cancer immunity 1:7 (2001) - article(x-axis). tumor-bearing mice were treated with buffer, meth...

LATEST PAPERS SEARCH for PAPERS Printer-friendly PDF Comment(s)

>Abstract >Introduction >Results >Discussion >References >Materials & methods >Contact authors

Cancer Immunity, Vol. 1, p. 7 (27 April 2001) Submitted: 4 March 2001. Accepted: 22 March 2001.Communicated by: Pramod K. Srivastava

Determinants of efficacy of immunotherapy with tumor-derived heat shock protein gp96

Joseph T. Kovalchin1, Ananth S. Murthy2, Mark C. Horattas2, Daniel P. Guyton2, and Rajiv Y. Chandawarkar3

1Department of Biomedical Sciences, Kent State University2Department of General Surgery, Akron General Medical Center and Northeastern Ohio University College of Medicine, Akron, OH 443073Division of Plastic Surgery, Akron General Medical Center and Northeastern Ohio University College of Medicine, Akron, OH 44307

Keywords: mice, metastasis, heat shock protein, gp96, immunotherapy, adjuvant therapy

Abstract

Immunotherapy with gp96 was highly effective in mice bearing methylcholanthrene-induced fibrosarcomas (MethA tumors) when treatment began 7 days or less after tumor challenge, but significantly less effective if thetreatment began 9 days after challenge. Immunotherapy of pre-existing tumors showed all the hallmarks ofspecificity of gp96 and dose-restriction observed previously with prophylactic studies. When mice with largeprimary Meth A tumors were treated with surgery alone, or with surgery followed by therapy with Meth A-derivedgp96, the mice that received surgery and immunotherapy did significantly better than those receiving surgeryalone. The relationship between the time of initiation of immunotherapy with gp96 and its efficacy was also testedin a metastatic model of the Lewis lung carcinoma. In this model, immunotherapy with gp96 was very effective iftreatment began up to 31 days after tumor challenge, but significantly less so if therapy was initiated day 33post-tumor challenge. These observations suggest that the regulatory phenomena that interfere withimmunotherapy gather momentum with surprising speed.

Introduction

Heat shock proteins (HSPs) isolated from cancer cells have been shown be immunogenic specifically against thecancers from which the HSPs were isolated (see 1,2 for reviews). HSPs isolated from normal tissues or fromnon-autologous tumors are not immunogenic. The specificity of immunogenicity has been shown to derive fromthe antigenic peptides associated with cancer-derived HSPs but not with normal tissue-derived HSPs (3), asdemonstrated in murine (4) and now in human cancers (5). The mechanism of immunogenicity involvesinteraction of the HSPs with an HSP-receptor CD91, on the antigen presenting cells (APCs), followed byre-presentation of selected chaperoned peptides by the MHC I molecules of the APCs (6,7). The MHC I-peptidecomplexes then stimulate the cognate CD8+ T lymphocytes. It appears that the HSP-chaperoned peptides arealso routed to presentation by the MHC II molecules and the consequent stimulation of cognate CD4+ T cells (8).Most of these aspects have been demonstrated in both the murine and human systems. The principlesdeveloped from these studies have also been extended to immunotherapy of viral infections (9,10,11).

Autologous tumor-derived preparations of the HSP gp96 are now being tested in a number of clinical trials(12,13). While tumor-derived HSPs have been shown to be effective in prophylaxis against and in therapy of anumber of murine (14,15) and rat tumors (16,17), the optimum parameters have not been defined systematically.These include the optimal dosage, regimen, the stage of tumor growth, primary versus metastatic tumor, andsynergy of immunotherapy with surgical excision of the tumor mass, to name a few. As an example, twoimmunizations have been shown to be effective in immunizing rats (16), mice (14), and frogs (18)

Cancer Immunity 1:7 (2001) - ARTICLE

http://www.cancerimmunity.org/v1p7/010307.htm (1 of 9)

http://www.cancerimmunity.org/PHP/latest_papers.phphttp://www.cancerimmunity.org/statics/search.htmhttp://www.cancerimmunity.org/v1p7/010307.pdfhttp://www.cancerimmunity.org/PHP/comments.php?DocID=010307http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9865680&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9655479&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8189059&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8376942&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11196165&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=7545313&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10839810&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11274422&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8280719&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9334371&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9480978&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11004674&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11062489&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=3458189&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=6698641&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10425272&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=6698641&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=3458189&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11265634&dopt=Abstract

prophylactically against a subsequent cancer challenge, but smaller and larger numbers of immunization havenot been tested. Therapy has been carried out with four or more treatments (15,17) but the effects of shorter orprolonged treatments have not been measured. A dose-restriction of immunogenicity of gp96 has been observedin the prophylactic setting (14,19) but its applicability has not been tested in therapy.

An examination of these and other parameters is of crucial significance for translation of this method ofimmunotherapy from animal experimentation to clinical reality. This study examines some of these parametersusing the murine models of the methylcholanthrene-induced BALB/c mouse fibrosarcoma Meth A as anon-metastatic primary tumor, and the spontaneous Lewis lung carcinoma of C57BL/6 mice, as a metastatictumor.

Results

Immunotherapy of early and late primary Meth A tumors

BALB/c mice were inoculated with 100,000 Meth A cells intradermally. The tumors were allowed to grow for 5, 7or 9 days at which time the tumors were typically 2.0, 5.1 and 8.4 mm in diameter respectively. Mice bearingthese tumors were then treated either with saline, Meth A-derived gp96, or as a negative control, BALB/cliver-derived gp96. Gp96 was administered intradermally and 0.5, 1 or 5 µg gp96 per injection wereadministered. Each mouse received four immunizations given on alternate days. As an example, mice thatbegan treatment on day 5 were immunized on days 5, 7, 9 and 11 (Fig. 1).

Figure 1. Immunotherapy of early and late primary Meth A tumors with gp96. Mice withpre-existing tumors of different sizes were treated with gp96 as shown in the left panel. Tumorrejection assays are shown in the other columns comparing tumor diameter (Y-axis) with time(X-axis). Tumor-bearing mice were treated with buffer, Meth A-derived gp96 at 0.5, 1.0 or 5.0µg/day, or liver-derived gp96 at 0.5 or 1.0 µg/day as indicated. Each line shows the kinetics oftumor growth in a single mouse.

It was observed that mice that began treatment with Meth A gp96 5 or 7 days after tumor cell implantationresponded vigorously to immunotherapy (Figs. 1 and 2). While tumors continued to grow for up to 10 days after



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10425272&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=3458189&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10224283&dopt=Abstract

tumor cell implantation in all groups of mice, the tumors of Meth A gp96-treated mice began to regress at aboutday 10-12 and the regression continued for the next several weeks. Most mice in these groups were substantiallyor completely cured of visible disease. Immunization with gp96 derived from normal liver had no influence on thekinetics of tumor growth (Figs. 1 and 2) as reported previously in models of prophylactic immunity to Meth A (3).Further, immunization was effective in mice that received 1 µg per immunization, but not in those that receivedless (0.5 µg per immunization) or more (5 µg per immunization) gp96 (Figs. 1 and 2). Thus, the activity of gp96was dose-restricted as has also been reported previously in models of prophylactic immunity to Meth A (14,19).

Figure 2. Immunotherapy of early, intermediate and late primary Meth A tumorsdemonstrating antigen specificity of gp96, timing of treatment and dosage - Summary of thedata in Figure 1. (A) Tumor-bearing mice were treated with tumor-derived gp96, liver-derived gp96or buffer alone. Tumor diameters were monitored. (B) Tumor-bearing mice received gp96-adjuvanttherapy starting on day 5 (early), day 7 (intermediate) or day 9 (late). Tumor diameters weremonitored. (C) Tumor-bearing mice were treated with varying doses of Meth A-derived gp96.Amounts indicated in the legend represent the dose given per injection 4 times on alternate days.Tumor diameters were monitored. Each line is an average of the data from 5 animals.

In contrast to mice that were treated beginning 5-7 days after tumor cell implantation and that responded well toimmunotherapy, mice that began treatment 9 days after tumor cell implantation responded significantly butrelatively poorly to immunotherapy with Meth A-derived gp96. Tumors of these mice began to undergo shrinkagewith kinetics initially similar to that of mice which began treatment early; however, by day 12-15, the tumors oflate-treated mice began to grow again but remained significantly smaller than the tumors of saline-treated mice(Figs. 1 and 2).

Influence of immunotherapy as an adjunct to partial surgical excision of established Meth A tumors

As mice bearing 9 day old Meth A tumors were relatively refractory to immunotherapy with gp96, furtherexperimental treatments were attempted with them. They were treated by surgery alone or by surgery followedby immunization with 5 injections of Meth A-derived gp96 on alternate days (see Fig. 3 for experimental design).Immunization was carried out with the 1 µg per injection dose determined to be optimal in the experiments shownin Figure 2, as well as in previous studies (19). It was observed that while tumors resumed growth in mice whichhad undergone surgery alone (Fig. 4 B), tumors of mice that had received immunotherapy with gp96 as anadjunct to surgery remained relatively stable for nearly 20 days after surgery (Fig. 4 C, D). At that point, tumors insome of the gp96-treated mice resumed growth. However, there remained a significant difference in the size of



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8189059&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=3458189&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10224283&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10224283&dopt=Abstract

tumors in mice that were treated with surgery alone versus those that received immunotherapy as an adjunct tosurgery.

Figure 3. Study design for tumor-gp96-adjuvant therapy of post-surgical residual disease.Mice bearing late tumors underwent partial resection on day 9, with 60% residual tumor volume,and were treated with tumor-derived gp96 thereafter, as indicated.

Figure 4. Results of tumor-derived gp96 adjuvant therapy. Tumor volumes from mice thatunderwent surgical excision followed by treatment with buffer (A) or 5 doses of tumor-gp96 (1µg/dose starting on day 9) after surgical incision (B). Data from (A) and (B) are summarized in (C).

Immunotherapy of metastatic Lewis lung carcinoma

Highly metastatic lines of the spontaneous Lewis lung carcinoma have been used as models of aggressive andmetastatic spontaneous human cancers. This model has been used in several types of immunotherapy includingimmunotherapy with autologous tumor-derived gp96 (15). Syngeneic C57BL/6 mice were inoculated with100,000 cells of the D122 line of Lewis lung carcinoma and the tumor was allowed to grow until day 24post-tumor cell implantation. At this time, the tumor was typically 3-4 mm in diameter and was surgically excisedby amputation of the foot. Untreated, these mice succumb to metastatic tumor growth in the lungs. Therapy ofthese mice was begun on day 29, 31 or 33 after tumor cell implantation (or day 5, 7 or 9 after surgery) (see Fig. 5



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstract

for experimental design). Mice were treated with 4 immunizations of 1 µg D122-gp96 per intradermal injectiongiven on alternate days. Survival of mice was monitored on day 33 post-tumor cell implantation. Two significantpatterns were observed (Table 1). While nearly all mice treated with buffer succumbed to tumor growth (27/28),100% of the 20 mice that began treatment with D122-gp96 on day 29 and 31 post-tumor cell implantation (day 5or 7 post-surgery) remained tumor-free. In contrast, of the mice that began treatment only 2 days later, i.e. onday 33 after tumor cell implantation (or day 9 post-surgery), 40% (4/10) succumbed to metastatic tumor growth.Figure 6 is a set of representative photographs that demonstrate the tumor burden within the thoraces of micetreated with gp96 at various time points.

Figure 5. Study design of post-operative adjuvant therapy of metastatic disease using gp96derived from the completely resected primary tumor. Primary tumors, inoculated in the foot,were removed from mice by amputation of the foot, and tumors were used as a source of gp96.Mice with subsequent tumor metastasis were treated early, intermediate or late depending on theday of commencement of post-operative treatment. Control animals received either buffer or gp96derived from liver.

Table 1. Gp96-therapy of metastatic disease.

Start of treatmentpost-tumor challenge

(day)

Disease-incidence in mice treated with

Buffer D122-gp96 Liver-gp96

29 89% (8/9) 0% (0/10) 44% (4/9)

31 100% (10/10) 0% (0/10) 55% (5/9)

33 100% (9/9) 40% (4/10) 100% (9/9)

The second notable pattern concerns the immunological activity of liver-gp96. Significant protection from tumorgrowth was observed in mice that began treatment with liver-gp96 on days 29 or 31 post-tumor cell implantation.Exactly 50% of such mice (9/18) remained tumor-free. While this is significantly lower than 100% of theD122-gp96-treated mice that remained tumor-free, it is also significantly higher than the absence of protection inthe buffer-treated mice. However, liver-gp96 afforded no protection among mice that began to receive treatmenton day 33 post-tumor transplant.



Figure 6. Representative photographs of thoracic cavities of D122-bearing mice treated withbuffer or tumor-derived gp96. Refer to the legend to Figure 5 and in Table 1. Thoracotomieswere performed via a midline sternal incision, subsequently exposing the heart and the lungs.

Discussion

Our observations confirm and extend the previous studies on the immunological activity of tumor-derived heatshock protein gp96. In addition, they uncover novel aspects of the activity of gp96 in immunotherapy ofpre-existing cancers and these have a significant bearing on the use of gp96 for immunotherapy of humancancer. Previous observations confirmed by our results include the ability of tumor-derived gp96 to mediateanti-tumor activity in primary and metastatic tumor models (15). Two other major facets of immunogenicity ofgp96 are also reproduced here: tumor-derived but not normal tissue-derived gp96 preparations elicit anti-tumoractivity (3,4) and the dose-restricted immunogenicity of gp96 (14,19).

The novelty of our observations lies in two major areas. We demonstrate a dramatic correlation between the timeof initiation of treatment and the consequence of immunotherapy with gp96. Immunotherapy is very effectivewhen begun 7 days post-tumor cell implantation (Meth A) or 31 days post-tumor cell implantation (D122). A delayof two additional days in either model makes the tumors refractory to immunotherapy. In the case of Meth A, thetiming of relative resistance to immunotherapy (approximately day 9 post-tumor cell implantation) correlates wellwith the previously reported timing of appearance of suppressor T lymphocytes (20). It is particularly noteworthythat even at 9 days post-tumor cell implantation, the mice respond to immunotherapy with gp96 until day 15 atwhich time tumor growth takes over sharply and uniformly in all mice. Surgical intervention on day 9, coupledwith immunotherapy with gp96, stabilizes tumor growth further. These observations indicate that althoughdown-regulatory events begin to take the lead somewhere between days 9 and 15, these events are still notirreversible. Surgical excision of the tumor tilts the balance in favor of the host such that immunotherapy coupledwith surgery still enables immunological activity against the tumor, resulting in prolonged stabilization of tumorsize. These observations highlight the dynamic and fragile interplay between immunogenic and down-regulatoryevents between days 9-15 in Meth A and between days 31-33 in D122. The sharpness with which the micebecome relatively refractory to immunotherapy suggests that the down-regulatory immunological activities areactive, dominant and gain-of-function events rather than passive, co-dominant and loss-of-function events.

The second novel aspect of our results lies in the observed efficacy of liver-derived gp96, in the D122 model(Table 1). In this model, liver-gp96 is clearly more active than PBS and clearly less active than D122-gp96. Theimmunological activity of gp96 has been repeatedly shown to be present solely in the autologous tumor-derived



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8189059&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8376942&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=3458189&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10224283&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=6232335&dopt=Abstract

gp96 preparations (3,4,15). However, if one examines the data carefully, one notes that although thetumor-derived gp96 preparations are far more efficacious than control gp96 preparations, the latter have someactivity above the saline controls (4). Indeed, in the D122 model, Tamura et al. (15) noticed and commentedupon this fact during a statistical analysis of the anti-tumor activity in mice immunized with saline versus normaltissue-derived gp96. The immunological basis of this activity may lie in the activation of the innate components ofthe immune response stimulated by interaction of gp96 with APCs as demonstrated recently (21,22). Suchactivation may provide a more favorable microenvironment for amplification of the adaptive anti-tumor immuneresponse elicited by the tumor itself.

Our observations highlight the need for the examination of a number of issues, including new opportunities, forthe translation of the HSP approach to human cancer immunotherapy. These include the identification of amechanistic basis for the resistance to immunotherapy that develops in hosts with tumors growing for longerperiods, the detailed and systematic analysis of the effective dosage and regimen in a number of animal modelsof cancer, and the use of autologous (i.e. individual tumor-derived) and generic gp96 preparations and possiblytheir combinations in the therapy of experimental cancers.

Abbreviations

HSP, heat shock protein; Meth A, methylcholanthrene-induced fibrosarcoma

Acknowledgements

We thank Robert J. Binder of the University of Connecticut Medical Center, Center for Immunotherapy of Cancerand Infectious Disease for reviewing our manuscript and for his insights, James Lehman Jr. and James M. Lewisof Akron General Medical Center, Division of Plastic Surgery for their helpful comments, and Nairmeen Hallerand Lisa Treen of the Kenneth L. Calhoun Research Laboratory at Akron General Medical Center forexperimental help.

References

1. Menoret A, Chandawarkar R. Heat-shock protein-based anticancer immunotherapy: an idea whose time has come. Semin Oncol 1998; 25: 654-60.(PMID: 9865680)

2. Srivastava PK, Menoret A, Basu S, Binder RJ, McQuade KL. Heat shock proteins come of age: primitive functions acquire new roles in an adaptiveworld. Immunity. 1998; 8: 657-65. (PMID: 9655479)

3. Udono H, Srivastava PK. Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol 1994; 152:5398-403. (PMID: 8189059)

4. Udono H, Srivastava PK. Heat shock protein 70-associated peptides elicit cancer-specific immunity. J Exp Med 1993; 178: 1391-6. (PMID:8376942)

5. Castelli C, Ciupitu AM, Rini F, Rivoltini L, Mazzocchi A, Kiessling R, Parmiani G. Human heat shock protein 70 peptide complexes specificallyactivate antimelanoma T cells. Cancer Res 2001; 61: 222-7. (PMID: 11196165)

6. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995; 269: 1585-8.(PMID: 7545313)

7. Castellino F, Boucher PE, Eichelberg K, Mayhew M, Rothman JE, Houghton AN, Germain RN. Receptor-mediated uptake of antigen/heat shockprotein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J Exp Med 2000; 191:1957-64. (PMID: 10839810)

8. Matsutake T, Srivastava PK. The immunoprotective MHC II epitope of a chemically induced tumor harbors a unique mutation in a ribosomal protein.Proc Natl Acad Sci U S A 2001; 98: 3992-7. (PMID: 11274422)



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8189059&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8376942&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=8376942&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11058573&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=11086034&dopt=Abstract

9. Blachere NE, Udono H, Janetzki S, Li Z, Heike M, Srivastava PK. Heat shock protein vaccines against cancer. J Immunother 1993; 14: 352-6.(PMID: 8280719)

10. Blachere NE, Li Z, Chandawarkar RY, Suto R, Jaikaria NS, Basu S, Udono H, Srivastava PK. Heat shock protein-peptide complexes, reconstitutedin vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med 1997; 186: 1315-22. (PMID: 9334371)

11. Ciupitu AM, Petersson M, O'Donnell CL, Williams K, Jindal S, Kiessling R, Welsh RM. Immunization with a lymphocytic choriomeningitis viruspeptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J Exp Med 1998; 187: 685-91.(PMID: 9480978)

12. Janetzki S, Palla D, Rosenhauer V, Lochs H, Lewis JJ, Srivastava PK. Immunization of cancer patients with autologous cancer-derived heat shockprotein gp96 preparations: a pilot study. Int J Cancer 2000; 88: 232-8. (PMID: 11004674)

13. Srivastava PK. Immunotherapy of human cancer: lessons from mice. Nat Immunol 2000; 1: 363-66. (PMID: 11062489)

14. Srivastava PK, DeLeo, AB, Old LJ. Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc Natl Acad Sci U S A 1986; 83:3407-11. (PMID: 3458189)

15. Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science 1997; 278: 117-20. (PMID: 9311915)

16. Srivastava PK, Das MR. The serologically unique cell surface antigen of Zajdela ascitic hepatoma is also its tumor-associated transplantationantigen. Int J Cancer 1984; 33: 417-22. (PMID: 6698641)

17. Yedavelli SP, Guo L, Daou ME, Srivastava PK, Mittelman A, Tiwari RK. Preventive and therapeutic effect of tumor derived heat shock protein,gp96, in an experimental prostate cancer model. Int J Mol Med 1999; 4: 243-8. (PMID: 10425272)

18. Robert J, Menoret A, Basu S, Cohen N, Srivastava PK. Phylogenetic conservation of the molecular and immunological properties of thechaperones gp96 and hsp70. Eur J Immunol 2001; 31: 186-95. (PMID: 11265634)

19. Chandawarkar RY, Wagh MS, Srivastava PK. The dual nature of specific immunological activity of tumor-derived gp96 preparations. J Exp Med.1999; 189: 1437-42. (PMID: 10224283)

20. North RJ, Bursuker I. Generation and decay of the immune response to a progressive fibrosarcoma. I. Ly-1+2- suppressor T cells down-regulatethe generation of Ly-1-2+ effector T cells. J Exp Med 1984; 159:1295-311. (PMID: 6232335)

21. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partialmaturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol 2000; 12: 1539-46. (PMID: 11058573)

22. Binder RJ, Anderson KM, Basu S, Srivastava PK. Cutting edge: heat shock protein gp96 induces maturation and migration of CD11c+ cells in vivo.J Immunol 2000; 165: 6029-35. (PMID: 11086034)

23. Srivastava PK. Purification of heat shock protein-peptide complexes for use in vaccination against cancers and intracellular pathogens. Methods1997; 12: 165-71. (PMID: 9184380)

Materials and methods

Purification of gp96

Gp96 was purified from normal liver, Meth A ascitic cells and D122 Lewis lung carcinoma as described (23).

Immunization and tumor challenge studies

Mice were challenged with Meth A or D122 cells and were immunized with gp96 as described (15,19).

Surgical techniques

BALB/c mice were inoculated with 100,000 live Meth A cells intradermally on their backs. On day 9 followinginoculation, these tumors were measured in three axes, including the diameters in two planes and the thickness.After deeply anesthetizing them, surgery was performed under sterile conditions approved by the Animal CareCommittee at the Akron General Medical Center. Tumors were excised tangentially in a plane parallel to theunderlying skin, taking care to leave at least 60% residual tumor volume. The tangential excision was preferredto create a new tumor surface that would be equidistant from the tumor bed, ensuring an equal nutrient supply tothe outer peripheral surface. Hemostasis was achieved with pressure alone, thus preventing any confoundingimpact of thermocoagulation on tumors. Mice were then housed in separate cages for 2-3 hours and theirrecovery closely monitored. Residual tumor volumes as well as those of the tumors excised were recorded.



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9184380&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=9311915&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pubmed&list_uids=10224283&dopt=Abstract

Contact

Address correspondence to:

Rajiv Chandawarkar, M.D.Division of Plastic SurgeryAkron General Medical Center400 Wabash AvenueAkron, OH 44307Tel: + 1 330 384-6000E-mail: [email protected]

Copyright © 2001 by Rajiv Chandawarkar



mailto:[email protected]

cancerimmunity.orgCancer Immunity 1:7 (2001) - ARTICLE

cancer immunity 1:7 (2001) - article(x-axis). tumor-bearing mice were treated with buffer, meth...

Documents