Idiotypic vaccination in B-cell malignancies

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Idiotypic vaccination in B-cell malignancies Alberto Bianchi and Massimo Massaia

Immunoglobulins contain unique portions, collectively termed idiotypes, that can be recognized by the immune system. Idiotypes expressed by tumor cells in B-cell malignancies can be regarded as tumorspecific antigens and targets for vaccine immunotherapy. Haptens and adjuvants, including cytokines, have been used in several animal models to increase idiotype immunogenicity and establish protective anti-idiotype immunity. These results have been extended by the use of DNA technology, and this has led to the development of a new generation of immunogens, namely fusion proteins and naked-DNA vaccines. The central role of antigen-presenting cells as initiators of anti-idiotype immune responses has also been recognized. Guided by the experimental data, idiotypic vaccination has come into medical use in patients with lymphoma and multiple myeloma.

Alberto Bianch i MD Assistant Member Massimo Massaia* MD Associate Member Divisione Universitaria di Ematologia, Dipartimento di Medicina e Oncologia Sperimentale, Azienda Ospedaliera San Giovanni Battista di Torino, Via Genova 3, 10126 TOrino, Italy. Tel : +3911 6633012 Fax: +39 11 6963737 *e-mail: maxmass@iol. it

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B- ELL Iymphoproliferative di order are clonal expan. ion of lymphoid cell. that hav rearranged immunoglobulin genes. Immunoglobulin. are heterodimeric gl oprotein. con i. ting of heavy and light chain. : both chains contain variable ( ) and con tant (C) region . . which are di tingui hed according to the degree of amino acid variability at each po it ion of the linear . equence. The V region. are further divided into framework (Fr) and complementaritydetermining regions ( DR. ). DR. them. elve. displa) the most hypervariable amino acid diversit and act a. antigenic determinants (termed idiotope ). and contribute to the fine-tuning of immune re. pon e . ultiple independent idiotope. in a ingle immunoglobulin molecule are collectivel termed idiotype (Id) and are . 0 . pecificall y expressed by any B cell that they are onl y ob cr.'ed in it. clonal progeny. The Id that i expre. ed on the immunoglobulin produced by neopia. tic B cells i. c10nally re tricted to tumor cell. and thus can be exploited a. a target for va cine immunotherapy. Depending on their maturation tage, neopla. tic B ell. an expre s differ nt immunoglobulin gene product.. from cytopla mic i. otype-re. tricted heavy cha in ( Il) to fully a~~embled !lurface immunoglobulin~ (. mig). Any n opla. tic B cell can potentially be recognized by T helper cell s and killed by cytoto ic T lymphocytes ( ): immunoglobulin~ are endogenously synthe iLcd and Id-deri ed peptide~ are expressed on the cell ~urface in association with major histocompatibility complex (M il ) molecules ' (Fig. I). Furthermore . . mlg' B cell. can be atta ked b antibodie re ognizing onformational epi tope. on immunoglobulin (Fig. I). The main ob taclc in • uch a favorable scenario (e.g. a tumor-specifi antigen available to the immune . y. tem in different form) is that Id belongs to elf and. as such, is under the protection of elf-tolerance mechani. m.,. Furthermore, T ce ll in cancer patients have lost the ability to recognize and eliminate tumor ell, owing to . everal mechani~ms su h as tumor-derived suppres. or factors, abnormal cytokine production. and inadequate interactions with tumor ell and antigen-pre. enting cell.

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Figure 1. Pathways of idiotype (Id) presentation and recognition by immune cells. (a) Direct recognition: helper (CD4') and cytotoxic (COB' ) T cells recognize Idderived peptides expressed on tumor cells together with major histocompatibility complex (MHC) class I (CDa- cells) or class II (CD4' cells) molecules. B cells efficiently process their endogenous immunoglobulin and present Id-derived peptides. (b) Indirect recognition: Id-derived peptides result from antigen processing in macro phages (M~) or dendritic cells (DC) . These are professional antigen-presenting cells that capture and process Id shed from tumor cells and convert it into MHC associated Id fragments. (c) Activation of CD4' cells, through further functional differentiation into T helper (Thl or Th2) cells, leads to humoral and cellular responses against antIgenic idiotypic determinants on the tumor cells. Activation of resting CDB' cells into cytotoxic T lymphocytes (CTL) might be triggered by direct interaction with the processed antigen, but is greatly enhanced by the collaborative action of soluble cytokines, mainly provided by activated CD4' cells. The humoral response to tumor antigeniC determinants IS sustained by antibody production. Th2 cells help B cells to differentiate and divide into antibody-forming cells (plasma cells, PC). Interaction between T and B cells is a complex two-way process requiring both soluble signals provided by cytokines and cell-surface co-stimulatory molecules.

(APC )2. The fir t move to exploit Id pecificitie a targets for vaccination i to render them immunogenic. Lynch and Ei en were the first to demonstrate in mice that thi can be achieved 3.4.

Pre-clinical experimental model : building up an immunogenic self-antigen Syngeneic BALB/c mice, immunized with purified immunoglobulin derived from mineral-oil-induced pia macytoma (MOPC), were pecifically protected again t the malignant plasma cell bearing the corre ponding Id on the cell urface3.4. The e animals pecifically rejected a tumor challenge of the parental tumor, but were not re istant to other MOP tumor , demon trating that anti-Id immunity was pecific. A number of subsequent report have hown that Id vacci nation induce resi tance to tumor growth in everal model of yngeneic tumor . In mo t of the e model , anti-Id immunity was dependent on T cell rather than anti-Id antibodies. Conjugation to 436

trong immunogenic protein carriers [such as keyhole limpet hemocyanin (KLH) or thyroglobulin] and emulsification in adjuvants were both required for optimal immunization.

linical studies using Id-KLH conjugates and adjuvants Guided by the experimental results, a pioneering study was carried out in patients with follicular lymphomas. Thi type of lymphoma is characterized by relatively slow tumor growth and low levels of circulating Id, which is a favorable etting for vaccination . ine patients in complete remis. ion, or with minimal residual disea e, were immunized with ubcutaneou injections of autologou purified tumor-derived immunoglobulin, conjugated to KLH and emulsified in the non-ionic block polymer adjuvant l. pecific anti-Id humoral and/or cellular response were observed in even out of nine patients. Two patients with measurable di ease showed a clinical improvement.

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These results have recently been confirmed in a larger series of patients6, Following standard chemotherapy, 41 patient with B-cell lympha oma received subcutaneou injection of autoloId gous Id-KLH conjugate mixed with an immunologic adjuvant containing threonylmuramyl b + dipeptide. Approximately 50% of the patients Hapten generated specific anti-Id responses. Their median duration of freedom from cancer progresc Adjuvant sion was ignificantly prolonged and re ulted in 6 a survival advantage . Anti-ld immunity was dependent on T-cell effector mechanisms other Cytokine d + than humora l responses: patients who had a significant increase in the frequency of tumorFusion protein specific CTL precursors remained without evidence of disease or with stable minimal disease 7• Multiple myeloma is another B-cell IymphoDendritic cell e + + proliferative disorder for which Id vaccination is under investigation. A number of experimental data indicate the presence of an Id-reactive T-cell population that can be exploited a specific antiFigure 2. The evolution of protein vaccines. (a) Soluble idiotype (Id) is conjugated to hapten and emul· tumor effector cellsS,9. In a pilot study, five tage sified in adjuvant. This strategy has proved effective In human clinical trials. (b) Soluble Id is conjuI-Ill multiple myeloma patients were repeatedly gated to hapten, but cytokines are used instead of adjuvants. This strategy is under clinical investiimmunized with autologous Id precipitated in alugation. (e) Fusion proteins are assembled by combining Id and cytokines. Experimental models indicate that fusion proteins induce protective anti-Id immunity without haptens and soluble cytokines. minium phosphate suspen ion. Four patients were (d) Dendritic cells are pulsed ex vivo with autologous Id and used as antigen-presenting cells to inlti· previously untreated and one patient was in table ate anti-Id immunity. This approach is currently under clinical investigation. (e) The same strategy as part ial rem is ion following chemotherapy. Three in (d) is used, but cytokines are added to facilitate triggering of anti-Id immunity. patients developed specific anti-Id T- and B-cell responses, and in two of them a decrease in the number of circulating CO 19+ B cells was observed during the immunization procedure JO • However, anti-Id immun- intraperitoneally or ubcutaneously. together with Id-KLH conjuity was transient and it amplitude was low, indicating the need for a gate. The protective anti-Id immunity was significantly increa ed by more effective immunization schedule to achieve a long-Ia ting T-cell GM-CSF and appeared to be dependent upon effector C04+ and C08+T cells. Immunization of mice with Id-KLH and GM-C Fled anti-Id immunity (Fig. 2). to a ignificant increase in tumor-free survival compared with Improving the formula: from adjuvant to cytokine Id- KLH alone. Adjuvant great ly facilitate active immunization because they induce The importance of local cytokine action in increa ing antigen proa slow and sustained release of antigens and stimulate APCs through cessing and recognition was formally hown by Tao and Levy in the the production of innammatory cytokine . Complete Freund ' ad- 3 13 mou e lymphoma modeJi b. They a embled a fu ion protein juvant is genera lly used in experimental models, but is unsuitable by combining Id and GM-CSF. Mice were immunized with everal for clinical u e. Five groups of adjuvants are currently available: form of vaccine preparation (Id-GM-C F fu ion protein with aluminium-containing compounds (which are not alway effective human or murine GM-C F; Id + GM-CSF mixture; Id- KLH conjuand stimulate main ly humoral response); aponin (of plant origin); gate; Id alone) and the induction of anti-Id ant ibodie and tumor non-ionic block polymer (artificial); monopho phoryl lipid and mu- challenge protecti n wa evaluated. Only immunization with ramyl dipeptides (bacterial); and various cytokines". Cytokines have Id-GM-C F fusion protein induced anti -Id antibodie and protected the advantage of se lectively mediating the local recruitment of AP s from challenge with a lethal do e of tumor cell. Thi protection wa and driving their interaction with specific . ub ets of T cellsl ~. obtained without other carrier protein or adjuvants. The . ame re ult A number of experimental systems have shown the efficacy of were ob erved when IL-2 or IL-4 replaced GM-C F in the fusion interleukin 2 (I L-2) and granulocyt - macrophage colony-stimulating protein p . The advantage of antigen-cytokine fu ion proteins re t., factor (GM-C F) as adjuvant. In particular, GM- F directly affects at least partially, in the increa ed half-life of the cytokine and the antigen processing and presentation by regulating the growth and differ- more efficient Id targeting to APC . entiation of dendritic cells (0 s)" . Levitsky el 01. have hown in mice that immunization with lymphoma cell that have been genetically en- Clinical trial u ing Id-KLH conjugates and cytokine gineered to produce GM- F, and to a lesser extent cells engineered to The use of cytokines as immunoadjuvant ha. entered clini al pracproduce IL-4, lead. to the eradication of established tumors. ntitum r tice. Kwak el al. are currently u ing an improved vaccine formulaimmunity generated by GM - F- or IL-4-tran duced lymphoma was tion with GM- F in patient. with follicular lymphoma who ha e allargely dependent on both 04+and OW Id- pecific T cell 14. ready been treated with chemotherapy. After achieving a complete imilar results have been hown by Kwak el 01. in the 3 13 remission, they receive a . erie of . ubcutaneou vaccinati n with mouse B-cell tumor l~. Mice received GM - F, administered either autologous Id- KLII, together with locally admini. tered GM- F.

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Figure 3. (a) Photograph of skin tests on the forearms of a patient with multiple myeloma. Autologous idiotype (Id) alone [0.5 mg, without keyhole limpet hemocyanin (KLH); solid arrow] and unrelated Id alone (0.5 mg, without KLH; open arrow) were Injected intradermally; the picture was taken 24 h later. The patient was challenged at the end of the vaccination procedure, which consisted of seven subcutaneous Injections of Id-KLH conjugate plus granulocyte-macrophage colony-stimulating factor (GM-CSF) as Immunoadjuvant. A skin induration with erythema (a reddened area of Inflammation) developed at the site of autologous Id Injection, but not at the site of unrelated Id administration, indicating the development of a delayed-type hypersensitivity (OTH) reaction. This type of reaction IS usually sustained by macrophages, which are the main antigen-presenting cells in this scenario, and T cells, which are the effectors (lymphocytic infiltrate is a typical finding) . T cells (both C04- and COS') necessary for producing the OTH-like response are cells that have become specifically sensrtlzed to the challenging antigen and recruit other cell types to the reaction site. A skin reacllon of this kind suggests that the Id vaccine also elicited antigen-specific T cells via the Immune-enhancing effects of GM-CSF. However, it IS important to stress that a OTH-like reacllon does not necessarily imply the presence of protective Immunity. Histologic assessment (x!O magnification) of skin biopsies taken from the sites of unrelated (b) and autologous (e) Id administration show that lymphocytic perivascular In@rates (solid arrows) are present only in the area of autologous Id InJection.

Immunologic monitoring has shown that a local reaction can be elicited by the intradermal injection of unconjugated Id alone (without KLH) at the end of the vaccination cour. e. In five out of six patients, the re pon e appears to be specific, as demonstrated by the lack of reactivity again t a control i otype-matched Id. Two out of the three patients analyzed thus far, whose bone marrows contained re. idual tumor cell detectable by polymerase chain reaction (PCR) po. t-chemotherapy, have become PCR-negative after vaccination I ~ . 438

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Cytokines are also under investigation as immunoadjuvants in multiple myeloma. Osterborg el al. are focusing their efforts on earl y-stage multiple myeloma. The rationale is that Id-specific T cells are detected (at a frequency of about 1:5000 peripheral blood mononuclear cells) mainly in patients with early stage mulliple myeloma or monoclonal gammopathy of undetermined significance (MGUS). Most of these Id-reactive T cells are Th I-type cell (ecreting interferon -y and/or IL-2) in patients with indolent di ease (MGUS and multiple myeloma stage 1)_ whereas they are predominantly Th2-type in patient with multiple myeloma tage Will (Ref. 19). A better outcome is to be expected if vaccination is carried out in the pre ence of T cells that are able to exert antitumor cytotoxic activity. In thi eries, patients receive a series of subcutaneous injections of autologou -Id-alum suspension, together with free GM-CSF. Preliminary analysis has shown that long-lasting Id-specific T-cell responses have been induced in all five immunized patient. Moreover, one patient exh ibited a decrease in circulating Id upon immunization 20 . A different approach is under investigation at our institution 2'. An initial eries of eight high-risk multiple myeloma patients has hown that Id vaccination is totally ineffective in the pre ence of progressive disease. We therefore designed a study in which Id vaccination is administered as a maintenance treatment after intensive induction chemotherapy. Several reports have emphasized the importance of timing as .oppo ed to the tota l number of injection of vaccine. Many of the T-cell abnormalities observed in multiple myeloma have re olved in complete remission. Moreover, patient with multiple myeloma in complete remission have undetectable erum levels of circulating Id, thus preventing inhibition of anti- Id immunity by free Id protein. Lastly, the rate of succe sful vaccine immunotherapy is negatively correlated with the number of tumor cells, and bone marrow transplantat ion achieves maximal reduction in tumor burden. Integration of Id vaccinat ion into the transplant elting might retore immune re ponsivene s to autologous Id by shaping the posttransplant Tcell receptor repertoire. Thu far, eight patients in complete or partial remission after intensive chemotherapy (four after double autologous transplants wit h peripheral blood progenitor cells) have been treated with subcutaneous injections of Id-KLH conjugate. Three patients received subcutaneou IL-2, whereas the other received . ubcutaneous GM-CSF as immunoadjuvant. even patients have comp leted the ~reatment and have remained in a remis ion lasting 15- 30 months. kin testing ha been performed with autologou unconjugated Id and isotype-matched unrel ated Id and a local reaction has only been observed at sites of autologous Id injection (Fig. 3). Skin biopsies in selected patients have shown a perivascular lymphocyte infiltration, which was more evident in patients receiving GM- SF. These preliminary data indicate that Id vaccination is worth in vestigating as a maintena nce therapy in patients with mulliple myeloma in complete remission or in those with stable di, ease after induction chemotherapy.

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More recent approache have appli ed D A technology to the development of a new generation of immunogen. , namely D A vaccines (Fig. 4). Thi: approach might be particu larly advantageous in lymph oma, where use of protein vaccines encounters two major drawbacks. The fir. t is the time and labor required to obtain sufficient Id for a personalized vaccine. Using 0 A technology, this step is acco mpli shed

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by creating somatic cell hybrids between the lymphom a B cell and a murine nonimmunoglobulin-producing myeloma cell. The second drawback to the use of protein vaccines is the need to obtain formal proof that the heterohybridoma culture contains the relevant immortalized lymphoma B cell. An alternative is to generate D A vaccines containing the V region gene sequences of both heavy and light chains assembled together with a flexible peptide linker. This heterodimeric ingle-chain Fv fragment (scFv) can be used to produce recombinant scFv protein in bacteria or as a naked-D A vaccine. The latter i assembled into an expression plasmid and directly injected into mu cle cells 2c . A preliminary comparison between protein and D A vaccines howed the latter was more effective in a mouse lymphoma modeF A Phase I trial i currently in progre in patients with chemoresistant or advanced lymphoma to test the safety and fea ibility of this genetic approach2.'.

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Improvement of DNA vaccines by adding genetic adjuvant Preliminary results have hown that D A Figure 4. The evolution of DNA vaccines. (a) Unique Idlotype sequences are assembled Into an expression vaccines induce weak protection in mice. plasmid and directly Injected Into muscle cells. (b, c) Sequences codlllg for cytokilles or foreign proteins are A much stronger response, however, is added to the construct to enhance plasmid Immunogenicity. The human immunoglobulin constant region and fragment C of tetanus toxin have been used in experimental models. (d-~ The same strategies as outlined in required in patients to generate long-last(a-c) are used, but DNA vaccines are injected Into dendritiC cells. ing anti-Id immunity and overcome possible tolerance. This goal can be achieved by constructing D A pia mids containing genes for additional proteins a genetic adjuvants. Syrengela. el at. vaccine preparation cannot be predicted a priori: the final outcome have shown in the 38C13 mouse lymphoma model that a plasmid of protein versu 0 A vaccine i influenced more by the complex containing the sequence~ of Id, GM-C F and the human im- interplay among host. cytokine and antigen than by the preparation munoglobulin C region protected mice against tumor challenge~4. The itself. presence of the human region was essential for pia mid immunogenicity, and thi& wa further enhanced by GM-C F. tevenson el The earch for better antigen-pre enting cell : dendritic af.22 have incorporated a gene encoding fragment (FrC) of tetanu cell toxin into a fusion protein with the scFv equence. Using an scFv Given the fact that T-cell activation requires effective antigen pre enfrom a patient's lymphoma, they have shown that mice respond tation, AP s playa central role in the generation and regulation of poorly to scFv alone. However, when the . cFv i~ fused to Fr . the anti-Id immunity. D s are very goo I candidates as initiators of antiantibody response to scFv i~ markedly increased. These re. ults indi- tumor response. c/o. They are de cribed as nature'. adjuvant because cate that D A could be a simple and effective tool to generate im- they are strategically located at sites of antigen exposure and expre . mune re ponse. again t a weakly immunogenic or tolerized tumor high levels of MH class I and MH cia .. II molecule. a. well a. antigen, provided that genetic adjuvants are included in the construct. C040, ORO and DR6 co-stimulatory molecules. Pre-clinical exome findings , however, caution against the general conclusion perimental modeb have shown that a specific anti-Id immunity can that D A vaccines are superior to protein vaccines. study in the be generated in the B L I mou. e lymphoma . ystem by immunization 3RCI3 mouse B-celllymphoma m del has shown that D A vaccines with 0 ' Ioaded 'ill l'iltO with the Id protein . Thi. anti-Id immunity do not alway~ elicit anti- Id immunity/I. In this study, scFv proteins, confers protection again. t a sub equent hallcnge with a lethal do e produced in bacteria, and naked DNA encoding scFv were compared. of tumor cells 27 • The reasibilit and cfficacy of Id-pulsed D vaccination has rescFv was tested alone or fused to GM- F or to an immunoenhancing peptide derived from IL- If~. scFv- GM - F was effective only cently been te. ted in patient. with B- ell lymphoma''. our patients when injected as a protein, not as a D A vaccine. In contrast, both affccted by follicular B-cell lymphoma and with residual disea. e after conventional chemotherapy, received a . erie. of three to four ~cFv- 1 L-If) peptide fusion protein and naked 0 A induced protective anti- Id immunity. These data indicate that the efficacy of a given infusions of lei-pulsed 0 s, followed by subcutaneous injections of 43 9

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Glossary Adjuvant - A substance that nonspecifically enhances the immune re- _ Idiotype (I d) - A collection of unique light and/or heavy chain V regionsponse to an antigen. associated structures of a single immunoglobulin molecule, which can be recognized by the immune system. Antigen-presenting cells (APCs) - A variety of cells that process and express antigens in a form that enables T and B cells to encounter and Monoclonal gammopathy of undetermined significance (MGUS) recognize the antigenic molecules. The presence of a monoclonal immunoglobulin in a person without evidence of multiple myeloma or related diseases. B-cell lymphoma - Neoplastic disease of B-Iymphocytic lineage that usually arises in one or more organs of the hematopoietic system (lymph Multiple myeloma - A neoplastic disease of B-cell lineage arising nodes, liver, spleen, bone marrow). B-cell lymphomas are a diverse in the bone marrow. It is associated with tumor cells of plasma cell group of diseases with a complex classification, and varying clinical morphology, monoclonal immunoglobulin production and lytic bone presentations, responses to therapy and prognoses. Follicular lymphoma lesions. denotes a subset of B-cell lymphomas that usually express idiotype. Naked DNA - DNA freed from the protein with which it is usually comCytokine - A soluble protein or peptide that mediates interactions be- plexed. tween immune cells and mainly exerts its effects in the immediate vicinPolymerase chain reaction (PCR) - A method for gr.eatly amplifying ity of its source. specific known DNA sequences using cyclic rounds of transcription diCytotoxic T lymphocytes (CTLs) - A functional subset of T cells (usu- rected by synthetic oligonucleotides. The chain reaction of repeated ally expressing CDB antigen) that kill target cells (tumor cells, altered synthesis typically results in million-fold amplifications of the starting autologous cells) following recognition of antigen-derived peptides and sequence. major histocompatibility complex (MHC) class I determinants. T-cell receptor - The T-cell antigen receptor, consisting of either an aB Dendritic cells (DCs) - A set of cells present in most tissues that are dimer or a y6 dimer associated with the CD3 molecular complex. considered to be the most effective antigen-presenting cells for the iniT helper (Th) cells - A functional subset of T cells (expressing the tial activation of resting CD4+T cells. CD4 antigen) that, following recognition of antigen and major histoGene transduction - A gene insertion into another cell under regu- compatibility complex class" determinants, help to generate cytotoxic lation of an active promoter. Several types of transfer vehicles are T cells and cooperate with B cells in the generation of antibody reused, viral vectors being the most widely employed. A tumor cell can be sponse. Two main types of T helper cells exist, termed Th1 and Th2 transduced to express major histocompatibility complex or co-stimulatory according to their particular functional and cytokine secretion profiles molecules, or secrete cytokines as the gene product. upon activation.

oluble Id 2 weeks later. All patient developed pecific anti-Id T-cell re pon e ,a a se sed by proliferation assay. Tumor re ponse were also observed: one patient experienced complete tumor regression, a second patient had a part ial tumor regression, and in a third patient, molecular evidence sugge ted that the di ea e had re olved itself. The use of Id-pulsed DCs is also currently under inve. tigation in patient with multiple myeloma a. a maintenance therapy after autologou periph eral-blood tem-cell transplant2Q • o s pre ent tumor antigens for both the induction and elicitation of antitumor responses. Their effi cacy, however, depends on the local cytokine concentration and the duration of antigen pre entation. The use of DCs transfected with Id and cytokine genes might be the next step towards improving the condition for triggering anti -Id immunity. Genetica ll y engineered DC. or macrophages have already been u ed to generate crL~ aga inst viral antigens1l' .

hifting from the autologou to the allogeneic setting A maj or concern about autologous Id vaccination is the state of host immunocompetence . Allogeneic vaccination in the peritransplant . etting is, however, immunologica ll y sound. First, immunization is ca rried out in hea lthy people before any inappropri ate interaction with the tumor antigen, which may be toleri zing. Recent work by Bogen11 in transgenic mice . howed that periph eral T-ce ll tolera nce to Id could represent a mechani sm of tumor e ape, owing to the deletion of Id-

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pecific 04+T cells induced by the exposure to increasing number of Id-secreting tumor ce lls. Second, the graft anti-Id effect can be exploited a an adoptive immunotherapy again t residu al tumor cells. Indeed, pro tective anti- Id immunity cou ld be transferred with bone marrow tran. pl ants in the 3 C 13 murine B-celll ymphoma model 12 . A pecific anti-Id immun ity has also bee n successful in the human sellingll. A sibling marrow donor was jmmunized wi th allogeneic Id isolated from the transplant recipient who had multiple myeloma. Id wa. conjugated to KLH and emulsified in an adjuvant. Donor immunization was documented by the induction of specific humoral and cellul ar anti -Id response . Transfer of anti -Id immunity to the recipient was demonstrated by the appearance of a proliferati ve anti -Id T-cell response. This T-cell -medi ated anti -Id response lasted for 60 days aft er the transplant. Expan ion and re-stimu lation of rec ipient peripheral blood mononuclea r ce lls then gave way to an Id-reactive, MHC cl ass II-restricted, CD4' T-cell linen In con lusion, the induction of protective anti -Id immunit y in many experim ental and clinical . ellings suggests that idiotypic vaccination will soon ex tend the therapeutic arm amentarium ava ilahle to clinicians fo[ the treatment of B-celll ymphoproliferative disorders. Acknowledgements. Thi\ ",lIrk wa, ~ upporIcd hy i\ssocia71one lIaliana Rlccrca w i Cancro (Mltan). and Con\lglio Na710nalc delle Rlcerchc, Progelto Finahllato 95.0041 0 PF39 (Rome).

MOLECULAR MED I CINE TODAY, OCTOBER 1997

The outstanding questions • Because many idiotype vaccination approaches induce an efficient antitumor respon e in experimental models, which vaccine formulation is best able to effectively recruit antitumor pathways in patients with B-ce\l malignancies? • What is the actual role of active specific immunotherapy with regard to the conventional treatments for B-cell neoplasms? • Should active pecific immunotherapy be given at a very early stage of the disease or as a maintenance therapy after intensive induction treatment? • Becau e current vaccine strategie for anti-idiotype immunotherapy still need to be highly individualized, how can we identify the patients who are mo t likely to benefit from this kind of treatment? • What duration and administration chedule of vaccine immunotherapy (lifelong treatment?) i be t able to improve the clinical outcome of patients with B-cell malignancies? Will prolonged administration of idiotype vaccine ultimately re ult in tolerance to tumor antigen ? Will tumor re istance to immunotherapy ever develop? • How can we develop reliable in vitro a ay to a e the effect of an immunotherapy regimen and predict it outcome in the clinical etting?

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