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BACKGROUND
Differences exist within the literature as to what constitutes the
important variables in the design of hybrid toxins with altered receptor
specificity. As originally conceived, the binding chain (B chain) of
diphtheria toxin or ricin was replaced with an alternate binding protein
modified to form a disulfide linkage with the toxin A chain. The
theoretical advantage of this scheme was that toxin A chains have
undetectable binding to cell membranes, and therefore the specificity of
the hybrid would be directed entirely by the new binding moiety. Initial
results with both diphtheria toxin and ricin A chain hybrids of peptide
hormones were disappointing; under conditions which tested selectivity
between target and non-target cells, potency of the hybrid relative to the
parent toxin was markedly reduced. Certain antibody-toxin A chain hybrids
showed good selectivity but were still less toxic than the parent toxin.
Subsequent discoveries showed the feasibility of altering the receptor
specificity of intact toxins such as ricin by attaching a new binding
moiety and inhibiting the usual ricin entry route by competition with
lactose; U.S. Ser. No. 186,735 filed Sept. 12, 1980; U.S. Ser. No. 199,781
filed Oct. 23, 1980; and U.S. Ser. No. 341,572 filed Jan. 21, 1982. These
hybrids in the presence of lactose were as toxic as ricin in the absence
of lactose when assayed after a three-hour exposure to cells. These
hybrids required a functional galactose binding site on the ricin B chain
for optimal specific toxicity. Recently, however, several A chain hybrids
have been reported which show a high degree of selectivity and are as
potent as the parent toxins when assayed after 18-26 hours in tissue
culture.
The present invention shows the rates of inactivation of protein synthesis
between 2 and 24 hours for two A chain hybrids with widely differing
affinities for the alternate receptor. The alternate binding proteins are
monoclonal antibodies which bind the Thy 1.1 antigen present on AKR cells.
Ricin B chain properties are studied by adding the B chain to mixtures of
the A chain hybrids and target cells.
The protein inhibition rate is much lower for Thy 1.1 ricin A chain hybrid
than for ricin alone. Additionally, the rate of inhibition by the hybrid
can be increased by adding excess ricin B chain with no change in the
amount of A chain bound to the target cells. In other words, ricin B chain
facilitates entry of the A chain into the cytosol compartment of the cell
by a process independent of the amount of A chain bound to the surface
membrane.
PRIOR ART
Neville, D. M., and R. Youle, Monoclonal Antibody-Ricin or Antibody-Ricin A
Chain Hybrids: Kinetic Analysis of Cell Killing for Tumor Therapy,
Immunology Reviews, Vol. 62, p 135, (1981).
Roitt, Ivan, Essential Immunology, 4th Edition, Boston, Mass.: Blackwell
Scientific Publications, 1980.
Youle, R. J., and D. M. Neville, Jr., Anti-Thy-1.2 Monoclonal Antibody
Linked to Ricin is a Potent Cell Type Specific Toxin, Proc. National
Academy of Science, USA, Vol. 77, p 5483 (1980).
Youle, R. J., G. T. Murray, and D. M. Neville, Jr., Studies on the
Galactose Binding Site of Ricin and the Hybrid Toxin Man-6P-Ricin, Cell,
Vol. 23, p. 551 (1981).
Vallera, Daniel A., Richad J. Youle, David M. Neville, Jr., John H. Kersey,
Bone Marrow Transplantation Across Major Histocompatibility Barriers, (in
press).
U.S. Pat. No. 4,359,459 (Neville et al.), Anti-Thy 1.2 Monoclonal
Antibody-Ricin Hybrid Utilized as a Tumor Suppressant.
U.S. Pat. No. 4,356,117 (Neville et al.), Chemical Modifications of
Proteins Which Induce New Receptor Specificities and Therefore Elicit New
Effects in Cells.
U.S. Pat. No. 4,397,843 (Neville et al.), Ricin and Modeccin Reagents
Effective as Tumor Suppressive Cytotoxic reagents (cip of Ser. No. 199,781
filed Oct. 23, 1980).
UTILITY STATEMENT
A method of utilizing protein target cells by mammalian cells which contain
Thy 1.1 antigen receptor sites. This is accomplished by incubating
anti-Thy 1.1-ricin A chain hybrids with target protein cells, such as AKR
mouse thymocytes and separately adding ricin B chain to the mixture. The
tumor suppressive cytotoxic effect is measured by normal pharmacological
tests.
METHODS AND MATERIAL
Ricin, known in the literature, was prepared as has been previously
reported in patent applications Ser. No. 199,781 (filed Oct. 23, 1980) and
Ser. No. 186,735 (filed Sept. 12, 1980).
Ricin B chain was prepared from ricin by the method of Olsnes and Pihl
(Biochemistry, Vol. 12, pp. 3121-3126, 1973), as modified by Cawley et al
(Arch. Biochem. Biophys., Vol. 190, pp. 744-755, 1973).
Ricin A chain was purified from ricin by the method of Cawley et al, Cell,
22:563-570, 1980.
Anti-Thy 1.1 monoclonal antibody, OX-7 was purchased from Accurate Chemical
and Scientific Co., Westburg, NY and Anti-Thy 1.1 monoclonal antibody
19E12 was obtained from hybridoma cells provided by R. C. Nowinski, grown
as acites in mice and purified over protein A Sepharose. F(ab).sub.2 was
prepared as described in Nisonoff et al, Nature, 189:293-295 (1961).
AKR-SL2 cells were also given by R. C. Nowinski. These hybridoma cells and
AKR-SL2 cells are available to scientific researchers on request.
Hybrid Synthesis. OX-7-ricin A chain and 19E12-F(ab)'-ricin A chain were
synthesized with N-succinimidyl(4-azidophenyldithio)propionate (SPDP) as
follows. OX-7 IgG, 1.55 mg in 250 .mu.l, was mixed with 10 .mu.l of 4 mM
SPDP in ethanol and incubated at room temperature 30 min. Then 10 .mu.l of
0.625 M DTT in 5.1 M sodium acetate pH 4.7 was added to the IgG for 30
min. The mixture was passed over a 0.75 cm.times.5 cm G25F column and the
IgG was pooled. To the IgG, 40 ul of 0.1 M 5,5'-dithiobis(2-nitrobenzoic
acid (DTNB) in 0.78 M sodium borate pH 8.5 and 1.25 mM EDTA, was added.
After 60 min. the IgG was freed from excess DTNB by passing over G25F in
0.4 M sodium borate pH 8.0 and this was added to 0.76 mg of freshly
reduced ricin A chain in 0.5 ml and incubated at room temperature for 3
hrs and then dialyzed against PBS. Approximately fifty percent of the IgG
was linked to ricin A chain and the hybrid mixture was used as such
without further purification. 19E12-F(ab').sub.2, 9.6 mg in 2 ml, was
incubated with 200 .mu.l of 1 M DTT for 30 min. then passed over a 1.8
cm.times.18 cm G25F column. F(ab)' was pooled and mixed with 320 .mu.l of
0.1 M DTNB in 0.78 sodium borate pH 8.5 and 1.25 mM EDTA for 30 min. The
F(ab)' solution was passed over a 1.8 cm.times.18 cm G25F column and the
F(ab)' was mixed with 6.5 mg of freshly reduced ricin A chain. The mixture
was incubated at room temperature for 3 hrs then dialyzed against PBS. The
hybrid was purified from unreacted ricin A chain by HPLC as described by
Youle and Neville, Proc. Natl. Acad. Sci., 77:5483-5486 (1980).
RESULTS AND DISCUSSION
Ricin A Chain Hybrids. Ricin, the ricin A chain, or a variety of hybrids
formed between these two toxins and anti-Thy 1.1 monoclonal antibodies
were incubated with murine lymphoma cells for 20 hours and their effect on
protein synthesis was determined (see the Figure). The least toxic hybrid,
the ricin A chain disulfide linked to the F(ab)' fragment of 19E12 IgG,
(19E12-F(ab)'-A) was 7-fold more toxic than ricin A chain on Thy 1.1
expressing AKR cells.
Ricin Hybrids (the hybrid formed with ricin and the F(ab)' fragment of the
low affinity antibody 19E12, 19E12-F(ab)'-R). After 20 hrs in the presence
of 50 mM lactose (which partially blocks the ricin entry route), the
hybrid formed with ricin and 19E12-F(ab)' is 5 times more toxic than ricin
plus 50 mM lactose (see the Figure). The rate of protein synthesis
inactivation for 200 ng/ml 19E12-F(ab)'-ricin in the presence of lactose
is 14-fold faster than 200 ng/ml ricin in the presence of lactose. The
FIGURE shows that 240 ng/ml 19E12-F(ab)'-A chain has little toxicity after
20 hrs. The B chain of ricin causes the enhancement of the Thy 1.1
specific entry rate.
Lactose has a definite effect on reducing the toxicity towards the target
cells; however, the hybrid is still 10.sup.3 times more toxic than ricin
plus lactose and 10.sup.4 fold more toxic to Thy 1.1 AKR cells than to
non-target Thy 1.2 EL-4 cells (the Figure). Comparing the rate of
inactivation of OX-7-ricin with that of OX-7-A at saturation, it would
appear that the presence of the B chain has a relatively small effect. It
is possible that the enhancement of entry rate produced by the B chain in
low affinity ricin hybrids or when added to high affinity A chain hybrids
is not operative for OX-7-ricin as a result of some interplay between the
very tight binding to the Thy 1.1 receptor and the noncleavable character
of the thioether linkage between ricin and OX-7 IgC.
Ricin A Chain Hybrids Plus Ricin B Chain. The effect of the ricin B chain
on enhancing protein synthesis inactivation rates can be demonstrated when
B chain is added to mixtures of OX-7-A and target cells. The enhancement
of the inactivation slopes is 2-fold for the addition of 170 ng/ml of B
and 5-fold for the addition of 1700 ng/ml of B. The maximum effect of
ricin B does not seem to have been reached. The target cell specificity in
the presence of B is maintained. No toxicity could be detected on EL-4
cells or on AKR cells in the presence of 10 ug/ml of competing OX-7
antibody for 35 ng/ml of OX-7-A plus 1700 ng/ml of B chain. The addition
of B chain to high affinity ricin A chain hybrids is a way to increase the
entry rate of the A chain without unduly sacrificing target cell
specificity.
In the presence of 1700 ng/ml of B chain, OX-7-A toxicity still saturates
at 176 ng/ml of OX-7-A. This indicates that the B chain only facilitates
entry of A chain bound specifically to the Thy 1.1 antigen. To show that
the ricin B chain increased the rate of A chain passage to the cytosol
without increasing the number of A chains bound to the cell, OX-7-A chain
above saturation was incubated with two series of cells for 1 hour. To one
series OX-7-A chain remained in the medium and the incubation was
continued for 3 hrs; then the rate of protein synthesis was determined. To
another set the medium containing OX-7-A chain was removed and replaced
with fresh medium lacing OX-7-A chain but containing ricin B chain. In
these washed wells, the total amount of OX-7-A chain in the well equals
the amount of OX-7-A chain bound to cells in the unwashed wells. Ricin B
chain added to the washed cells accelerated the protein synthesis
inactivation.
Although OX-7-A exhibits first order inactivation kinetics over 20 hours,
OX-7-A is not required in the medium past 2 hours. Saturating OX-7-A chain
was added to cells for 2 hours and replaced with fresh media lacking
OX-7-A chain for 18 hours. The final rate of protein synthesis was
essentially as low as in cells incubated with the same amount of OX-7-A
chain over the whole 20 hours (Table 1 below). The OX-7-A chain inhibits
protein synthesis only to 80% after 2 hours, thus prebound hybrid
continues to inactivate protein synthesis over the 20 hours time course.
It should be pointed out that there is a major difference in the transport
process between ricin, which exhibits a dose dependent lag period, and
OX-7-A or OX-7-A plus B chain which show no detectable lag. OX-7-ricin at
low doses sometimes displays a lag but always less than ricin for similar
rates of inactivation of protein synthesis.
The high affinity A chain hybrid shows selectivity in dose response curves
at 24 hours relative to a chain alone or to the non-target EL-4 cells of
10.sup.5, yet target cell killing is not greater than 99%, a fact due to
the relative low rate of entry of the A chain into the cytosol via the Thy
1.1 receptor. Killing rates are limited by the number of occupied
receptors and the efficiency of the entry process. Entry efficiency can be
increased by the presence of the ricin B chain.
EXAMPLE 1
Super-saturated OX-7-A chain was incubated with two series of cells for one
hour. To one series, OX-7-A chain remained in the medium and the
incubation was continued for 3 hours after which the rate of protein
synthesis was determined. To the other series, the medium containing
OX-7-A chain was removed and replaced with fresh medium lacking OX-7-A
chain, but containing ricin B chain. By replacing the medium in these
wells, the total amount of OX-7-A chain in the well equals the amount of
OX-7-A chain bound to cells in the unwashed wells. Ricin B chain added to
the washed wells accelerated protein synthesis inactivation beyond that in
unwashed wells without ricin B (Table 1). This shows that ricin B
increases the rate of A chain passage to the cytosol without increasing
the number of A chains bound to the cell.
EXAMPLE 2
AKR tumor cells, in 0.1 ml medium, were first incubated as noted in column
1, Table 1. They were then aspirated, placed in fresh medium, and
incubated as noted in column 2. [.sup.14 ]leucine was added to the cells
for 1.5 hours, then the cells were harvested and their protein synthesis
rate determined, as noted in column 3.
TABLE 1
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Addition to Addition to Relative Rate of
AKR Cells AKR Cells Protein Synthesis
______________________________________
(1 hr incu- (1 hr incu-
bation) bation)
None None 100
880 ng/ml 880 ng/ml 73
OX-7-A Chain OX-7-A Chain
880 ng/ml 1.2 .mu.g/ml
49
OX-7-A Chain Ricin B
880 ng/ml 12 .mu.g/ml 29
OX-7-A Chain Ricin B
None 12 .mu.g/ml 100
Ricin B
2 hr incu- 2 hr incu-
bation bation
None None 100
200 ng/ml 200 ng/ml 5
OX-7-A Chain OX-7-A Chain
200 ng/ml None 6
OX-7-A Chain
200 ng/ml 1.5 .mu.g/ml
2
OX-7-A Chain Ricin B
None 1.5 .mu.g/ml
107
Ricin B
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