POTENTIATION OF ANTI-CD38-IMMUNOTOXIN CYTOTOXICITY

Abstract: The present invention is directed to the use of agents that induce high levels of cell surface molecules to provide targets for immunotoxins directed against the same cell surface molecules. A specific example is given in which all-trans-retinoic acid (RA) is used to induce high levels of CD38 cell surface antigen expression in several myeloid and lymphoid leukemia cells. CD38 was then used as target for delivering plant toxin (gelonin) to leukemia cells. Treatment of leukemia cells with RA induced high levels of CD38 in those cells that otherwise had low CD38 expression. The RA-induced leukemia cells then became exquisitely sensitive to an immunotoxin constructed from an anti-CD38 monoclonal antibody conjugated to the plant toxin gelonin.

Description: POTENTIATION OF ANTI-CD38-IMMUNOTOXIN CYTOTOXICITY BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the fields o f immunology and tumor biology. More specifically, the present invention relates to treatment for the enhancement of CD38 protein expression in target tumor cells to increase the cytotoxicity of anti- CD38 based immunotoxins. Description of the Related Art The use of monoclonal antibodies for delivering drugs o r toxins to distinct molecular structures expressed on the surface o f unwanted tumor cells represents an attractive and potentially useful strategy. Theoretically, such a targeted approach to cancer therapy could offer a major advance in the selective elimination of tumor cells while reducing the toxicity of treatment towards normal non-target tissues. Nevertheless, in practice many problems exist that need to b e addressed before immunotoxin or antibody-drug therapies can be truly effective in vivo. One potential limitation to the success of any targeted approach to therapy is the heterogeneity of target antigen expression within a population of tumor cells. It follows that if a small number of cells within a tumor were negative for the target antigen o r expressed the antigen only very weakly, then these cells could possibly escape destruction due to a failure of antibody-mediated delivery of the cytotoxic agent to those particular cells. A possible means o f overcoming this problem would be to identify agents that induce high levels of cell surface target molecules, in the expectation that target tumor cells which were antigen negative would express these target molecules in abundance. All-trαns-retinoic acid (RA) is an agent that induces high levels of CD38 cell surface antigen expression in several myeloid an d lymphoid leukemia cells. Retinoic acid-induced expression of CD38 in these cells is specific, rapid, dose-dependent, and highly sensitive, with 4-fold induction at as low a dose of retinoic acid as 10 13 M. The induction of CD38 expression by retinoic acids has been shown t o involve the RAR retinoid receptor. RAR receptors form heterodimers with RXR receptors; the RXR/RAR heterodimer then interacts with DNA sequences known as retinoic acid response elements (RARE's) which are involved in retinoid-induced transcription. CD38 is a 45-kDa cell surface protein which is primarily expressed by early progenitor and mature activated cells of th e hematopoetic system. It is a transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain. The extracellular domain has been shown to be a bifunctional enzyme having ADP-ribosyl cyclase as well as ADP-ribosyl hydrolase activities in that it catalyzes the conversion of NAD+ to cADPR (cyclase) and can further hydrolyze it to ADP-ribose (hydrolase). cADPR is involved in the mobilization of calcium from intracellular stores which is a second messenger activity important for cellular proliferation, differentiation, and apoptosis. CD38 is believed to act as a receptor for an unidentified ligand and to act as a cell adhesion molecule by interacting with CD31. Experiments in which CD38 function was activated by monoclonal antibodies directed against it have implicated CD38 in proliferation of mature B lymphocytes an d myeloid leukemia cells, rescue of germinal center cells from apoptosis , and growth suppression of stroma-supported cultures of B-cell progenitors as well as induction of the cytokines IL-6, IGN-g, GM-CSF, and IL-10. In addition, it has been shown to signal an increase in TNF-α, IL-1, IL-6, and IL-8 transcription in myeloid leukemia cells. The prior art is deficient in the lack of a method to induce the expression of a target molecule for immunotherapy of tumor an d other disease-causing cells. The present invention fulfills this longstanding need and desire in the art. SUMMARY OF THE INVENTION The present invention demonstrates the potential o f retinoid-induced CD38 expression to serve as a target for delivering th e immunotoxin anti-CD38-gelonin. The results obtained suggested that retinoic acid treatment of leukemia cells, even at very low concentrations (subnanomolar) makes these cells exquisitely sensitive to immunotoxin-induced killing. The current invention comprises a method of treating a n individual having a pathophysiological state, comprising the step o f administering to said individual a pharmacologically effective dose o f an agent which upregulates the expression of a cellular target and also administering a pharmacologically effective dose of a n immunotoxin directed against the upregulated cellular target. The current invention also comprises a method of treating an individual having a pathophysiological state responsive to retinoid treatment, comprising the step of administering to said individual a pharmacologically effective dose of a retinoic acid metabolite and a pharmacologically effective dose of an immunotoxin. Other and further aspects, features, and advantages of th e present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, m ore particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part o f the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention an d therefore are not to be considered limiting in their scope. Figure 1 shows a dot blot of mRNA from a variety o f human tissues after hybridization with a radiolabeled human CD38- specific nucleic acid probe. Relatively low CD38 mRNA expression was observed only in thymus tissue [from both adult (E5) and fetal (G6)] while a lesser level of expression was seen in normal prostate (C7). Figure 2 shows the effects of 5 nM all-trαns-retinoic acid (RA) on the cytotoxicity of the immunotoxin an d the effects of adding increasing concentrations of the unconjugated anti-CD38 monoclonal antibody (IB4). Point C indicates the effect o f immunotoxin alone. +IT (+RA) shows the effect of 5 nM all-trans- retinoic acid (RA) on the cytotoxicity of the immunotoxin. In the res t of the samples, increasing concentrations of IB4 were added along with immunotoxin and 5 nM all-trαns-retinoic acid (RA). After 3 days o f incubation, cell viability was determined with the MTS assay. The results are represented in terms of % surviving cells. Figure 3 shows effect of retinoic acid pretreatment on th e cytotoxicity of anti-CD38 immunotoxin on HL-60 cells. HL-60 cells were incubated overnight in the presence or absence of retinoic acid. After removal of the media and twice washing the cells, the cells were reincubated with increasing concentrations of immunotoxin (represented as ng/well) in the presence or absence of 100-fold excess of unconjugated anti-CD38 monoclonal antibody IB4. After three days, the MTS assay was used to determine cell viability which is represented in terms of % surviving cells. Figure 4 shows the effect of treatment with either immunotoxin or gelonin on the viability of HL-60 cells. HL-60 cells were incubated for three days in increasing concentrations of either immunotoxin or gelonin (represented as ng/well toxin) in the presence or absence of 5 nM retinoic acid. Cell viability was determined by th e MTS assay and is represented here in terms of percent cell survival relative to the control sample (no toxin). Figure 5 shows the effect of increasing concentrations o f all-trαns-retinoic acid (RA) (in nM) on cell survival. HL-60 cells were incubated with either immunotoxin or unconjugated anti-CD38 monoclonal antibody in either the absence or the presence o f increasing concentration of retinoic acid (shown in nM). After three days, cell viability was determined by MTS assay and is shown i n terms of percentage of cell survival relative to an untreated control. Figures 6A and 6B show the effect of increasing concentrations of immunotoxin (shown in ng/well) in either th e presence or absence of 5 nM all-tr n^-retinoic acid (RA) on cell survival of different cell lines including Daudi, THP-1, K562 (which is resistant to RA-induced expression of CD38), and a RARα-expressing variant of HL60. Cell viability was measured by the MTS assay after three days and is represented in terms of percent cell survival relative to an untreated control. Figure 7 shows the immunotoxin induced killing of Doxo- resistant HL-60 cells which are resistant to adriamycin-induced killing. The cells were incubated with increasing concentrations o f immunotoxin (shown in ng/well) in either the presence or absence of 5 nM RA. Cell survival was assayed after 3 days using the MTS assay. Figure 8 shows the immunotoxin mediated killing of the non-Hodgkin lymphoma cell line MZ(NHL) that has a high basal expression of CD38 antigen. The cell were incubated with increasing concentrations of immunotoxin (shown in ng/well) in the presence o r absence of 5 nM retinoic acid. After three days, cell viability was assayed with the MTS assay and is shown in terms of % viable cells. Figure 9 shows the immunotoxin mediated killing of th e retinoic acid-resistant variant of the HL60 cell line (HL60R). These cells are resistant to retinoic acid-induced expression of the CD38 antigen due to a point mutation in the retinoic acid receptor alpha (RARα) gene. The cell line was cultured with increasing concentrations of immunotoxin (in ng/ml) under different conditions. After 3 days , cell viability was assayed b y the MTS assay and is represented i n terms of O.D. The presence of retinoic acid failed to promote immunotoxin-induced killing of these cells due to their inability t o express CD38 antigen in response to retinoic acid treatment. DETAILED DESCRIPTION OF THE INVENTION An immunotoxin is defined as any immunological molecule such as an antibody which has been conjugated with a toxin, preferably a cytotoxin. The present invention is directed to a method of treating a n individual having a pathophysiological state, comprising the step o f administering to said individual an a pharmacologically effective do s e of an agent which upregulates the expression of a cellular target. This administration is followed by the administration of a pharmacologically effective dose of an immunotoxin directed against the cellular target. Preferably, the administered agent is selected from the group consisting of differentiating agents, cytokines, interleukin-2, tumor necrosis factor, interferon-α, interferon-γ and peptide hormones. In one embodiment, the invention comprises th e administration of a pharmacologically effective dose of a retinoid. Preferably, the retinoid induces expression of CD38 antigen in cells. If this is the case, a pharmacologically effective dose of an anti-CD38 immunotoxin is administered. Representative pathophysiological states which may be treated using the methods of this embodiment of th e invention include RARα selective acute myeloid leukemia, acute promyelocytic leukemia, lymphomas, and myelomas. Representative retinoic acid metabolites which may be u sed in the methods of th e present invention include all-trans - retinoic acid (RA); 9-cis retinoic acid (9-cis RA); (E)-4- [2- (5 ,6,7 , 8-tetrahydro-5,5, 8 , 8-tetramethyl-2-nephthalenyl)- l - propenyljbenzoic acid (TTNPB); (E)-4- [2-(5 ,6,7, 8-tetrahydro-3 ,5 ,5 , 8, 8- pentamethyl-2-nephthalenyl)- l -propenyl]benzoic acid (3-met TTNPB); and other retinoids that can bind and activate the RARα receptor. Preferably, the retinoid is administered in a dose of from about 0. 1 mg/kg to about 2 mg/kg. The immunotoxin used in the methods of the present invention specifically target cells expressing the CD38 antigen. Preferably, the immunotoxin comprises a monoclonal antibody directed against the CD38 antigen conjugated to a toxin molecule. Although a person having ordinary skill in this art could substitute any toxin, a preferred toxin useful in these methods is gelonin. Although a person having ordinary skill in this art could substitute any monoclonal antibody specific for the CD38 antigen, IB4 or IB6 antibodies were u sed herein to demonstrate the present methods. Preferably, th e immunotoxin is administered in a dose of from about 0.05 mg/kg t o about 2 mg/kg. The following examples are given for the purpose o f illustrating various embodiments of the invention and are not meant t o limit the present invention in any fashion. EXAMPLE 1 CD38 expression in normal tissues is limited mainly to the thymus. The tissue specificity of CD38 was examined by th e hybridization of a radiolabeled CD38 nucleic acid probe against a commercial (CLONTECH) tissue specific mRNA dot blot. The results o f the hybridization are shown in Figure 1. It was observed that CD38 is mainly expressed in the thymus with significantly lower levels of expression in the prostate. EXAMPLE 2 Retinoic acid (RA) augments the cytotoxic effect of i mmu notox i n through enhanced expression of CD38. HL-60 cells were incubated with either immunotoxin alone or in the presence of 5 nM retinoic acid (RA). Increasing concentrations of unconjugated IB4 monoclonal antibody were added to the cells incubated with immunotoxin and retinoic acid. After three days, the cells were assayed for viability with the MTS assay. Briefly, 6.5 mg/ml MTS solution [(3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and 0.5 mM PMS (phenazine methosulfate) solution were mixed at a ratio o f 20: 1. 20 μl of the combined MTS/PMS solution was placed in each well of a 96 well plate containing samples of the cells to be tested. The plate was incubated for 1-4 hours at 37°C in a 5% C0 2 atmosphere, after which time, the amount of formazan produced by live cells from cellular reduction of MTS was measured by reading the absorbance a t 490 nm. The results are shown in Figure 2. Immunotoxin alone had little effect on the viability of th e cells (C). However, when the cells were incubated with immunotoxin in the presence of 5 nM retinoic acid, a significant reduction in cell viability was observed. Increasing concentrations of unconjugated IB4 monoclonal antibody blocked the cytotoxic effect of immunotoxin an d retinoic acid. The fact that unconjugated IB4 blocked the ability of th e immunotoxin to kill the cells demonstrates that the immunotoxin is specifically interacting with the CD38 surface marker and that th e effect of the retinoic acid is to increase the expression of the CD38 antigen. EXAMPLE ! A 11-tn7n..v-retinoic acid (R A) presentment enhances the induced killing of HL-60 Cells. HL-60 cells were preincubated overnight in either th e presence or absence of 5 nM all-trαns-retinoic acid. The cells were washed twice and incubated in increasing concentrations o f immunotoxin in either the presence or absence of IB4 unconjugated anti-CD38 MoAb. After three days, the cell were assayed for viability. The results are shown in Figure 3. Preincubation with all -trans- retinoic acid followed b y immunotoxin treatment resulted in more cell death than treatment with immunotoxin alone. The presence of 100 fold excess of th e unconjugated anti-CD38 monoclonal antibody IB4 blocked the toxicity of the immunotoxin in both cases by competing with the immunotoxin for access to the CD38 markers on the cells. These results demonstrate that the all-trαn.y-retinoic acid (RA) was causing some change in the cells which render them more susceptible to the immunotoxin rather than playing a direct role in the death of the target cells. EXAMPLE 4 Gelonin must be conjugated to the anti-CD38 antibody to have a toxi c effect on the target cell HL-60 cells were incubated for three days with increasing concentrations of either immunotoxin or gelonin in either the presence or absence of 5 nM retinoic acid. Afterwards, the cells were assayed for viability using the MTS assay. As seen in Figure 4, gelonin alone had no toxic effect in either the presence of absence of 5 nM. Thus, the toxic effect of gelonin depends on it being conjugated t o the anti-CD38 monoclonal antibody in order to deliver the toxin to th e cell. EXAMPLE 5 Even nominal levels of all-tr^ .v-retinoic acid (RA) lead to i n crea sed toxicity of the immunotoxin. HL-60 were incubated with either immunotoxin o r unconjugated IB4 monoclonal antibody in increasing concentrations o f monoclonal antibody. Figure 5 shows that even the lowest level of all- trans -retinoic acid (RA) (1 nM) lead to almost complete killing of th e target cells by the immunotoxin. This effect was not observed with th e unconjugated monoclonal antibody. This result indicates that it is th e gelonin conjugated to the monoclonal antibody in the immunotoxin that leads to the increased cell death rather than some effect of th e antibody itself. EXAMPLE 6 Retinoic acid can induce expression of the CD38 marker in a variety o f cell lines. The Daudi, THP-1, K562, and HL60-RARα cell lines were treated with increasing concentrations of immunotoxin in either th e presence or absence of 5 nM all-trαfts-retinoic acid (RA). After three days, the viability of the cells was examined using the MTS assay, which is shown in Figure 6. In the THP-1 and HL60-RARα cell lines, all-trans- retinoic acid induced cell death while the cell which were cultured in the absence of all-trans- retinoic acid were mostly unaffected by the immunotoxin. In th e Daudi cells, which have a high basal expression of CD38, the immunotoxin resulted in almost complete cell death regardless of whether retinoic acid was present. On the oth er hand, K562, which are resistant to RA-induced CD38 expression, were unaffected by the immunotoxin regardless of the presence of retinoic acid. EXAMPLE 7 Tmrrmnotoxin induced cell death in HL-60 cells resistant to Adriamycin. HL-60 subcloned cells, resistant to adriamycin-induced killing were cultured with immunotoxin either alone or in the presence of 5 nM all-trαns-retinoic acid. After three days, the MTS assay was used to test cell viability. Figure 7 shows the results obtained. Some cell death was observed in the presence of immunotoxin alone which was greatly augmented by the addition of 5 nM all-trαns-retinoic acid. EXAMPLE 8 Cells which have high basal expression ol CD 8 are killed b_y immunotoxin regardless of the presence or absence of al -trans- retinoic acid (R A). MZ, a non-Hodgkin lymphoma cell line which has a high basal expression of CD38, was treated with increasing amounts o f immunotoxin in either the presence or absence of 5 nM all-trans- retinoic acid. The addition of immunotoxin resulted in a high level of cell death regardless of the presence or absence of retinoic acid (Figure 8). This is strong evidence that retinoic acid is increasing the toxicity of immunotoxin by enhancing the level of CD38 on oth er cell lines which do not have a high basal level of CD38. EXAMPLE 9 Retinoic increases C!P38 expression in a number of lymphoid tu or cell s. Table I lists the potential targets for anti-CD38 bound toxin treatment A number of different lymphoid tumor cell lines were treated with 5 nM all-trαrcs-retinoic acid (RA). Afterwards, th e expression of CD38 in untreated versus treated cell was measured by flow cytometry. A significant rise in CD38 expression was observed in acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), lymphoma, and myeloma tumor cells. The increase in CD38 expression ranges from 2.5 to 20 fold. Thus, retinoic acid can be used in all of these tumor types to increase the vulnerability of the tumor cells t o immunotoxin treatment. TABLE 1 Potential Targets for Anti-CD38 Bound Toxin Treatment Cell Target Basal CD38 CD38 after RA Treatment AML 50 ± 10 180 ± 20 AFL 6 ± 4 120 ± 30 Lymphomas 80 ± 20 210 ± 10 Myelomas 60+ 20 180 ± 25 SUE Myesthenia gravis B cells producing self reactive ab Rheumatoid arthritis Self reactive T lymphocyte Organ transplantation EXAMPLE 10 Tmmunotoxin does not affec cells resistant to a11-tπ γ-retinoic aci d ( A) HL-60 cells with a mutated RARα gene that renders the cells resistant to the effects of retinoic acid were treated with immunotoxin in either the presence or absence of 5 nM retinoic acid. In these cells, the addition of retinoic acid had no effect on the toxicity of the immunotoxin. As shown in Figure 9, no appreciable cell death was observed in the cells treated with all-trαn.s'-retinoic acid (RA), with unconjugated IB4 and gelonin, or with gelonin alone. This is further proof that the immunotoxin kills cells which are affected by retinoic acid because of a retinoid induced increased in expression of CD38 target of the immunotoxin. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which th e invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to b e incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments , molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are n o t intended as limitations o n the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by th e scope of the claims.

Claims: WHAT IS CLAIMED IS: 1 . A method of treating an individual having a pathophysiological state, comprising the steps of: a), administering to said individual a pharmacologically effective dose of a agent which up-regulates the expression of a cellular target; and, b). administering to the same individual a pharmacologically effective dose of an immunotoxin directed against the up-regulated cellular target. 2. The method of claim 1, wherein the administered agent is selected from the group consisting of differentiating agents, cytokines, interleukin-2, tumor necrosis factor, interferon-α , interferon-γ and peptide hormones. 3 . The method of claim 1 , wherein said agent is a retinoid and wherein said cell target is the CD38 antigen. 4. The method of claim 3, wherein said pathophysiological state is selected from the group consisting of acute myeloid leukemia, acute promyelocytic leukemia, lymphomas, and myelomas . 5 . The method of claim 3, wherein said retinoid is selected from the group consisting of all-trαns-retinoic acid (RA); 9-cis retinoic acid (9-cis RA); (E)-4-[2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-nephthalenyl)-l-propenyl]benzoic acid (TTNPB); (E)-4-[2-(5, 6,7, 8-tetrahydro-3, 5,5,8, 8-pentamethyl-2-neρhthalenyl)-l- propenyljbenzoic acid (3-met TTNPB). 6. The method of claim 5, wherein said retinoid is administered in a dose of from about 0.1 mg/kg to about 2 mg/kg. 7. The method of claim 3, wherein said immunotoxin specifically targets cells expressing the CD38 antigen. 8. The method of claim 7, wherein said immunotoxin comprises a monoclonal antibody directed against the CD38 antigen conjugated to a toxin molecule. 9. The method of claim 8, wherein said toxin is gelonin. 10. The method of claim 8, wherein the monoclonal antibody is selected from the group consisting of IB4 or IB6. 11. The method of claim 1, wherein said immunotoxin is administered in a dose of from about 0.05 mg/kg to about 2 mg/kg.

Inventors: MEHTA KAPIL, ROSENBLUM MICHAEL G, MEHTA, KAPIL, ROSENBLUM, MICHAEL, G.
Applicants: RES DEV FOUNDATION, RESEARCH DEVELOPMENT FOUNDATION
Application year: 2000
Main classification: A61K39
IPC codes: A61K31, A61K38, A61K39, A61K45, A61K47, A61P35, A61P43