the Ki-1 antigen (CD30) were 
used in this study: HRS-1, HRS-3, HRS-4, Ber-H2 and Ki-1, of which the 
preparation of HRS-1, Ber-H2 and Ki-1 have been previously described 
(31,32,23). The characteristics of these antibodies are summarized in 
Table 1. HRS-1, Ber-H2 and Ki-1 were separated from the ascitic fluid of 
hybridoma bearing BALB/c mice by affinity chromatography on Staphylococcal 
protein A-Sepharose. HRS-3 and HRS-4 were purified from ascitic fluid by 
ammonium sulfate precipitation and ion exchange chromatography on 
DEAE-Sepharose. 
MRC OX7, a mouse IgGl monoclonal antibody recognizing the Thy-1.1 antigen, 
was prepared from ascitic fluid as described by Mason and Williams (33) 
and was used as a nonspecific control antibody. The antibody preparations 
were &gt;90% pure when analyzed by SDS-PAGE. 
The preparation of the HRS-3 antibody proceeded in a manner similar in many 
respects to that of HRS-1 and HRS-2 described in reference 31, with a few 
modifications. BALB/c mice (4-6 weeks old) were immunized against HD cell 
line L540 cells according to the protocol referred to in reference 31, and 
the resultant lymphoid cells were fused with X63Aj8.635 cells. The fusion 
products were selected by standard techniques, and about 1114 positive 
growing clones were obtained. The supernatants of these clones were 
initially screened to identify those reactive with surface antigens (POK) 
and intracellular (APAAP) antigens, and from this screening 116 clones 
were selected for further screening. After histological screening to 
exclude those reactive with tonsils, only about 37 candidates remained. A 
second screening was then undertaken using other Hodgkin's disease cell 
lines as targets, after which 6 candidates remained. Of these 6 clones, 
two clones, designated HRS-3 and HRS-4, were characterized and employed in 
further studies. 
TABLE I 
______________________________________ 
Characteristics of CD 30 Antibodies 
Antibody 
Subclass Immunogen Reference 
______________________________________ 
HRS-1 IgG2a L 428 Pfreundschuh et al, 1988 
HRS-3 IgG1 L 540 Pfreundschuh et al, 1988 
(Unpublished) 
HRS-4 IgG1 L 540 Pfreundschuh et al, 1988 
(Unpublished) 
Ber-H2 IgG1 L 428 Schwarting et al, 1987 
Ki-1 IgG3 L 428 Schwab et al, 1982 
______________________________________ 
E. Preparation of Fab' fragments 
HRS-3 and HRS-4 were dialysed into 0.1 M citrate buffer, pH 8.0, and 
concentrated by ultrafiltration (Amicon, PM10 membrane) to 7.5 mg/ml. The 
pH was reduced to 3.7 by addition of 1M citric acid and the antibody 
solutions were subsequently incubated for four hours at 37.degree. C. with 
pepsin (enzyme:protein ratio, 1/6 by weight). The digestion was terminated 
by raising the pH to 8.0 with 1M Tris buffer. The F(ab').sub.2 fragments 
were isolated by gel filtration on columns of Sephacryl S-200HR 
equilibrated in PBS, pH 7.5. F(ab').sub.2 fragments were reduced to Fab' 
monomers with 1-5 mM DTT. Residual DTT was removed by gel filtration on 
Sephadex G-25. 
F. Preparation of Immunotoxins 
IgG immunotoxins were prepared using the SMPT linker as described by Thorpe 
et al. (34). Briefly, SMPT dissolved in DMF was added to the antibody 
solution (7.5 mg/ml in borate buffer, pH 9.0) to give a final 
concentration of 0.11 mM. After 1 hr the derivatized protein was separated 
from unreacted material by gel chromatography on a Sephadex G25 column and 
mixed with freshly reduced ricin A-chain. The solution was concentrated to 
about 3 mg/ml and allowed to react for 3 days. Residual disulfide groups 
were inactivated by treating the immunotoxin with 0.2 mM cysteine for 6 
hours. The solution was then filtered through a Sephacryl S200 HR column 
in 0.1M Phosphate buffer, pH 7.5 to remove unreacted ricin A, cysteine and 
aggregates. Finally, the immunotoxin was separated from free antibody by 
chromatography on a Blue Sepharose CL-6B column equilibrated in 0.1M 
sodium phosphate buffer, pH 7.5, according to the method of Knowles and 
Thorpe (24). 
Fab' immunotoxins were prepared according to Ghetie et al. (35). Briefly, 
Fab' fragments (5 mg/ml in 0.1 M sodium phosphate buffer, pH 7.5 
containing 1 mM EDTA) were derivatized with DTNB (Ellman's reagent) at a 
final concentration of 2 mM. Unreacted DTNB was removed by gel filtration 
on a Sephadex G25 column equilibrated in PBS. The derivatized Fab' 
fragments which contained 1-2 activated disulfide groups were allowed to 
react with a 1.5 fold molar excess of freshly reduced A-chain for 2 hours 
at room temperature. The Fab'.dgA immunotoxins were subsequently purified 
on Sephacryl S200 HR and Blue Sepharose columns as described for IgG 
immunotoxins. 
The A-chain component of all the immunotoxins fully retained its ability to 
inhibit protein synthesis in rabbit reticulocyte lysates (36) after the 
A-chain had been released from the immunotoxins by reduction with DTT. 
G. Radioiodination 
Monoclonal antibodies were labeled with carrier free .sup.125 I using the 
IODO-GEN reagent to a specific activity of approximately 1 uCi/ug as 
described (37). Briefly, 500-750 ug of antibody in 100 ul borate buffer 
were incubated with 0.7 mCi of Na .sup.125 I in glass tubes coated with 8 
ug of IODO-GEN for 10 minutes at room temperature. Free iodide was removed 
by gel chromatography on a Pharmacia PD 10 column. The radioiodinated 
antibodies fully retained their capacity to bind to L540 cells, as shown 
by their ability to compete equally with unlabeled antibodies for binding 
to cell antigens when applied at saturating concentrations (33). 
H. Crossblocking Experiments 
Triplicate samples of L540 cells (5.times.10.sup.6 cells/ml, 100 ul) in PBS 
containing 2 mg/ml BSA and 0.2% (w/v) NaN.sub.3 (PBS/BSA/N.sub.3.sup.-) 
were mixed with .sup.125 I-labeled antibody (ug/ml, 100 ul) and a 100 fold 
excess of various unlabeled antibodies. The samples were then incubated 
for 30 minutes at 4.degree. C. The cells were washed three times with PBS 
and the radioactivity of the pellet was measured using a Packard 
Multi-Prias 1 gamma counter. Blocking of labeled antibody was calculated 
as follows: 
##EQU1## 
I. Scatchard Analysis 
.sup.125 I-labeled antibody (50 ul) at various concentrations (0.25-32 
ug/ml) was mixed for one hour at 4.degree. C. with L540 cells 
(2.times.10.sup.6 cells/ml, 50 ul) in PBS/BSA/N.sub.3.sup.-. The cells 
were separated from the supernatant by centrifugation (12,000 x g, 1 min) 
through 75 ul of a mixture of 8.8% (v/v) Dow Corning silicone fluid 
200/1CS, 7.2% 200/5CS and 84% Dow Corning 550. The Eppendorf tubes were 
then snap-frozen and the tips containing the cell pellets were cut off. 
The radioactivity in the cell pellet and in the supernatant were measured. 
The amount of radiolabeled nonspecific OX7 antibody that bound to the 
cells under identical conditions was substracted from the total amount of 
radiolabeled specific antibody that was associated with the cells to 
obtain the amount of specific antibody that was attached to cell antigens. 
The dissociation constant (K.sub.d) and the number of molecules of 
antibody bound per cell under equilibrium conditions were calculated by 
analysing the data according to the method of Scatchard (38). 
J. FACS Analyses 
L540 cells (10.sup.6 cells/ml, 100 ul) in PBS/BSA/N.sub.3.sup.- were 
incubated in the cells of a rigid u-bottomed 96 well microtitre plate for 
15 minutes at 4.degree. C. with antibodies, Fab' fragments or immunotoxins 
(100 ul) at concentrations ranging from 0.001 to 100 ug/ml. The cells were 
washed three times with PBS/BSA/N.sub.3.sup.- and were treated with 
FITC-labeled goat anti-mouse immunoglobulin (30 ug/ml, 100 ul) in 
PBS/BSA/N.sub.3.sup.- for 15 minutes. The cells were then again washed 
three times with PBS/BSA/N.sub.3.sup.- and analyzed on a FACS IV 
(Becton-Dickinson, Oxnard, USA). The molar concentrations of antibody and 
immunotoxin which gave 50% of the maximal fluorescence (i.e., under 
saturating conditions) were determined. 
K. Cytotoxicity Assays 
L540 cells suspended at 4.times.10.sup.5 cells/ml in complete medium were 
distributed in 100 ul volume into the wells of 96-well flat-bottomed 
microtitre plates. Immunotoxins in the same medium were added (100 
ul/well) and the plates were incubated for 24 h at 37.degree. C. in an 
atmosphere of 5% CO.sub.2 in humidified air. After 24 hours, the cells 
were pulsed with 1 uCi [.sup.3 H]-leucine for another 24 hours. The cells 
were then harvested onto glass fibre filters using a Titertek cell 
harvester and the radioactivity on the filters was measured using a liquid 
scintillation spectrometer (LKB, Rackbeta). The percent reduction in 
[.sup.3 H]-leucine incorporation, as compared with untreated control 
cultures, was used as the assessment of killing (35). 
L. Immunoperoxidase Staining of Human Tissues 
Cryostat sections of normal human tissues were treated with antibodies and 
stained using the ABC immunoperoxidase method, the three layer 
immunoperoxidase method or the APAAP technique that have been described in 
detail elsewhere (23,26,31,39). 
II. Results 
A. Crossblocking of CD30 MoAbs 
These studies were conducted to determine whether the five antibodies 
recognize the same or different epitopes on the CD30 antigen. The results 
are shown in FIG. 1. HRS-1, HRS-3, HRS-4 and Ber-H2 cross-blocked each 
others' binding to L540 cells but were not blocked by Ki-1. HRS-1 was 
slightly less effective at blocking HRS-3, HRS-4 and Ber-H2 binding than 
were HRS-3, HRS-4 and Ber-H2 at blocking each others' binding, probably 
because it has lower affinity. Ki-1 was only blocked by itself and did not 
block the other four antibodies. 
Thus, there appear to exist at least two epitopes on the CD30 antigen, one 
of which is recognized by HRS-1, HRS-3, HRS-4 and Ber-H2 and the other by 
Ki-1. 
B. Scatchard Analyses of the Binding of Intact Antibodies and Fab' 
Fragments 
Table 2 summarizes the results of the Scatchard analyses of the binding of 
the five intact antibodies and the two Fab' fragments tested. HRS-4 had 
the highest avidity for L540 cells (K.sub.d =7 nM). Ber-H2 and HRS-3 had 
the next highest avidity with K.sub.d values of 14 nM and 15 nM 
respectively. The most weakly binding antibodies were HRS-1 and Ki-1 which 
had K.sub.d values of 160 nM and 380 nM respectively. 
TABLE II 
______________________________________ 
Scatchard Analyses of CD 30 
Antibodies and Fab' Fragments 
Antibody K.sub.d (nM .+-. sd).sup.1) 
No. of molecules at saturation 
______________________________________ 
HRS-1 160 .+-. 40 1.6 .+-. 0.3 .times. 10.sup.6 
HRS-3 15 .+-. 3 1.7 .+-. 0.4 .times. 10.sup.6 
HRS-3Fab' 
27 .+-. 10 3.2 .+-. 0.5 .times. 10.sup.6 
HRS-4 7. .+-. 4 1.7 .+-. 0.2 .times. 10.sup.6 
HRS-4Fab' 
17 .+-. 3 2.8 .+-. 0.4 .times. 10.sup.6 
Ber-H2 14 .+-. 4 1.6 .+-. 0.3 .times. 10.sup.6 
Ki-1 380 .+-. 90 1.7 .+-. 0.2 .times. 10.sup.6 
______________________________________ 
.sup.1) Values of K.sub.d are the arithmetic mean and standard deviation 
of the results from 3 separate experiments 
The intact antibodies bound to L540 cells more avidly than their Fab' 
fragments. The difference was 1.8 fold for HRS-3 and 2.4 fold for HRS-4. 
This is due to the fact that intact antibodies can bind two antigens per 
cell, whereas the monovalent Fab' fragments bind to one antigen. In 
accordance with this, the number of Fab' molecules bound per L540 cell at 
saturation exceeds the number of intact antibodies by a factor of 1.6-2.0. 
The high absolute number of molecules bound (1.6-1.7.times.10.sup.6 for 
intact antibodies) may be because L540 cells are large, having 
approximately eight times the volume of, for example, human B lymphocytes. 
C. Cytofluorimetric Comparison of the Binding of Immunotoxins and Native 
Antibodies to L540 Cells 
The ability of the immunotoxins to bind to L540 cells was compared with 
that of the native antibodies and Fab' fragments using a cytofluorimetric 
assay. The concentrations of immunotoxin or antibody that gave half the 
maximal fluorescence are listed in Table III. These values allow the 
relative binding abilities of antibodies and immunotoxins to be compared 
(33) but are not themselves true measures of affinity/avidity because, 
unlike with the Scatchard analyses above, an indirect labeling technique 
is used. All the immunotoxins bound to L540 cells 1.6-3.8 fold more weakly 
than did their parental antibodies and Fab' fragments. The lower binding 
capacity of the immunotoxins can be explained either by steric hindrance 
by the A-chain moiety or by a loss of antibody affinity due to the 
chemical and physiochemical procedures used to prepare the immunotoxins. 
TABLE III 
______________________________________ 
Binding Capacity of CD 30 Antibodies and 
Immunotoxins Compared by Cytofluorimetric Analysis 
Concentration giving 50% 
maximal fluorescence (nM + sd) 
Unconjugated antibody 
Immunotoxin 
______________________________________ 
HRS-1 240 .+-. 140 900 .+-. 333 
HRS-3 3.0 .+-. 0.7 6 .+-. 1 
HRS-3Fab' 23 .+-. 9 57 .+-. 27 
HRS-4 1 .+-. 0.5 3 .+-. 1 
HRS-4Fab' 15 .+-. 6 33 .+-. 20 
Ber-H2 2.5 .+-. 1 4 .+-. 1 
Ki-1 310 .+-. 110 1000 .+-. 320 
______________________________________ 
D. Cytotoxicity of Immunotoxins to L540 Cells 
A representative cytotoxicity experiment is shown in FIG. 2 and the results 
of several experiments are summarized in Table IV. The IgG immunotoxins 
fall into two groups. The immunotoxins derived from the high affinity 
antibodies, HRS-3, HRS-4 and Ber-H2, were powerfully toxic and inhibited 
protein synthesis by L540 cells by 50% at concentrations (IC.sub.50) of 
0.9, 1.0 and 2.0.times.10.sup.-10 M respectively; by contrast, the 
immunotoxins derived from the low affinity antibodies, HRS-1 and Ki-1, 
were weakly effective with IC.sub.50 values of 0.8-1.0.times.10.sup.-8 M 
respectively. The potency of the two most powerful immunotoxins, HRS-3.dgA 
and HRS-4.dgA, was only 15-fold less than that of ricin itself. The 
cytotoxic effect of all the immunotoxins was specific since the native 
antibodies and OX7.dgA, an immunotoxin that does not bind to L540 cells, 
were not toxic at 10.sup.-6 M. 
The immunotoxins prepared from the Fab' fragments of HRS-3 and HRS-4 were 
also highly toxic to L540 cells, with IC.sub.50 values of 7.0 and 
3.0.times.10.sup.-10 M respectively. These immunotoxins were therefore 
only 7.8 and 3.0-fold less toxic, respectively, than their intact IgG.dgA 
counterparts. The lower activity of the Fab' immunotoxins is consistent 
with the findings of others (35,40,41) and is explained by the 
1.8-2.4-fold lower affinity of the monovalent Fab' fragments compared with 
their divalent IgG counterparts. 
TABLE IV 
______________________________________ 
Cytotoxicity of CD 30 Immunotoxins on L 540 Cells 
Material IC.sub.50 (M) No. of experiments 
______________________________________ 
HRS-1.dgA 8.0 .+-. 2.0 .times. 10.sup.-9 
3 
HRS-3.dgA 9.0 .+-. 0.8 .times. 10.sup.-11 
5 
HRS-3Fab'.dgA 
7.0 .+-. 1.5 .times. 10.sup.-10 
3 
HRS-4.dgA 1.0 .+-. 0.4 .times. 10.sup.-10 
7 
HRS-4Fab'.dgA 
3.0 .+-. 0.7 .times. 10.sup.-10 
3 
Ber-H2.dgA 2.0 .+-. 0.5 .times. 10.sup.-10 
3 
Ki-1.dgA 1.0 .+-. 0.5 .times. 10.sup.-8 
3 
Ricin 6.0 .+-. 2.0 .times. 10.sup.-12 
4 
Ricin A 8.0 .+-. 3.2 .times. 10.sup.-7 
2 
OX7.dgA &gt;1 .times. 10.sup.-6 
2 
______________________________________ 
E. Immunohistological Staining Pattern of Normal and Malignant Human Tissue 
As shown in Table V, the pattern of reactivity of the five CD30 antibodies 
was very similar with the exception of HRS-4 which unexpectedly stained 
normal pancreatic tissue. They all strongly stained Hodgkin's disease 
tissue although there was a tendency in the lymphocyte dominant subtype to 
give weaker staining. 
All the antibodies reacted with a few rare cells in the bone marrow, liver, 
lymph nodes, skin, spleen and thymus. These cells appeared to be large 
mononuclear cells in accordance with the finding that the antibodies stain 
activated lymphocytes (26). No staining was seen in the colon, kidney or 
lung. 
TABLE V 
______________________________________ 
Reactivity of CD 30 Antibodies with Normal 
and Malignant Cells of Various Tissues 
HRS-1 HRS-3 HRS-4 Ber-H2 
Ki-1 
______________________________________ 
Bone Marrow -/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
Colon - - - - - 
Kidney - - - - - 
Liver -/+.sup.b) 
-/+.sup.b) 
-/+.sup.b) 
-/+.sup.b) 
-/+.sup.b) 
Lung - n.d. n.d. - - 
Lymph Nodes and 
+.sup.c) 
+.sup.c) 
+.sup.c) 
+.sup.c) 
Tonsil 
Pancreas - - +++ - - 
Skin -/+.sup.d) 
-/+.sup.d 
-/+.sup.d) 
- - 
Spleen -/+.sup.e) 
-/+.sup.e) 
-/+.sup.e) 
-/+.sup.e) 
-/+.sup.e) 
Thymus -/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
-/+.sup.a) 
Breast carcinoma 
- - - - - 
Colon carcinoma 
- - - n.d. n.d. 
Hodgkin's disease 
+++ +++ +++ +++ +++ 
Lung carcinoma 
- - - n.d. n.d. 
Ovary carcinoma 
n.d. - - n.d. n.d. 
Pancreas carcinoma 
- - - n.d. n.d. 
Stomach carcinoma 
- - - n.d. n.d. 
Thyroid carcinoma 
n.d. - - n.d. n.d. 
______________________________________ 
-: no staining; +: weak staining; ++: moderate staining; +++: strong 
staining 
-/+: mixed staining pattern as follows: 
.sup.a) very few positive cells 
.sup.b) few positive Kupffer cell like cells 
.sup.c) few positive large cells around, between and at the inner rim of 
the follicular mantles 
.sup.d) few positive Histiocyte like cells 
.sup.e) few positive large cells in the white pulp 
The strong staining of pancreatic tissue by HRS-4 alone may preclude the 
use of this antibody for therapy. Evidently, antibodies that recognize the 
same epitope on the immunizing antigen differ in primary sequence in a way 
that can lead to spurious cross-reactivity with normal tissues. Similar 
unpredictable cross-reactivity has been observed with CD22 antibodies by 
Li et al. (42). 
III. DISCUSSION OF EXAMPLE I STUDIES 
The cross-blocking studies performed on the five CD30 antibodies set forth 
in the present example indicate that there are at least two epitopes on 
the CD30 antigen. One epitope is recognized by HRS-1, HRS-3, HRS-4 and 
Ber-H2 and the other is recognized by Ki-1. This accords with the findings 
of Schwarting et al. (32) who demonstrated by FACS analyses that Ki-1 and 
Ber-H2 recognize different epitopes and of Pfreundschuh et al. (31), who 
found no cross-blocking between HRS-1 and biotinylated Ki-1. 
HRS-3, HRS-4 and Ber-H2, which bound most strongly to L540 Hodgkin cells 
(K.sub.d =15 nM, 7 nM, 14 nM), formed the most potent IgG.dgA 
immunotoxins. All three immunotoxins killed 50% of L540 cells at 
0.9-2.0.times.10.sup.-10 M which is only 15-30 fold greater than is needed 
for an equivalent effect with ricin itself. HRS-1 which binds to the same 
epitope 11-23 fold more weakly was 40-90 times less active as an 
immunotoxin. Ki-1, which recognizes a different epitope and has a low 
affinity comparable to that of HRS-1, also yielded a relatively 
ineffective immunotoxin. Thus, it can be deduced that the affinity 
(avidity) of the CD30 antibodies rather than the epitope they recognize is 
the primary determinant of their potency as ricin A containing 
immunotoxins. Different conclusions about the importance of epitope 
location have been drawn from other studies. Shen et al. (43) concluded 
that both antibody affinity and epitope location determined the potency of 
CD22 immunotoxins. By contrast, Press et al. (44), in a study of three CD2 
immunotoxins, concluded that epitope location critically influenced 
immunotoxin potency: immunotoxins recognizing one epitope on the CD2 
molecule were rapidly transported to lysosomes and degraded, whereas an 
immunotoxin recognizing another epitope lying closer to the membrane 
remained in peripheral endocytic compartments and was powerfully toxic. 
The Fab' fragments of HRS-3 and HRS-4 yielded immunotoxins that were only 
7.8- and 3-fold less potent respectively than their IgG.dgA counterparts. 
Their lower activity can be explained by the fact that they can bind to 
only a single antigen on the cell surface and so bound 1.8-2.4 fold more 
weakly than their divalent counterparts. Fab' immunotoxins and IgG 
immunotoxins have different advantages that recommend their use for 
therapy. The stronger affinity, greater cytotoxic activity and longer half 
life in vivo are the major advantages of IgG immunotoxins over Fab' 
immunotoxins (43). The Fab' immunotoxins, on the other hand, may penetrate 
better into solid tumors (43) and have lower immunogenicity in man because 
they lack the relatively immunogenic Fc portion of the antibody (45). 
EXAMPLE II 
Antitumor Effects of Ricin A-Chain Immunotoxins Prepared from Intact 
Antibodies and Fab' Fragments on Solid Human Hodgkin's Disease Tumors in 
Mice 
I. Materials and Methods 
A. Materials 
Blue Sepharose CL-6B, Sepharose G25 (fine grade), Staphylococcal protein-A 
Sepharose, DEAE Sepharose and Sephacryl S-200 HR were obtained from 
Pharmacia Ltd. (Milton Keynes, England). Pepsin was purchased from Sigma 
(Poole, England). Tissue culture medium RPMI 1640 and fetal calf serum 
were from Gibco-Biocult Ltd. (Paisley, Scotland). Falcon tissue flasks 
were purchased from Becton Dickinson (Lincoln Park, USA). .sup.3 H-Leucine 
was obtained from Amersham International (Aylesbury, UK). 
B. cells 
The human Hodgkin's disease-derived cell line L540 (46) and the sublines 
which were obtained by reestablishing L540 tumors in culture were 
maintained in RPMI 1640 supplemented with 20% (v/v) fetal calf serum, 4 mM 
L-glutamine, 200 U/ml penicillin and 100 ug/ml streptomycin (`complete 
medium`). 
C. Antibodies 
The mouse monoclonal antibodies used in this study were HRS-3, Ber-H2 (32), 
and IRac (47,48). All are of the IgGI subclass. HRS-3 and Ber-H2 recognize 
the CD30 antigen which has been shown to be composed of two nonreducible 
subunits of 105 and 120 kDa antigen on Hodgkin and Reed-Sternberg cells 
(47). 
Ber-H2 and IRac were separated from the ascitic fluid of hybridoma-bearing 
BALB/c mice by affinity chromatography on Staphylococcal protein 
A-Sepharose. HRS-3 was purified by ammonium sulfate precipitation and ion 
exchange chromatography on DEAE-Sepharose. 
The mouse IgG1 monoclonal antibody MRC OX7 which recognizes the mouse Thy 
1.1 antigen (20) was used as a nonspecific control antibody. 
All the antibodies were more than 90% pure when analyzed by SDS-PAGE using 
the Pharmacia pharmphast system. 
D. Immunoperoxidase Staining of Human Tissues 
Cryostat sections of normal human tissues were treated with antibodies and 
stained using indirect immunofluorescence and immunoperoxidase techniques 
as described elsewhere (65). 
E. Crossblocking Experiments 
Radioiodination and cross-blocking experiments were performed as described 
in Example I. 
F. Preparation of Immunotoxins 
Deglycosylated ricin A-chain immunotoxins were prepared essentially as 
described in Example I. 
G. Cytotoxicity Assays 
Cytotoxicity assays were performed essentially as described in Example I. 
H. Mice 
The mice used in the treatment experiments are N:NIH outbred stocks 
carrying a different combination of genes: the nude (nu) gene from N:NIH 
background, the xid from CBA/N and the beige (bg) gene from C 57 BL/6N. 
These so called `triple beige` nudes (nu/nu/bg-xid) have a B-cell 
deficiency in addition to the NK and T cell defect known from beige nude 
mice (52). 
Monogamous pairs of homologous males and heterozygous females for the nu 
gene were mated. The offspring were weaned after 21 days. Four to six week 
old homologous females weighing of 18-22 grams were used for the 
experiments. 
I. Antitumor Experiments 
For the establishment of solid tumors, 2.5.times.10.sup.7 L540 cells in 200 
ul complete medium were injected subcutaneously (s.c.) into the right 
posterior gluteal region of the triple beige mice. Tumors usually became 
visible after 5-7 days in more than 90% of the animals injected and grew 
to 1 cm diameter, corresponding to a volume of approximately 700 mm.sup.3, 
within 30 days. Antitumor experiments were started when the tumors reached 
60-80 mm.sup.3, (approximately 0.5 cm diameter). Tumor diameters were 
recorded twice a week and the tumor volume was calculated as follows: 
##EQU2## 
Tumor bearing animals were randomly divided into groups of 8-10. 
Immunotoxins or antibodies were injected intravenously (i.v.) under 
sterile conditions into the tail vein in a volume of 200 ul PBS containing 
2 mg/ml BSA. The doses of immunotoxins that were administered represented 
the same proportion of the LD.sub.50 (about 40%) for both the intact 
antibody immunotoxins and the Fab' immunotoxins. The doses in terms of 
total protein were 48 ug for IgG immunotoxins and 206 ug for Fab' 
immunotoxins. This corresponds to 8 ug ricin A-chain for intact and 77.6 
ug A-chain for Fab' immunotoxins. The doses of unconjugated antibodies or 
Fab' matched those of the immunotoxins: 40 ug for intact Abs and 129 ug 
for Fab' fragments. 
The antitumor experiments were terminated 30 days after the animals were 
treated in order to keep tumor diameters less than 1.5 cm in accordance 
with British Home Office requirements. The antitumor effects of different 
treatments were compared by the `growth index` which is calculated by 
dividing the mean tumor volume per group at day 30 by the mean tumor 
volume per group at the day of treatment (day 1). The statistical 
significance of the treatment results was calculated by the student's t 
test. 
J. Establishment and Characterization of Recultures 
Tumors were removed under sterile conditions, rinsed in complete medium and 
finely minced with a scalpel. Tumor cell-containing medium was then 
carefully transferred into 25 ml Falcon tissue flasks with a syringe, and 
incubated in complete medium. When the cultures appeared to be homogenous 
for L540 cells (about 2 weeks later) the cells were retreated with 
immunotoxins in vitro (see cytotoxicity assays). Sublines that were less 
susceptible to the immunotoxins were checked for changes of antigen 
expression by FACS analysis. The technique used for the FACS analyses has 
been described in Example I. 
II. Results 
A. Crossblocking of HRS-3, Ber-H2 and IRac 
Crossblocking experiments showed that neither HRS-3 nor Ber-H2 blocked the 
binding of IRac to L540 cells and vice versa. (FIG. 3). This is consistent 
with the finding that IRac recognizes a 70 kd Mr antigen on Hodgkin cells, 
whereas HRS-3 and Ber-H2 recognize the 105/120 kd Ki-1 (CD30) antigen. 
HRS-3 and Ber-H2 completely crossblocked each other's binding and seem 
therefore to recognize the same or at least two closely linked epitopes on 
the CD30 antigen, as also shown in Example I. The fact that IRac binds to 
a different antigen from HRS-3 and Ber-H2 indicates that IRac.dg and 
either HRS-3.dgA or Ber-H2.dgA may be useful as a `cocktail` in vivo to 
maximize tumor cell kill. 
B. Staining of Normal Human and Hodgkin's Disease Tissue 
Immunoperoxidase staining of 28 different human tissues with HRS-3, Ber-H2 
and IRac revealed no major cross-reactivity (Table VI). HRS-3 and Ber-H2 
stained a few large lymphoid cells in the colon, lymph nodes, tonsils, and 
tissue of autoimmune thyroiditis. These cells were probably activated 
lymphocytes since CD30 is known to be expressed on such cells (26). IRac 
did not cross-react with any of the normal tissues tested as judged by the 
immunoperoxidase method. 
All three antibodies strongly bound Hodgkin's disease derived cell lines. 
When tested on sections of Hodgkin's disease tissue from 30 patients, 
HRS-3 and Ber-H2 bound to more than 90% of the cases and stained &gt;90% of 
those cells that could be morphologically identified as Hodgkin or 
Reed-Sternberg cells. By contrast, IRac preferentially stained nodular 
sclerosis and the mixed cellularity subtype and gave a more moderate 
labeling. 
TABLE VI 
______________________________________ 
Tissue Staining of Three Monoclonal 
Antibodies Recognizing HD/RS Cells 
Tissue HRS-3 Ber-H2 IRac 
______________________________________ 
Adrenal - - - 
Brain (cortex) - - - 
Brainstem - - - 
Breast - - - 
Cerebellum - - - 
Cervix - - - 
Colon -* -* - 
Gall bladder - - - 
Heart - - - 
Kidney - - - 
Liver - - - 
Lung - - - 
Lymph node -* -* - 
Mucosa (nasal) - - - 
Oesophagus - - - 
Ovary - - - 
Parathyroid - - - 
Prostate - - - 
Spleen - - - 
Stomach (antrum) 
- - - 
Stomach body - - - 
Testis - - - 
Thyroid - - - 
Thyroid (AI.sup. 1) 
-* -* - 
Thyroid (Hashimoto's) 
- - - 
Tonsils -* -* - 
Uterus - - - 
Vagina - - - 
Hodgkin's disease.sup.2 
+++ +++ (+++) 
______________________________________ 
.sup.1 autoimmune Thyroiditis 
.sup.2 Primary material and cell lines 
*rare cells within lymphoid tissue stain positively 
C. Cytotoxicity to L540 cells in vitro 
The most potent immunotoxin was that prepared from intact IRac antibody 
(Table VII). It had an IC.sub.50 of 1.times.10.sup.-11 M which is similar 
to ricin itself under the same experimental conditions. The next most 
potent immunotoxins were HRS-3.dgA and Ber-H2.dgA which were 9 times and 
20 times less effective than IRac.dgA, with IC.sub.50 values of 
9.times.10.sup.-11 and 2.times.10.sup.-10 M respectively. The IRac Fab' 
immunotoxin (IC.sub.50 =6.times.10 M) was 60 fold less potent than the 
intact IRac.dgA immunotoxin whereas the HRS-3 Fab'.dgA (IC.sub.50 
=7.times.10.sup.-10) was only 7.8 times less potent than the intact HRS-3 
immunotoxin. 
The cytotoxic effect of all the immunotoxins was specific since the native 
antibodies and OX7.dgA, an immunotoxin that does not bind to L540 cells, 
were not toxic at 10.sup.-6 M. 
TABLE VII 
______________________________________ 
Cytotoxicity of Immunotoxins in vitro 
IMMUNOTOXIN IC.sub.50 (M).sup.1 
______________________________________ 
Ber-H2.dgA 2.0 .+-. 0.5 .times. 10.sup.-10 
HRS-3.dgA 9.0 .+-. 0.8 .times. 10.sup.-11 
HRS-3Fab'dgA 7.0 .+-. 1.5 .times. 10.sup.-10 
IRac.dgA 1.0 .+-. 0.2 .times. 10.sup.-11 
IRacFab'.dgA 6.0 .+-. 1.2 .times. 10.sup.-10 
______________________________________ 
.sup.1 Average of at least 3 separate experiments .+-. standard deviation 
D. Antitumor Effects on Solid L540 Tumors in vivo 
1Intact Antibody Immunotoxins 
In Table VIII are listed the detailed results of a series of antitumor 
experiments in which HRS-3, Ber-H2 and IRac immunotoxins were administered 
at various doses to triple beige nude mice bearing solid L540 tumors of 
various dimensions. FIG. 4a shows a typical experiment in which intact 
antibody immunotoxins were administered to mice with tumors of 60-80 
mm.sup.3 (0.5 cm diameter). All three immunotoxins had impressive 
antitumor activity. IRac.dgA was the most powerful (growth index 0.8), 
followed by HRS-3.dgA (growth index 1.4) and then BerH2.dgA (growth index 
4.6). The difference in antitumor activity between IRac.dgA and HRS-3.dgA 
did not reach statistical significance whereas the difference between 
IRac.dgA and Ber-H2.dgA was statistically significant (P&lt;0.02). There were 
several complete remissions: 17/24 for IRac.dgA, 11/16 for HRS-3.dgA and 
3/8 for Ber-H2.dgA. Of these, five IRac.dgA- and four HRS-3.dgA-treated 
animals had relapses after 5-15 days. No relapses were observed after 20 
days of complete remission. In contrast, the tumors grew progressively in 
untreated animals (growth index 9.7) and in animals treated with an 
immunotoxin of irrelevant specificity, OX7.dgA (growth index 8.7). 
It is possible that part of the antitumor activity of the immunotoxins was 
mediated through the antibody component alone, since the native 
antibodies, when administered at doses equivalent to those in the 
immunotoxins, appeared slightly to retard tumor growth. The growth indices 
were 7.5 for HRS-3, 8.8 for Ber-H2 and 7.5 for IRac as compared with 9.7 
for the untreated control group. None of these differences was, however, 
statistically significant. In each group of eight mice treated, with the 
antibodies there was one lasting complete remission. 
TABLE VIII 
__________________________________________________________________________ 
Treatment of Solids L540 Tumors with Different Immunotoxins in Triple 
Beign Mice 
Average tumor size 
Dose (mm3) Growth index 1) 
Number of 
Response 
Treatment 
(ug Protein) 
Day 1 Day 30 
(day 30/day 1) 
mice treated 
CR Relapse 
PR or 
__________________________________________________________________________ 
NR 
PBS -- 69 667 9.7 .+-. 1.6 
.sup. 24 2) 
1 -- 23 
PBS -- 306 1854 5.8 .+-. 0.7 
8 -- -- 8 
IRac.dgA 48 15 0 0.0 .+-. 0.0 
10 10 -- -- 
IRac.dgA 48 79 65 0.8 .+-. 0.2 
.sup. 24 2) 
12 5 7 
IRac.dgA 48 407 360 0.9 .+-. 0.4 
8 1 2 5 
IRac 40 54 407 7.5 .+-. 1.5 
8 1 -- 7 
IRacFab'.dgA 
206 53 422 8.0 .+-. 2.9 
8 2 -- 6 
IRacFab' 129 75 796 10.6 .+-. 1.9 
8 -- -- 8 
HRS-3.dgA 
48 99 134 1.4 .+-. 0.5 
.sup. 16 3) 
7 4 5 
HRS-3 40 62 464 7.5 .+-. 1.8 
8 1 1 6 
HRS-3Fab'dgA 
206 70 189 2.7 .+-. 0.7 
8 2 3 3 
HRS-3Fab' 
129 82 651 7.9 .+-. 1.5 
8 -- -- 8 
Ber-H2.dgA 
48 63 291 4.6 .+-. 1.8 
8 3 -- 5 
Ber-H2 40 61 529 8.8 .+-. 1.7 
8 1 -- 7 
OX7.dgA 48 68 589 8.7 .+-. 1.1 
8 -- -- 8 
__________________________________________________________________________ 
1) arithmetic mean .+-. one standard error (s.e.) 
2) average of 3 separate experiments 
3) average of 2 separate experiments 
CR = complete remission 
PR = partial remission 
NR = no response 
2. Fab' Immunotoxins 
The Fab' immunotoxins were administered to the mice in doses that 
represented the same proportion of the LD.sub.50 (i.e., 40%) as for the 
intact antibody immunotoxins. The HRS-3 Fab' immunotoxin was only slightly 
less effective at inhibiting the growth of 60-80 mm.sup.3 (0.5 cm 
diameter) tumors than was the intact HRS-3 immunotoxin (Table VIII and 
FIG. 4c). The tumor growth index was 2.7 in the HRS-3 Fab'.dgA recipients 
as compared with 1.4 in the recipients of the corresponding intact 
antibody immunotoxins. This difference is not statistically significant 
(P&gt;0.05). The overall number of complete remissions was similar (5/8 for 
HRS-3 Fab'.dgA versus 11/16 for HRS-3.dgA, respectively), but there was a 
higher proportion of relapses in the HRS-3 Fab'.dgA treated group (3/8 
versus 4/16 respectively). 
In contrast, the IRac Fab' immunotoxin was substantially (P&lt;0.002) less 
effective than its intact antibody counterpart (growth index: 8.0 versus 
0.8; permanent CR: 2/8 versus 12/24). 
The Fab' fragments of HRS-3 and IRac alone had no antitumor effect (FIG. 
4d). 
E . Dependence of the Antitumor Effect on Tumor Size at the Time of 
Treatment 
To evaluate the effect of tumor size on the responsiveness to the 
treatment, the effects of IRac.dgA on the rate of tumor growth in groups 
of mice with small tumors (10-20 mm.sup.3 ; approx. 2 mm in diameter) when 
compared with groups with large tumors (400-600 mm.sup.3 ; approximately 1 
cm in diameter) (see FIG. 5). All animals with small tumors had lasting 
complete remissions. By contrast, only 3/8 animals with large tumors had 
complete remissions and, of these, 2 animals subsequently relapsed. 
F Emergence of Immunotoxin-Resistant Tumor Mutants in vivo 
Tumors from mice which had complete remissions after treatment with 
HRS-3.dgA or IRac.dgA but which subsequently relapsed were re-established 
in tissue culture and their immunotoxin-sensitivity was determined. All 
four sublines established from relapsed HRS-3.dgA recipients were as 
sensitive as the original L540 line to HRS-3.dgA and IRac.dgA. By 
contrast, three of four sublines that originated from relapsed IRac.dgA 
recipients were 40, 60 and 200 times less sensitive to IRac.dgA than the 
original L540 line (Table IX). The degree of resistance of the sublines to 
IRac.dgA correlated with the decrease in their ability to bind IRac 
antibody as measured by FACS analyses. The mean fluorescence intensity 
(MFI) of the sublines was 13%, 24% and 33% of the MFI of the parental L540 
line. 
These results indicate that the L540 tumor originally implanted into the 
mice contained a few IRac antigen-deficient mutants which were not killed 
by IRac.dgA and which after a period of complete remission regrew into 
solid tumors at the original tumor site. Importantly, the 
IRac.dgA-resistant sublines were approximately as sensitive to HRS-3.dgA 
as the original L540 line indicating that treatment of the mice with a 
cocktail of IRac.dgA and HRS-3.dgA would reduce the likelihood of mutant 
tumor cell escape. 
TABLE IX 
______________________________________ 
Characteristics of L540 Sublines Derived from IRac.dgA-treated 
Mice which had Complete Remissions but Subsequently Relapsed 
Resistant Immunotoxin sensitivity 
or Antigen density 
IC.sub.50 (M) 
Subline 
sensitive 
(% MFI.sup.1) 
IRac.dgA 
HRS-3.dgA 
______________________________________ 
1 Sensitive 
87 2 .times. 10.sup.-11 
1 .times. 10.sup.-10 
2 Resistant 
33 4 .times. 10.sup.-10 
6 .times. 10.sup.-11 
3 Resistant 
24 6 .times. 10.sup.-10 
1 .times. 10.sup.-10 
4 Resistant 
13 2 .times. 10.sup.-9.sup. 
4 .times. 10.sup.-10 
L540 Sensitive 
100 1 .times. 10.sup.-11 
9 .times. 10.sup.-11 
______________________________________ 
.sup.1) MFI of the sublines expressed as percentage of the MFI of origina 
L540 cells stained with IRac at saturating concentrations 
In a further study, tumors from mice which showed relatively little 
response (i.e., did not reach CR) after IRac.dgA or HRS-3.dgA therapy were 
re-established in tissue culture and their immunotoxin sensitivity was 
determined. All three sublines derived from HRS-3.dgA recipients and both 
sublines derived from IRac.dgA recipients were as sensitive to 
immunotoxins as the original L540 line. Thus, the relatively poor 
responsiveness of these tumors to immunotoxin therapy was not because the 
tumor cells themselves were resistant to the immunotoxins. 
III. DISCUSSION OF EXAMPLE II STUDIES 
The major findings to emerge from this study were: i) a single intravenous 
injection of the intact immunotoxins, HRS-3.dgA or IRac.dgA, cured up to 
44-50% of mice with solid Hodgkin tumors of 60-80 mm.sup.3 size; ii) the 
HRS-3 Fab' immunotoxin was slightly less potent in vitro and in vivo than 
the intact HRS-3 immunotoxin, whereas the IRac Fab' immunotoxin was much 
less potent compared with the intact IRac immunotoxin; iii) tumors that 
regrew after IRac.dgA treatment in mice consisted mainly of mutants with a 
reduced sensitivity to IRac.dgA but not to HRS-L 3.dgA; iv) since 
HRS-3.dgA (or HRS-3 Fab'.dgA) and IRac.dgA combine highly specific 
cytotoxicity in vitro, potent antitumor effects in vivo, little 
cross-reactivity with normal human tissue and recognize different 
antigens, they could be used as a cocktail for the treatment of patients 
with Hodgkin's disease. 
The immunotoxins used in this example exhibited surprisingly good antitumor 
effects in a solid Hodgkin's disease xenograft model. The growth index 
(ratio of tumor volume per group on day 30:day 1) was 0.8 for IRac.dgA, 
1.4 for HRS-3.dgA and 4.6 for Ber-H2.dgA as compared with 9.7 for 
untreated control animals. With IRac.dgA recipients, tumors of 
approximately 1 cm diameter were smaller on average 30 days after 
treatment than on the day of treatment. In addition, 100% of small (10-20 
mm.sup.3) tumors were destroyed by a single IRac.dgA injection, indicating 
the importance of tumor size on complete remission rates. Possible 
explanations for the high in vivo efficacy of the present immunotoxins are 
that deglycosylated ricin A-chain, the SMPT linker, and a final 
purification step on Blue Sepharose were employed when manufacturing the 
immunotoxin. These procedures enable the preparation of `second 
generation` immunotoxins that have higher purity, higher in vivo 
stability, and which avoid liver entrapment better than immunotoxins of 
the first generation, resulting in substantially improved antitumor 
activity in mouse tumor models. 
The intact HRS-3 and IRac immunotoxins had superior antitumor effects to 
their Fab' counterparts. With the HRS-3 immunotoxins the differences were 
small: HRS-3.dgA treatment resulted in lasting complete remissions in 7/16 
mice and a tumor growth index of 1.4 as compared with 2/8 mice and a tumor 
growth index of 2.7 in the recipients of HRS-3 Fab'.dgA. These differences 
were more marked with the IRac immunotoxins; treatment with IRac.dgA 
produced lasting complete remissions in 12/24 mice and a growth index of 
0.8 as compared with 2/8 mice and a growth index of 8.0 in the recipients 
of IRac Fab'.dgA. The difference in the degree of superiority of the two 
intact antibody immunotoxins over their Fab' counterparts correlated with 
their relative cytotoxic potency in vitro. The HRS-3 Fab'.dgA was only 7.8 
fold less potent at killing L540 cells in vitro than the intact antibody 
immunotoxin, whereas the IRac Fab'.dgA was 60-fold less potent. The higher 
cytotoxicity of IgG over Fab' immunotoxins in vitro is well established 
and due to the superior affinity of the bivalent intact immunotoxin. 
Several tumors that resisted immunotoxin treatment or which responded but 
subsequently regrew were reestablished in vitro and their sensitivity to 
immunotoxins was determined. The 5 sublines established from tumors that 
were resistant to HRS-3.dgA in mice were as sensitive to the immunotoxins 
as the original L540 line in vitro, suggesting that the amount of 
immunotoxin that reached the solid tumor was not sufficient to kill all 
sensitivity tumor cells. 
By contrast, three of four tumors that relapsed after IRac.dgA treatment 
were 40, 60 and 200 times less sensitive to IRac.dgA in vitro than the 
original L540 cells. The three tumors all had a reduced expression of the 
IRac antigen suggesting that the immunotoxin killed the majority of cells 
in the tumor with normal levels of antigen, leaving a few 
antigen-deficient mutants that later regrew into sizeable tumors. 
Importantly, the sublines were still sensitive to HRS-3.dgA. This strongly 
suggests that the problem of antigenic heterogeneity can be overcome, at 
least in part, by the administration of immunotoxin cocktails. 
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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