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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Berlex Biosciences, Richmond, 2 Medarex, Milpitas, California; 3 Innsbruck Medical University, Innsbruck, Austria; and 4 Washington University School of Medicine, St. Louis, Missouri
Requests for reprints: Renate Parry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94806. Phone: 510-669-4734; Fax: 510-669-4270; E-mail: renate_parry{at}berlex.com.
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75 µCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 µCi dose at day 49. We conclude that [90Y]-CHX-A''-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients. |
Introduction |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of mindin/RG-1 protein expressed in baby hamster kidney cells. A cDNA with the mindin/RG-1 coding region was obtained from Incyte (Palo Alto, CA). The coding sequence was PCR-amplified with forward (5'-TCCCTCTAGAGCCACCATGGAAAACCCCAGCCCGGC-3') and reverse (5'-AAGGCATCACGTGTTAGACGCAGTTATCAGGGACG-3') primers for 35 cycles (annealing temperature 60°C, extension time of 120 seconds) with Pfu1 Turbo polymerase (Clontech), and ligated into pSIN rep21 (9). Similarly, a plasmid containing full-length mindin/RG-1 with GGSS-6xHIS added to the carboxyl terminal (pPEG-8) was obtained using the same PCR forward primer, with the reverse primer being 5'-AGTAAGGCATCACGTGTTAATGGTGATGGTGATGATGGCTGCTGCCGCCGACGCAGTTATCAGGGACGCAC-3'. Expression plasmids pPEG6, for the carboxyl-terminal 6xHis-tagged mindin/RG-1, and pPEG8, for the untagged mindin/RG-1, were transfected into baby hamster kidney (BHK)-21 cells (American Type Culture Collection, Rockville, MD) using LipofectAMINE-2000 (Invitrogen, Carlsbad, CA). Expression of mindin/RG-1 protein into the culture medium was monitored by Western blot analysis using either an anti-6xHis antibody or a rabbit polyclonal anti-mindin/RG-1 antibody plus horseradish peroxidase–labeled second antibody.
Mindin/RG-1 protein purification. (a) Untagged mindin/RG-1 protein was purified from serum-free medium from BHK cells expressing mindin/RG-1 protein by diluting the medium 1:1 in 20 mmol/L sodium acetate buffer (pH 6.5) and loading to a 5 mL Q-Sepharose ion exchange column. Fractions were monitored by Western blot, and those containing mindin/RG-1 were pooled and further purified using a SuperDex-75 size exclusion column. (b) Mindin/RG-1 protein containing the 6-histidine expression tag was purified from BHK cell conditioned medium using NiNTA-Sepharose affinity chromatography. Peak fractions were pooled and dialyzed into PBS and stored at –80°C.
Generation, cloning and expression of fully human anti-mindin/RG-1 antibodies in Chinese hamster ovary cells. Antibodies were originally produced by hybridomas using Medarex's UltiMAb Human Antibody Development System (Medarex, Inc., Milpitas, CA). For Chinese hamster ovary (CHO) expression, the human antibody 19G9 VH and VL sequences were cloned into an expression vector pIE-Sr 
1fa [pIE] (Medarex) and transfected into DG44 CHO cells. Transfection was carried out with LipofectAMINE-2000 following the manufacturer's instruction. Selective conditions were applied at 48 hours posttransfection. Coselection for G418 and dihydrofolate reductase was first carried out with 
-MEM supplemented with 5% heat-inactivated dialyzed FCS, 400 µg/mL of G418, and 20 nmol/L of methotrexate in 96-well plates. A series of antibody-expressing CHO cell clones were selected and subjected to further amplification in iteratively increasing concentrations of methotrexate. The final selections were based on quantification using analytic protein A chromatography and A280 absorbency.
Immunoreactivity of anti-mindin/RG-1 antibodies. The immunoreactivity was tested by (a) 6-His Capture ELISA, (b) Biacore analysis using BHK expressed protein, and (c) RIA. Kinetic analysis of binding affinity of purified antibodies to native mindin/RG-1 was done using a method in which soluble BHK-mindin/RG-1 was captured by immobilized monoclonal antibody. Anti-human Fc antibody was covalently attached to the Biacore sensor chip via amine coupling. Association and dissociation data were fit to a 1:1 Langmuir model.
Immunohistochemistry. Frozen and paraffin-embedded tissue samples were obtained from PhenoPath (Seattle, WA) and from the pathology department at Innsbruck, Austria. Low-density human tissue arrays were obtained from Innogenex (San Ramon, CA; cat. TS-4201-05). Binding of the primary antibodies (RG-1/3c; 19G9) was visualized using a Streptavidin kit (Zymed, San Francisco, CA) with 3,3'-diaminobenzidine as chromogen.
In vivo characterization of human anti-mindin/RG-1 antibodies. Male, nude (nu/nu) mice, 6 to 8 weeks of age were obtained from Simonsen Laboratories (Gilroy, CA). Mice were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care, and all experiments were conducted in accordance with principles and procedures approved by the Animal Care and Use Committees at Berlex Biosciences and Washington University. Mice were implanted s.c. with LNCaP cells (107 cells/mouse). Approximately 4 to 6 weeks after receiving the cell implant, mice bearing tumors of 50 to 400 mm3 were selected and randomized into groups (n = 15 for biodistribution, n = 20 for efficacy studies). Antibodies were radiolabeled with 111In for biodistribution studies, 86Y for positron emission tomography (PET) studies, or 90Y for efficacy studies and given i.v. through the lateral tail vein.
Biodistribution studies. At selected time points (days 1, 3, 6, and 9 postinjection) animals treated with [111In]-CHX-A''-DTPA-19G9 or a radiolabeled, isotope-matched human IgG control antibody ([111In]-CHX-A''-DTPA-IgG), were exsanguinated under anesthesia. All organs, including blood and tumor, were collected, weighed and radioactivity was measured in a gamma counter (Auto-Gamma Cobra II, Packard, Downers Grove, IL) and expressed as the percentage of injected dose per gram (% ID/g).
Positron emission tomography imaging studies. The 86Y was produced on the CS15 cyclotron by the 86Sr(p,n)86Y reaction (10). The imaging of 86Y-CHX-A''-DTPA-19G9 was done at Washington University on the micro-PET-R4 tomograph (Concorde Microsystems, Inc., Knoxville, TN). Coregistration of the PET images was achieved in combination with a micro-CAT-II camera (Imtek Inc., Knoxville, TN). The image registration between micro-CT and PET images was accomplished by using a landmark registration technique AMIRA image display software (AMIRA, TGS Inc., San Diego, CA).
In vivo efficacy studies. LNCaP-bearing nude mice (50-400 mm3) were randomized (n = 20) to different treatment and control groups (antibody alone, radiolabeled human, isotope-matched IgG control antibody, or no treatment). Tumor volumes and general toxicity as measured by body weight and general clinical appearance was monitored for up to 30 days (study 1) or 90 days (study 2). In study 1, [90Y]-CHX-A''-DTPA-19G9 was given at a single i.v. dose of 25, 50, 75, or 100 µCi. In study 2, tumor-bearing animals were given a single i.v. dose of 75 µCi of [90Y]-CHX-A''-DTPA-19G9 at day 0 and a second dose of 75 µCi at day 49.
Serum prostate-specific antigen determination. Human prostate-specific antigen (PSA) was quantified in the plasma from LNCaP-bearing mice by an ELISA kit from Diagnostic Systems Laboratories, Inc., (Webster, TX) and read on a Molecular Devices Versamax microplate reader with SoftMax Pro software (Sunnyvale, CA).
Statistical analysis. Values from biodistribution studies are averages of three individual mice ± SE. For the animal efficacy studies, the effect of treatment on weekly tumor volumes and final tumor weights was analyzed by nonparametric tests and deemed significant if P < 0.05. The overall effect of treatment was assessed by the Kruskal-Wallis test, whereas comparison between two treatment groups was analyzed by the Mann-Whitney U test. Reported P values are from the Mann-Whitney U test.
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Results |
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1.9 kb was detected at significantly higher levels in the prostate samples as compared with any RNAs from other tissues (Fig. 1B). In this set of tissue samples, mindin/RG-1 mRNA was expressed at 8-fold higher abundance in the prostate than any other tissue sample. A band with a significantly lower intensity at about 6 kb was also seen in the Northern blot and this may represent an alternatively spliced form of mindin/RG-1 mRNA. Furthermore, we have determined that mindin/RG-1 gene maps to chromosome 4p16.3 and the dominant mRNA is coded by six exons (data not shown).
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85% identity at the protein level. A polyclonal rabbit antiserum, RG-1/3C, raised against the peptide sequence shown in Fig. 2 reacted selectively with mindin/RG-1 in Western blots. By Western blot analysis, the protein is typically detected as a band that runs between the 38 and 47 kDa markers. This was seen in the prostate tumor cell line LNCaP, which express mindin/RG-1 endogenously, and in PC-3 cells transfected with cDNA coding for the full-length protein (PC-3 cells themselves do not express RG-1/mindin message; data not shown). RG-1/3c also detected the protein in paraffin-embedded tissue sections (Fig. 3), and staining of sections could be efficiently blocked with 10-fold excess of competing peptide (Fig. 3F). The same staining pattern of Mindin/RG-1 protein in human prostate tissue was seen with the human biotinylated antibody 19G9 (Fig. 3H).
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80% of primary prostate tumors (15 of 18) and in metastases to lymph node (8 of 10) and bone (18 of 23; Table 1). As most patients that present with advanced disease have tumors that are unresponsive to androgen ablation therapy, it was of interest to determine if mindin/RG-1 expression was maintained in tumors from this group of patients. As shown in Table 1, 80% of local recurrent tumors from androgen-independent patients (22 of 26) maintain expression of mindin/RG-1 protein. Thus, expression of mindin/RG-1 is seen in the majority of tumors from patients that are androgen-unresponsive as well as from patients that have early stage disease.
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Fully human antibodies against mindin/RG-1 show nanomolar affinity to mindin/RG-1 protein. To evaluate the option of targeting mindin/RG-1 for antibody-based therapy, mindin/RG-1 protein was expressed in BHK cells, purified as described in Materials and Methods, and used to immunize transgenic mice using Medarex's UltiMABSM technology. Following immunization, hybridomas were generated and culture supernatants from the hybridomas were screened by a capture ELISA assay to identify antibodies recognizing the native protein. Several monoclonal antibodies were identified and four were further characterized by using BIACore technology to determine kinetic variables. These four antibodies exhibited affinities in the nanomolar range (8.4-34 nmol/L). As an example, the IgG1 19G9 (Kd 8.4 nmol/L) bound to native but not denatured protein and detected mindin/RG-1 protein by Western blot analysis only on nonreducing gels (data not shown). Antibody 19G9 was cloned and expressed in CHO cells, and purified from the culture medium by Protein A chromatography. The CHO-expressed antibody and the hybridoma-produced antibody exhibited virtually identical affinity for mindin/RG-1 (data not shown).
19G9 antibodies can be conjugated to CHX-A''-DTPA and radiolabeled with 111In, 90Y, or 86Y. The chelator CHX-A''-DTPA was conjugated to the 19G9 antibody produced by CHO cells and to a control human IgG1 antibody through primary amine groups on antibody lysines as previously described (11). Typically, specific activities ranged from 0.25 to 1.0 mCi/mg. Specific activities of 8 mCi/mg were also experimentally achieved. A molar ratio of 3:1 (chelator to antibody) was achieved as determined by two methods: (a) a spectrophotometric method using Arsenazo III (12) or (b) a titration method using 111In (13). The absence of antibody aggregates in the conjugate preparation was confirmed by size exclusion chromatography (data not shown). The CHX-A''-DTPA-19G9 antibody conjugate was radiolabeled with either 111In for biodistribution studies, 86Y for small animal PET imaging studies, or 90Y for in vivo efficacy studies. The radiochemical yield was always in the order of 70% to 95%, and the radiochemical purity as determined by thin layer chromatography (11), was generally >97% and frequently >99%. It was shown by ELISA and RIA that the immunoreactivity of the antibody was maintained after conjugation and after radiolabeling, demonstrating binding of the antibody 19G9 to mindin/RG-1 with nanomolar affinity (data not shown).
Radiolabeled CHX-A''-DTPA-19G9 antibody shows a high and tumor-specific accumulation in a human prostate tumor (LNCaP) xenograft as determined in biodistribution and small animal positron emission tomography imaging studies. In order to monitor the tumor-specific accumulation of 19G9 in vivo, biodistribution studies were done using 111In-labeled CHX-A''-DTPA-19G9 and an 111In-labeled isotype-matched human antibody that does not bind to mindin/RG-1 (CHX-A''-DTPA-IgG1), thus serving as a negative control. As shown in Table 2, [111In]-CHX-A''-DTPA-19G9 showed a high and target-specific accumulation in the LNCaP xenograft, whereas no specific tumor accumulation was seen with the radiolabeled control antibody, CHX-A''-DTPA-IgG1. In order to visualize the tumor accumulation in LNCaP tumor–bearing mice, small animal PET studies were done using CHX-A''-DTPA-19G9 labeled with 86Y as a PET tracer. All animals (n = 4) showed high levels of activity in the blood at 1 hour, which rapidly cleared after 4 hours. At 4 hours, LNCaP tumors could already be delineated in all subjects. Over the 72-hour imaging period, exceptionally high tumor to background contrast was noted in all animals, demonstrating the excellent targeting properties of radiolabeled 19G9 antibody recognizing mindin/RG-1 protein (Fig. 4).
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Discussion |
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The tissue distribution of mRNA for mindin/RG-1 in the human seems to be significantly different from that reported for mouse mindin, where expression of mindin is seen in the lung, lymphoid tissues, and heart as well as a number of other tissues (data not shown, ref. 8). It has been reported that mindin is expressed in mouse macrophages and that it may function as an opsonin for clearance of bacteria (8). Our preliminary analysis of mindin/RG-1 expression in human macrophages by Western blot has revealed that mindin/RG-1 is expressed in differentiated macrophages but not in circulating monocytes (data not shown). It is also notable that immunohistochemistry studies revealed expression in granulocytes infiltrating tumor tissue.
The abundant expression of mindin/RG-1 in the prostate in humans prompts the question of what its role might be in normal prostate tissue as well as in prostate tumors. In the rat, a mindin homologue has been reported to promote outgrowth of embryonic hippocampal neurons (6). In the mouse, a mindin homologue has been reported to be a pattern-recognition molecule for microbial pathogens (8). Although there is no information regarding its function in humans, it is likely that the role of mindin/RG-1 is distinct from any opsonizing role that the protein may have. Such alternate roles for extracellular matrix molecules in modulation of cell attachment, growth, and differentiation have been documented previously for other extracellular matrix molecules, and it is possible that mindin homologues have similar properties (14).
Mindin/RG-1 protein is abundantly expressed in both normal and tumor tissues with expression in tumors being generally higher than that in normal prostate. Significantly, expression of mindin/RG-1 is maintained in tumor metastases to distal sites including the bone and lymph nodes. Its expression is also maintained in recurrent tumors from patients that are unresponsive to androgen ablation therapy. The distribution of the protein, taken together with the fact that it seems to be secreted into the extracellular milieu, makes it a promising target for antibody based therapy of prostate cancer. This is supported by our studies with the mindin/RG-1 specific antibody 19G9, which was shown by biodistribution studies using [111In]-CHX-A''-DTPA-19G9 as well as PET imaging studies using [86Y]-CHX-A''-DTPA-19G9 to accumulate specifically and at high levels in LNCaP tumor xenografts in nude mice (15). The accumulation was target-specific as shown by the absence of accumulation of control antibodies in the tumors. These results were confirmed with a second mindin/RG-1–specific antibody, 34E1 (data not shown). It is important to note that neither 19G9 nor 34E1 bind to mouse protein, and consequently it was possible to conduct the biodistribution studies without concern for the tissue distribution of the mouse homologue, which is quite distinct from that of the human homologue.
The chelator CHX-A''-DTPA exhibits high affinity for several isotopes that are suitable for either diagnostic or therapeutic purposes, including 111In, 86Y, 90Y, and 177Lu. PET imaging studies with the positron emitter 86Y showed the ability of the mindin/RG-1–specific antibodies to delineate the LNCaP tumor with a high tumor to background ratio. Our therapeutic efficacy studies were done with 90Y, a pure ß-emitter. Highly significant antitumor effects were seen with 19G9 over a dose range of 25 to 100 µCi/mouse with inhibition of tumor growth being observed at all doses above 25 µCi. At doses of 75 µCi per mouse, complete inhibition of tumor growth was seen for up to 50 days, although residual tumor cells were still present at sacrifice. Significantly, at the 75 µCi dose, no long-term toxicity of the radiolabeled conjugate was detected as monitored by loss of weight, demonstrating that a clear therapeutic window can be established where tumor growth inhibition can be achieved without significant toxicity. Similar data was obtained with a second antibody, 34E1 (data not shown), supporting the suitability of mindin/RG-1 as a therapeutic target.
Forty-nine days after administration of a single dose of radiolabeled antibody, regrowth of the tumors became apparent. This, however, could be prevented by administration of a second dose of radiolabeled antibody at this time. Following administration of this second dose, no regrowth of tumors was detectable over the next 41 days of the study. When the study was terminated on day 90, all mice were alive bearing either no detectable tumor or small tumor masses at the site of implantation. Histologic analysis of the smallest of these tumors revealed variably sized necrotic regions inter-spacing small clusters of viable tumor cells. Often, collagen scarring and hemosiderin-ladened macrophages were present around the tumor cells.
The antitumor effects were also clearly shown by quantitation of PSA levels in the sera of mice. Highly significant reductions in the levels of circulating PSA were seen in mice treated with radiolabeled antibodies. The mean levels of PSA did not return to zero, consistent with the presence of some viable cells in the remaining small tumor masses. However, when the levels were evaluated on an animal by animal basis, some mice did not have any or extremely low levels of PSA, demonstrating that in some mice, virtual eradication of tumor may have occurred.
Several other cell surface or secreted proteins have been suggested as possible targets for immunotherapy of prostate cancer. These include PSMA (16), PSCA (17), TMEFF2 (18), prostein (19), STEAP2 (20), PSGR (21), Trp-p8 (22), and NGEP (23). Mindin/RG-1 is distinct from these in lacking a transmembrane domain and is secreted from tumor cells but can also loosely associate with the outer surface of the plasma membrane. Although this location precludes its use as a target for antibody-drug conjugate–based therapies that require internalization of antibody-drug conjugates, mindin/RG-1 is clearly an attractive target for therapeutic approaches which do not require internalization of the antibody conjugate, e.g., radioimmunotherapy. The location of the target at the cell periphery and the high expression level may provide increased access of antibodies to the target when compared with strictly membrane-associated proteins. Radiolabeled antibodies provide a significant bystander killing effect that may be extremely important in eradicating tumors where target expression may be heterogeneous throughout the tumor or tumor metastases. Whereas the location of mindin/RG-1 may be advantageous with respect to antibody access, we recognize that some protein may be shed from the tumor into the serum. Our preliminary analysis indicates that the levels of mindin/RG-1 in serum are low. However, more extensive analysis of this will be necessary to gain a better understanding of potential toxicities that could result from antibody binding to protein in the serum. Clearly, no evidence of systemic toxicity was seen in the animal model studies reported here.
Of the previously mentioned targets, most have been evaluated as targets for drug-based immunotherapy, e.g., maytansinoid-conjugated antibodies targeting PSCA or PSMA (17, 24) or Auristatin E-conjugates of antibodies targeting TMEFF (25). Recently, the use of TMEFF2/Tomoregulin targeted radioimmunotherapy was explored in a preclinical study (26). A radioimmunotherapy approach to prostate cancer is being pursued in the clinic with 90Y-labeled J591, an anti-PSMA antibody conjugated to 1,4,7,10-tetra-azacylododecane N,N['],N[d'],N-'J-tetraacetic (DOTA) as the chelator (27). Preclinical studies with [90Y]-DOTA-J591 have been reported in LNCaP cells propagated as xenografts in nude mice in a fashion similar to that described here. Although antitumor effects were reported, it was found that efficacy was achieved in this model only in conjunction with significant toxicity and animal death (28, 29). In contrast, the therapeutic window that we report here with [90Y]-CHX-A''-DTPA-19G9 is clearly superior to that reported for [90Y]-DOTA-J591 in the same xenograft model. Because signs of efficacy were observed with 90Y-labeled J591 in the clinic only at or above the maximal tolerated dose (27), the large therapeutic window observed with our mindin/RG-1–specific antibody may translate into the much needed improved efficacy and tolerability in the radioimmunotherapy of advanced prostate cancer.
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Acknowledgments |
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The authors gratefully acknowledge Dr. Martin Brechbiel (NIH, Bethesda, MD) for providing the CHX-A''-DTPA chelator; Drs. Richard Laforest, Ananya Ruagma, and J. Yoo for coregistration imaging (Washington University School of Medicine, St. Louis, MO), Dr. Xiao-Yan Zhao for scientific discussions, Miriam Schroeder and Mithra Mahmoudi for technical assistance in performing immunohistochemistry studies, Rhonda Humm and Jean McRobbie for cell culture support (Berlex Biosciences).
Received 4/11/05. Revised 6/ 8/05. Accepted 7/13/05.
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