New strategies for active immunotherapy engineered

tumor cells

Drew The Johns Hopkins

While

University

previous

clearcut

tumor

vaccine

has been

newer

approaches

have

tumor

cells

certain

genetically

cytokines. fashion

producing

significant

this approach of tumor

can

been

induce

systemic

have

to establish

developed

so that

can alter the

antigen-specific

strategies

difficult

Engineering

paracrine

Pardoll

School of Medicine,

efficacy

with genetically

they

shown in human

in animal

express

tumor

cells

powerful toxicity.

Baltimore,

new

to

local

of tumor

T lymphocytes,

resulting

USA

results,

trials. Recently, that

antigens

secrete

In addition

presentation

intriguing

systems

cytokine

Maryland,

modify

or secrete

cytokines effects

in

a

without

to local inflammation, antigen

or activation

in systemic

antitumor

immunity.

Current

Opinion

in Immunology

Introduction Tumor immunity is dependent on the existence of antigens within tumors that can be recognized as foreign by the host immune response. It is the tremendous diversity of the T- and B-cell receptors that endows this system with the ability to distinguish fine antigenic differences among cells. For the generation of an antitumor response, two criteria must be fulfilled. First, the tu mor must present novel antigens or neo-epitopes not found on normal cells. Second, the immune system must be appropriately activated to respond to these novel antigens. The classic studies of Boon and colleagues [1,2*] have demonstrated that tumor antigens recognized by T cells fall into one of two categories; (a) novel peptide sequences generated by point mutations in genes encoding various cellular proteins or (b) cases in which the gene encoding the tumor antigen is identical to the germline sequence, but is not expressed in any normal tissues. As a consequence the immune system need not be tolerized to the gene during development and peptides derived from the non-mutated form serve as a tumor-specific antigen. Two general strategies of cell-based immunotherapy have been developed for cancer. Adoptive immunotherapy involves the expansion of tumor-reactive lymphocytes in vitro followed by their reinfusion into the host. The alternate strategy, active immunotherapy, involves immunization with tumor cells in the hope of generating either novel or enhanced immune responses against tumor antigens, which can result in systemic responses against tu mor deposits.

1992, 4:619623

Specific active immunotherapy vaccines have already been used to treat cancer patients with solid tumors. These vaccines have been prepared in various ways, but typically, irradiated tumor cells have been mixed with adjuvants such as bacille Calmette-Guerin (BCG) in order to elicit new immune responses. While it is clear that irradiated tumor cells can generate protective immune responses in a number of tumor models, there has been little evidence that the inclusion of adjuvants enhances the immune response directed by the T-cell arm of the immune system. Furthermore, these immune responses have tended to be relatively weak and therefore ineffective against established tumors. Recently, there has been renewed interest in active immunotherapy approaches using genetically altered tu mors. The first studies demonstrating enhanced immunogenic&y of genetically altered tumors were performed 25 years ago, starting with Lindenman and Klein [3], who showed that vaccination with influenza virus infected tumor cells generated enhanced systemic immune responses against a challenge with the original wild-type tumor cells. The next generation of genetically altered tu mor cell vaccines involved the use of mutagenesis to generate immunogenic variants of non-immunogenic tumor cells that upon immunization could generate enhanced immune responses against the original non-immunogenic variants. As newer techniques of gene transfer have been developed, infection with infectious virus and mutagenesis have been replaced with specific gene transfer in an attempt to more carefully regulate the nature of the genetic alteration in the tumor. Most recently, there has been intense interest in the study of immune responses generated by tumor cells engineered to secrete various

Abbreviations CTL-cytotoxic MCP-monocyte

T lymphocyte; C-CSF-granulocyte colony stimulating factor; IFN-interferon; chemoattractant protein; MHC-major histocompatibility complex; TN&tumor @ Current

Biology

Ltd ISSN 0952-7915

IL-interleukin; necrosis factor. 619

620

Cancer

cytokines. This latter strategy does not involve inducing the expression of any foreign genes in tumor cells, but rather, seeks to locally alter the immunological environment of the tumor cell so as to either enhance antigen presentation of tumor-specific antigens to the immune system or to enhance the activation of tumor-specific lymphocytes. These various strategies are reviewed here, with emphasis on results from the more recent studies on cytokine gene transfer into tumor cells.

Mutagenesis

studies

Studies originally performed by Boon and colleagues [ 41, demonstrated that mutagenesis in tu’tro of tumorigenic cell lines could produce subclones that were non-tumorigenie, as defined by their inability to establish tumor takes in syngeneic mice at inoculum doses significantly larger than those that were tumorigenic in the original variant. It was hypothesized that these turn- variants were non-immunogenic on the basis of the expression of novel tumor antigens that increased the tumor’s immunogenicity. The molecu~dr basis for this increased immunogenicity was elegantly dissected using a ‘shot-gun’ cosmid transfection technique, followed by sib selection to identify DNA segments encoding antigens recognized by cytotoxic T lymphocyte (CTL) clones derived from mice immunized with the turn- variants [ 11. Comparison of the sequence of an identified gene in the turn ~ variant with the germline sequence indicated that a single amino acid change produced by a point mutation had conferred upon a peptide derived from that region of the gene the ability to bind to the Ld MHC class I molecule expressed by the tumor. Identification of the specific neoantigen that abrogated tumorigenicity in the turn- variants did not, however, explain how mice that had rejected the turnvariant were now immunized against challenge with the wildtype turn+ cell line. Nonetheless, this result suggests that the parental turn+ cell line possess antigens cdpable of being recognized by lymphocytes. Somehow, the immune response generated by the stronger antigens in the turn- variant resulted in an enhancement of an im mune response against the ‘weaker’ antigens in the turn+ cells.

Generation

of immunogenic

transfection

with foreign

variants

by

genes

A variant of the mutagenesis strategy involved the use of transfection to induce the expression of foreign genes in tumors. In the first reported study with this strategy, allogeneic class I MHC genes were introduced into tumors [ 51. When an Ld class I gene was introduced into an H2h Lewis lung tumor line, these cells were rejected in the sygeneic C57BL6 mice. Introduction of the Ld gene into these tumors reduced their tumorigenicity to less than 1/40th of that of the parental tumor cells. After inoculation and spontaneous regression of the viable Ld+

clones, the mice acquired resistance against challenge with the parental Lewis lung tumor. In that study, however, Ld+ and Ld- neo-resistant transfectants showed no statistical difference in their ability to generate a systemic immune response against the parental tumor. More recently, tumors have been transfected with viral genes encoding proteins known to be highly immunogenic in the antiviral response [6]. Thus, introduction of the influenza HA gene into BALB/c-derived CT26 colon tumor cells, followed by repeated FACS sorting of highexpressing variants, resulted in transfectants that were rejected by BALB/c mice at doses 100 times greater than the parental tumor. Again, these HA transfectants induced a systemic immune response against challenge m?th the parental ~~26 tumors. While only high HA expressors were able to be rejected by syngeneic animals and induce systemic immune responses against the original tumor, there was significant discrepancy between different subclones of HA transfectants expressing similar HA levels, suggesting that other factors besides simply HA expression contributed to immunogenic&y of the tumor.

introduction

of MHC

class II genes into tumor

cells While most epithelial tumors express MHC class I and can therefore present class I restricted antigens to CD8+ 7’cells, they are almost exclusively MHC class II- unless treated with cytokines such as interferon (IFN)-y. In certain tumor models the introduction of MHC class II genes into tumor cells results in a decrease in their tumorigenicity and generation of systemic immune responses against the parental tumor [ 71. It was postulated that the expression of MHC class II molecules on tumor cells allowed for the presentation of class II restricted, tumor-specific antigens to T helper cells, which ultimately provided in uizlo help for CTLs capable of eliminating the MHC class II parental challenge. This hypothesis remains to be proven, as epithelioid tumor cells are typically no? considered to be effective antigen-presenting cells for T helper cells. Nonetheless, a number of studies have clearly demonstrated that endogenous antigens can be presented via the class II pathway. Currently, a number of groups are attempting to cointroduce genes encoding MHC class II molecules and B7 into tumor cells. It has recently been demonstrated that B7 engages CD28 on 1‘ cells and that this signal represents a critical cost_imulato~ signal for T helper cell activation [8,9]. Thus, the combined expression of B7 and MHC class II on tumor cells may potentially enhance presentation .of class 11 specific tumor antigens to CD4+ T helper cells.

Introduction

of cytokine

genes into tumor

ceils

Without question, the greatest amount of research in the area of genetically altered tumor vaccines has been with

immunotherapy

the introduction of cytokine genes. Quite a few cytokine genes have been introduced into tumor cells with varying effects on both tumorigenicity and immunogenicity. In general, two sorts of antitumor responses have been analyzed in these systems. Many of the cytokines, when produced by tumors, induce a local inflammatov response which results in elimination of the injected tumor. As discussed below, this local inflammatory response is most often predominantly dependent on leukocytes other than classical T cells. These systems have been used to uncover irz !‘i?~oeffects of cvokines that arise with the activation of tumoricidal potential by various types of leukocytes. In a subset of the studies. systemic immune responses were generated against challenge with the wild-type parental tumor. In all cases in which systemic immunity against wild type challenge has been analyzed, it is mediated by 7‘ cells. An analysis of the various studies in which particular lymphokines were used reveals a number of critical variables that affect both the local inllammatoty response and the capacity to generate systemic immune responses against the parental tumor. (a) The many different transplantable tumor systems that are used in these studies are variable in the extreme. Furthermore, subclones of the same original tumor type can behave extremely differently when passaged independently for as little as a few months in culture. (b) There are significant differences in the dose of tumor cells used for immunization as well as challenge in the various studies, which can have significant effects on the characteristics of the immune response generated. (c) Iarge differences have been reported in the level of cytokine gene expression because most of the studies used different vectors for gene transfer, with different promoters and enhancers regulating gene expression. In a number of cases, the magnitude and characteristics of the local inflammatory as well as the systemic immune response were highly dependent on the levels of cytokine produced. (d) The location of immunization and challenge also dithered among studies. Three major routes of tumor cell immunization and/or challenge have been used, namely subcutaneous, intravenous or intraperitoneal. As the lymphatic drainage and circulatory characteristics of these diRerent compartments varies tremendously, it is not surprising that significantly different results have been obtained as a function of these different routes of cell administrdtion.

Interleukin-2

A number of groups have analyzed the tumorigenicity and immunogenic@ of tumors engineered to secrete interhkin (IL)-2 [l&12]. IIdtransduction was fkt USed in an attempt to bypass a putatively defective helper arm of the immune response and thereby directly provide a second signal to tumor-specific CTLs [lo]. In all cases, expression of high levels of IL-2 resulted in the regression of even large innocula of tumors. Histological evaluation of the rejecting tumors revealed a massive infiltrate of lymphocytic cells. In many of the tumor models, a systemic immune response against the parental tumor was indeed generated. Rejection of the IL-2 transduced

with genetically

engineered

tumor

cells Pardoll

tumor cells was dependent on CD8+ cells but not on CD4+ cells, suggesting that T helper cells are rendered irrelevant in the rejection response. Of note, however, was a significant delay in tumor formation in the mice depleted of CDS, suggesting that an additional cell type was important in the rejection of these IL-2 transduced tumor cells. Recent studies have indicated that locally activated natural killer cells are a critical local effector cell in the rejection of IL-2 transfected tumors. Gilboa and colleagues [ 1I] did a careful analysis of the correlation between levels of IL-~ production in different subclones of retrovirally transduced tumor cells and local rejection as well as induction of systemic immunity. They found a clearcut correlation between increased levels of local lL2 production and both systemic and local antitumor responses.

lnterleukin-4

The first indications that local production of IL-4 produces an antitumor inflammatory response were uncovered fortuitously by Tepper et al. [ 131. While studying the effects of potential autocrine stimulation of B-cell tumors by IL-4 secretion, they found that these tumors were, in fact, rejected after introduction into syngeneic animals. A histological analysis of the infiltrate revealed large numbers of macrophages and eosinophils. While no evidence of a systemic immune response wzs uncovered in the original study, a subsequent study using IL-4 transduced Renca tumor cells revealed that while the local inflammatory infiltrate was quite similar, a systemic immune response was generated against the parental tumor [14*]. In the case of Renca, small doses of established tumors can be cured after subsequent injection of IL-4 transduced cells subcutaneously at a distant site. Thus, the immune response generated by cytokine gene-trdnsduced tumor cells is powerful enough to eliminate an established tumor, albeit only small doses. In the Renca IL-4 transduction study, in ztzjo antibody depletions of CD4+ and CDS+ T cells were performed in an attempt to determine which T-cell subsets were important for the generation of systemic immune responses. It was found that elimination of CD8 completely abrogated the systemic immune response, indicating that class I restricted CTIs were critical for the effector phase of systemic im munity. Depletion of CD4+ cells also partly diminished systemic immunity. These results, together with the histological findings of large numbers of macrophages and veq small numbers of T cells at the site of the transduced tumor suggest that the generation of T-cell dependent systemic immunity by IL-4 transduced tumor cells may be at least partly due to the enhanced presentation of tumor antigens by influxing macrophages to CD4+ T helper cells as an intermediary in the ultimate stimulation of tumor-specific CD8+ CTIs;.

Interferon-y

A number of studies that introduced the IFN-y gene into tumor cells have demonstrated a concomitant induction of both MHC class I and MHC class II gene products

621

622

Cancer

and, in certain cases, the induction of rejection of the tumor [ 15-171. In one study, the systemic immune response generated against parental tumors was shown to be CDS-dependent. A recent study has correlated the enhanced ability of IFN-y transduced tumor cells to present MHC class I restricted antigens with the enhanced generation of tumor-reactive, CDS+ tumor-infiltrating lymphocytes and enhanced therapeutic efficacy in adoptive transfer [ 171, The effects of IFN-?( are, however, quite tumor system-dependent, and in certain cases the cotransduction of IFN-)I with other cytokine genes actu ally reveals an inhibitory effect of IFN-y in the generation of systemic immune responses.

Tumor

necrosis

factor

The gene encoding tumor necrosis factor (TNF) has also been introduced into tumor cells [ 18,191.Typically, TNFtransduced tumor cells grow more slowly in vitro and are often found to be rejected when injected into syngeneic animals. It is unclear whether the rejection phenomenon is due simply to the direct effects of TNF on the tumor cells or whether TNF has additional effects on inflammatory cells such as macrophages. Nonetheless, a number of groups have shown that animals that have rejected TNF-transduced tumor cells are immune to challenge with the parental tumor; as with other cytokines, CD8+ T-cell depletion abrogates the systemic immune response.

Interleukin-7

Similar generation of antitumor immune responses has been reported by immunization with tumor cells en@ neered to secrete IL-7 [20-l. It is interesting that the systemic immunity in one system of IL-7 transduced cells was independent of CD8+ T cells but dependent on CD4+ T cells. Furthermore, CF3+ cells, presumably macrophages, were also required for tumor rejection. As the tumor used in this study was an MHC class II+ plas macytoma line, it is possible that the antitumor response generated by CD4 + cells was a response to MHC class II restricted tumor antigens. The CD4+ cells responding to MHC class II restricted tumor specific antigens may have subsequently secreted cytokines which acted as macrophage-activating factors, thereby recruiting tumoricidal macrophages responsible for the ultimate tumor destruction. Two additional cytokines, monocyte chemoattractant protein (MCP)-1 and granulocyte colony stimulating factor (G-CSF), have shown to be associated with local inflammatory-mediated rejection of tumors engineered to secrete these cytokines locally. In the case of G-CSF, the local inflammatory infiltrate was characterized by large numbers of granulocytes [21 I, and in the case of MCP-1, the local inflammatory infiltrate was characterized by large numbers of macrophages [ 221. In neither of these cases was a systemic immune response demonstrated.

Conclusion One of the most important concepts regarding immunization with tumor cells engineered to secrete cytokines is that the engineering of these cells to produce cytokines locally may increase the ability of the cytokine to activate immune responses against tumor antigens in a paracrine fashion, while the toxicity from systemic cy tokines is minimal. This is because the production of the cytokine is localized to the region of the genetically transduced tumor cell. It is also important to determine whether the effects of local cytokine secretion are truly at the level of antigen presentation or whether there are direct effects on infiltrating lymphocytes. As a prelude to rational human therapy with genetically modified tumor cells, multiple cytokine genes are compared in poorly immunogenic tumor models to assess which among the panoply of cloned genes generates the most potent systemic immune response. Finally, before largescale expensive clinical trials are undertaken, it will be important to definitively demonstrate that genetically engineered tumor cells in a number of systems truly generate enhanced systemic immune responses in a comparison with simpler approaches such as the use of adjutants or irradiated wild-type tumor cells.

References

and recommended

Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.

reading

within the annual period of re-

LURQUIN C, VAN PEL A, MARL~MEB, DE PLAEN E, SZIKORAJo P, JANSSENSC, REDDEH~E M, LEJEUNEJ, BUN T: Structure

for the Gene Coding Encoded Cytolytic

for Turnantigen P91A a Peptide by the Mutated Exon is Recognized with L* by T Cells. Cell 1989, 58:2933303.

VAN DER BRLJGGENP, TRAVERSAKIC, CHOMEZ P, LURQ~JINC, DE PLAEN E, VAN DEN EYNDE B, KN~JTHA, BOON T: A Gene Encoding an Antigen Recognized Cytotoxic T Lymphocytes on a Human Melanoma. Science 1991, 254:1643-1648. ‘The antigen recognized by tumor-specific CTLs against a human* melanoma is shown to be derived from a non-mutated gene that is expressed in the melanoma but not in any normal tissues. 2. .

3.

LINDENMANNJ, KLEIN P: Viral Oncolysis:

genicity of Host Virus. J Eq Med

Increased CeII Antigen Associated with 1967, 126:93-108.

ImmunoInfIuenza

4.

VAN PKI. A, BOON T: Protection Against a Nonimmunogenic Mouse Leukemia by an Immunogenic Variant Obtained by Mutagenesis. Proc Natl Acad Sci USA 1982, 79:471%4722.

5.

ITAYA T, YAMAGI~A S, OKADA F, OIKAWA T, KIIRIJMAKI N, of TAKEICHI N, HOSOKAW M, KOBAY~SHI H: Xenogenization

a Mouse Lung Carcinoma (3LL) by Transfection with an AIIogeneic Class I Major Histocompatibihty Complex Gene (H-2L*). Cancer Res 1987, 47:3136-3140. ’ 6.

FEARON E, ITAYA T, HUNT B, VOGEISTEIN B, FROST P: Induc-

tion in a Murine Tumor of Immunogenic by Transfection with a Foreign Gene. 38:2975-2980. 7.

OSTFZANDROSENBERG S, THAK~JRA, CLEMENTSV: Rejection

Mouse Sarcoma Cells after Transfection Genes, J Immunol 1990, 144:406%4071. 8.

Tumor Variants Cancer Res 1988, of of MHC Class II

LINSLEY P, BRAINY W, GROSMAIRE L, ARUFFO A, DAMLE N, LEIIHETTERJ: Binding of the B CeII Activation Antigen B7

Immunotherapy

to CD28 Costimulates mRNA Accumulation. 9.

T CeU Proliferation and Interleukin-2 / f?@ Med 1991, 173:721-730.

IO.

11.

12.

13.

FEAKON E, PARIX)I.I.D, ITAYA T, G~LIIMBEK P, LEQTSKY H, SIM~NSJ, KARASWAMAII, VOC;EISTEINB, FROST P: Interleukin2 Production by Tumor Cells Bypasses T Helper Function in the Generation of an Antitumor Response. Cell 1990, 6O397-403. GANSRACHI:RB, %II~RK, DANIEIS B, CK~NIN K, BANNI:RJIK, GIWOA E; Interleukin-2 Gene Transfer into Tumor Cells Abrogates Tumorigenicity and Induces Protective Immunity. J E.q ‘Ifed 1990, 172:1217-1224.

TFPI’ER R, PATTENGALE P,

Displays Potent 57:503-512. 14. .

Anti-Tumor

ISIXR

P: Murine Interleukin-4 Activity In Viva Cell 1989,

G~LUMES P, MENBY A, IEVITSKYH, JAFFEE E, KARASUYAMA H,

BAKERM, PARD~LLD: Treatment of Established Renal Cancer by Tumor Cells Engineered to Secrete Interleukin4. Science 1991, 254:713-716. Tumors engineered to secrete IL-4 can generate CD8+ T~cell depend dent systemic responses capable of curing animals of small amounts of established tumor. 15.

WATANABE Y, KLlIunAyASHI K, MNATAKE S, NISHIHARA K, N~YM E, TANNAXA T, SAKATA T: Exogenous Expression of Mouse Interferon-y cDNA in Mouse Neuroblastoma Cl300 Cells Results in Reduced Tumorigenicity by Augmented Anti-tumor Immunity. Proc Nat1 Acad Sci USA 1989, 86:94569460.

tumor

cells Pardoll

GANSBACHERB, BANN~RJI R, DANIE~~ B, ZI~R K, C~~NIN K, GIIBOA E. RetroviraI Vector-mediated y-Interferon Gene Transfer into Tumor Cells Generate Potent and Long Lasting Antitumor Immunity. Cancer f&s 1990, 50:782O7825.

17.

RESTIFON, Spiess P, Icw’ S, MOLEJ, ROSENBERGS: A Nonim-

munogenic Sarcoma Transduced with the cDNA for Interferon y Elicits CD8+ T Cells Against the Wild-type Tumor: Correlation with Antigen Presentation Capability. J Exp Med 1992, 18.

175:1423-1431.

ASHER A, MIILEJ, K.&s10A, F%STII;ON, SAI.OJ. REICHERTC, JAFFE G, FENDIV B, K~~%IxR M, ROSENBERG S: Murine Tumor CeUs

Transduced with the Gene for Tumor Immunol 1991, 1463227-3234.

Necrosis

Factor-a.

,/

19.

BWVKENSTEINT, QIN Z. lJnERIA K, MLII,ER W, RCXIN H, VOLK HOD, DIAMANTSTEINT: Tumor Suppression after Tumor CeIItargeted Tumor Necrosis Factor a Gene Transfer. .I Exp .Ifcd 1991, 173:104’-1052.

20. .

HOCK H, Dobct~

21.

COLOMHO M, FER~RI G, STOPPACC~AKO A, PARENTSM, Ro~owo M, MA~ILIO F, P~IANI G: Granulocyte Colony-stimulating

BIJRENIKJ, SIMOL’A J. JANDI.O\‘AT: Immunotherapy

of Cancer Using Local Administration of Lymphoid Cells Expressing IL-2 cDNA and Constitutively Producing IL-2. Immunol l&t 1990, 23:287-292.

engineered

16.

HARDING F, MCARTHORJ, GRAMSJ, RALIIET D, ALLISONJ: CD28-

mediated SignaIling Co-stimulates Murine T CeIIs and Prevents Induction of Anergy in T-cell Clones. Nuture 1992, 356607609.

with genetically

M, DIA~~ANTSTEIN T, BLANKFNSI-EINT. Interleukin 7 Induces CD4+ T Cell-dependent Tumor Rejection. / &p Med 1991, 174:1291&1298. IL-7 is shown to mediate systemic antitumor immunitywhich, in contrast to other lymphokine genes. appears to depend on CD4+ rather than CDX+ T cells.

Factor Gene Transfer Supresses Tumorigenicity of a Murine Adenocarcinoma In Viva J Exl, Med 1991, 173:889-897. 22.

ROLLINSB, S~IN~AYM: Suppression of Tumor Formation In Vivo by Expression of the JE Gene in Malignant Cells. ,Mol Cell Rio1 1991, 11:3125-3131.

D Pardoll, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, 364 Koss Baltimore, Matykand 21205 2196, IJSA.

623