Biological Response Modifiers: The New Immunotherapy · Biological Response Modifiers: The New Immunotherapy Kenneth A. Foon1 Division of Clinical Immunology, Roswell Park Memorial

ICANCER RESEARCH 49, 1621-1639, April 1, 1989]

Perspectives in Cancer Research

Biological Response Modifiers: The New ImmunotherapyKenneth A. Foon1

Division of Clinical Immunology, Roswell Park Memorial Institute, and Department of Medicine, Stale University of New York at Buffalo, Buffalo, New York 14263

Immunotherapy is divided into two overlapping categories,active and passive. The goal of active immunotherapy is thestimulation of host ant Â¡tumorimmunity, either cellular or humoral. This can be accomplished in a direct or specific fashionby using tumor vaccines to generate an immune response totumor-associated antigens. Nonspecific antitumor immunitycan be propagated by compounds such as BCG.2 Passive im

munotherapy relies on the administration of biologically activeagents with innate antitumor properties, such as antibodiesreactive with growth factor receptors. In most instances, hostimmunity is an important cofactor in active immunotherapy.

In addition, some agents, such as antibodies, can exert anti-tumor effects via active or passive mechanisms. The compleximmune circuits that are set into motion by these therapiesaccount for the imperfect, but nonetheless useful division intoactive and passive types.

Immunotherapy is very effective in certain animal modelsystems and it has been used to treat human cancers for severaldecades (1). In the 1960s and 1970s trials of nonspecific im-munostimulants including BCG or specific allogeneic or au-tologous vaccines were evaluated with early promising reports(2-5). Unfortunately, none of these approaches has been shownto be effective in controlled trials (6, 7).

During the past decade, renewed interest in immunotherapyhas been stimulated by genetic engineering and mass cell cultureand improved techniques in protein and nucleic acid sequencing. These have made available highly purified molecules including interferons, interleukins, tumor necrosis factor, andhematopoietic growth factors. Also, hybridoma technology (8)has generated murine monoclonal antibodies directed againsttumor-associated antigens. The term "biological response modifiers" (9) is often used to refer to these newer approaches to

immunotherapy.

Interferons

Interferon was initially identified as a soluble factor able toinhibit infection of chick chorioallantoic membranes by influenza A virus (10). Subsequent studies have shown the interferons to be a family of closely related proteins and glycoproteins.In addition to their antiviral activity these molecules are potentregulators of cell gene expression, structure, and function. Theyalso exhibit direct antiproliferative activity. These propertiesunderlie the current interest in interferon as an anticancer agent.

Three major species of human interferon are identified: a, ÃŸ,and 7(11) (Table 1). a-Interferon is produced by leukocytes (B-cells, T-cells, null cells, and macrophages) following exposure

Received 7/15/88; revised 10/11/88; accepted 1/6/89.' To whom requests for reprints should be addressed, at Division of Clinical

Immunology, Roswell Park Memorial Institute, Elm and Carlton Sis.. Buffalo,NY 14263.

2The abbreviations used are: BCG, Bacillus Calmette-GuÃ©rin;IL-1, IL-2, IL-3, interleukins 1, 2, and 3, respectively; NK natural killer; (MI,, chronic myelog-enous leukemia; LAK, lymphokine-activated killer; TNF, tumor necrosis factor;i.a., intraarterially; TIL, tumor-infiltrating lymphocytes; CPU, colony-formingunit; GM-CSF, granulocyte-macrophage-colony-stimulating factor; G-CSF, gran-ulocyte-colony-stimulating factor, M-CSF, macrophage-colony-stimulating factor; ALL, acute lymphoblastic leukemia.

to B-cell mitogens, viruses, foreign cells, or tumor cells, ÃŸ-Interferon is produced by fibroblasts following exposure toviruses or foreign nucleic acids. 7-Interferon is produced by T-lymphocytes following stimulation of T-cell mitogens, specificantigens, or IL-2 (13). Complete nucleotide sequences for thea-, ÃŸ-,and 7-interferon genes are known, and amino acidsequences have been deduced (14-16). Sixteen distinct a-inter-feron genes are described; each encodes a protein of approximately 166 amino acids (14). Only a single 0-interferon genehas been identified encoding a protein of 166 amino acids;similarly there appears to be a single 7-interferon gene encodinga protein of 144 amino acids (15-17).

The a-interferon first used in clinical trials was obtained fromSendai virus-stimulated buffy coat leukocytes. Its purity wasabout 1% (IO6 units/mg protein; 1 unit of interferon is approx

imately the amount that reduces viral replication in cell cultureby one-half) (18). Use of high performance liquid chromatog-

raphy, two dimensional polyacrylamide gel electrophoresis, andimmune affinity chromatography has resulted in the purification of a-interferon to homogeneity (IO8 units/mg protein) (19,

20). Use of recombinant DNA technology can produce largequantities of pure a-interferon (21).

Large scale production of ÃŸ-and 7-interferons is more recentand clinical trials are limited. a-Interferon has been extensivelystudied for the past decade in both basic science and clinicalresearch and is among the most potent biological agents everadministered to humans. Although antitumor activity is detected in some solid malignancies in vitro and in vivo (22-24),the most impressive responses are in hematological malignancies (12).

The mechanisms of interferon-mediated antitumor effects inmurine models and humans are unclear. Putative mechanismsinclude a direct antiproliferative effect on the tumor, inductionor augmentation of a host effector mechanism, such as NK andmonocyte cytotoxicity, and induction or augmentation ofexpression of membrane antigens on tumor cells which facilitatesubsequent immune recognition by the host.

ii Interferon has moderate activity in a limited number ofsolid tumors (Table 2). It is active in bladder cancer wheninstilled directly at very high doses (57-59). It is also active inacquired immunodeficiency syndrome-related Kaposi sarcoma(45-47), renal cell carcinoma (40-44), malignant melanoma(27-32), and carcinoid tumors (90). There is little evidence ofactivity in the common cancers such as breast and colon cancerand only limited activity for squamous cell tumors of the headand neck and lung (33-39, 48-52, 48, 53, 54, 60, 61). Clearly,a-interferon as a single agent will not have a major impact in

the therapy of solid tumors. Current directions of interferonresearch include combinations with other biologicals and insome cases cytotoxic drugs.

a-Interferon has had its greatest impact in the treatment ofcertain hematological malignancies. Approximately 90% ofpatients with hairy cell leukemia respond to a-interferon (64-71) with a normalization of blood counts. Improvement innatural killer cell activity and immunological surface marker

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BIOLOGICAL RESPONSE MODIFIERS

Table l Interferons in clinical use"

Type

Subtype*

(newnomenclature) Source

Purity Amino aciddifferences

Leukocyte (IFN-a[LE])c

Lymphoblastoid (IFN-alfa-Nl), Wellferon* (Burroughs Wellcome Co.)

Recombinant a.1 (IFN-alfa-2b), Intron A* (Schering

Corp.)

Recombinant Â«A(IFN-alfa-2a), Roferon-AÂ®(Hoffmann-La Roche, Inc.)

Recombinant <>D(IFN-aD)Recombinant a2arg (II N-

alfa-2c)

Fibroblast (IFN-/3)

Recombinant 0cys (rlFN-0cys)

Recombinant /3ser (rlFN-

Immune (IFN-f)

Recombinant y (rIFN-y)

Leukocytes fromnormal blood

Lymphoblastoid(Namalwa) cellsin culture

Transformed Esche-richia coli

Transformed E. coli

Transformed E. coliTransformed E. coli

Fetal foreskin fibro-blast in culture

Transformed E. coli

Transformed E. coli

T-lymphocytesfrom normalblood

Transformed E. coli

<r

>95

>95

>95>95

>95

>95

>95

Arginine at position23; deletion at position 44 when compared to other a subtypes.

Lysine at position 23;deletion at position44.

29 variations from aAArginine at position

23.Arginine at position

34.

Cysteine at position17.

Serine at position 17.

" Reproduced with permission from Ref. 12.* New nomenclature was proposed at a joint meeting of the WHO and USAN council in May 198S.c IFN, interferon; rIFN, recombinant IFN.d These crude preparations can be purified to near homogeneity (see text).

Table 2 Clinical trials with a-interferon

Tumor

No. ofÃ©valuablepatients

Response rates

CR" PR MR

%oftotal

response Ref.

Solid malignanciesOsteogenic sarcomaMelanomaBreast cancerRenal cellKaposi's sarcoma (AIDS-related)

ColorÃ©ela!carcinomaCarcinoidLung

Small cellNon-small cell

Ovarian cancerBladder cancer (papillomatosis or superficial)Head and neck (squamous)NasopharyngealCervical cancer

Hematological malignanciesHairy cell leukemia*Non-Hodgkin's lymphoma

Low gradeIntermediate and high gradeHodgkin's diseaseCutaneous T-cell lymphomaChronic lymphocytic leukemiaMultiple myelomaChronic myelogenous leukemia

1518518725212065

9

10704255111314

158

10761214273

22468

0706

1400

005

20403

22

12108038

11414372226

013

16623

3784

141241'

46

21028

44

6223

7117

17363

67

01

1965911543

96

46151950161781

25,2627-3233-3940-4445-4748-5290

5348,5455,5657-5960,616263

64-71

74-7774,7575,7678-8076,77,81,82,9185-8788

" CR, complete response (absence of disease); PR, partial response (>50% decrease in disease); MR, minor response (less than a partial response); % of total

response, CR + PR/number of Ã©valuablepatients; AIDS, acquired immunodeficiency syndrome.* Complete response means absence of hairy cells in the bone marrow and normalization of peripheral blood leukocytes, platelets, and erythrocytes. Partial response

means a normalization of peripheral blood leukocytes, platelets, and erythrocyte counts and a >50% reduction in hairy cells in the bone marrow. Minor responsegenerally means improvement in hemoglobin to more than 10 g/dl or improvement in platelets to more than 100 x I(("/liter or improvement in neutrophils to morethan 1 x 1(("/liter. Percentage of total response for hairy cell leukemia includes minor responses.

'"Complete response and partial response not available from all trials; percentage of total response includes all responses.

expression parallels immune recovery (68). Complete responsesare rare with a-interferon and there are no cures. However,patients have durable responses. Side effects are few becauseonly low doses of interferon are required for response, a-Interferon can be discontinued in most patients after a response

is achieved. Most patients relapse over a period of 6 months to2 years (71) but respond to additional treatment with interferon.Pentostatin is also very effective therapy in hairy cell leukemiaand is an alternative in patients who fail to respond or becomeresistant to a-interferon (72). Studies to assess the standard

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low dose of a-interferon (3-4 x IO6units 3 times/week) with alower dose (0.3-0.4 x 106 units 3 times/week) are under way.

Preliminary results suggest efficacy for the lower dose (73).Although a-interferon should not necessarily replace splenec-tomy as primary therapy for hairy cell leukemia, it is useful inpatients who are not surgical candidates or who have failedsplenectomy.

o-Interferon is active in several other hematological malignancies. Approximately 50% of patients with low grade non-Hodgkin's lymphoma or cutaneous T-cell lymphoma respond

to a-interferon (74-80). Although it is not active in advancedchronic lymphocytic leukemia (76, 77, 81, 82, 91), results inpreviously untreated patients are more encouraging (83, 84).Modest activity is reported in patients with multiple myeloma(85-87). Over 80% of patients with chronic myelogous leukemia in the chronic phase respond to a-interferon with excellentcontrol of blood counts (88). In a small fraction of these patientsthe percentage of cells with the Philadelphia chromosome decreases or disappears, albeit transiently (88). Studies are underway to demonstrate whether a-interferon prolongs the durationof chronic phase and/or survival in CML. 7-Interferon also hasactivity in CML (92); trials of a- and 7-interferon in CML arein progress.

Recombinant 0-interferon recently entered clinical trials. Itsactivity seems similar to a-interferon (24). In contrast, 7-interferon has demonstrated limited antitumor activity (93-95) withthe possible exception of CML (92). However, 7-interferonenhances immune responses at low doses (96).

Interleukin 2

IL-2 is a Mr 15,000 glycoprotein of 133 amino acids. IL-2 isreleased following antigen recognition and presentation to Icells, and causes T-cell proliferation. It was originally referredto as T-cell growth factor. IL-2 is currently available as arecombinant molecule and has been used in many clinical trials.IL-2 causes lymphoid proliferation both in vitro and in vivo(97). It activates the lytic mechanisms of LAK cells for freshtumors and appears to activate cytolytic T-cells (98-100). Itenhances the effect of transferred LAK cells and cytolytic T-cells (101, 102). IL-2 affects the vascular endothelium andcauses emigration of lymphoid cells from the blood into tissues(103, 104). IL-2 causes the release of other lymphokines including 7-interferon and TNF which likely mediate additionaleffects (97, 105).

IL-2 is active during the early stages of tumor growth (day 3)in both immunogenic and nonimmunogenic murine tumors(106-108). IL-2 is active in the advanced stages of tumor growth(day 10) but only in animals with weakly immunogenic tumors.The antitumor effect in nonimmunogenic tumors is mediatedprimarily by LAK cells and in weakly immunogenic tumors byT-cells as well as LAK cells (109).

Infusion of IL-2 in humans is associated with increases inmature T-cells (97). It also induces expression of IL-2 receptorson both T-cells and monocytes and enhances tumor cell lysisby immune effector cells (110).

IL-2 as a single agent is active in renal cell cancer, melanoma,and non-Hodgkin's lymphoma (111). Studies are under way in

other tumor types. The toxicity of IL-2 administered systemi-cally is substantial and includes fever, chills, nausea, vomiting,diarrhea, hypotension, cutaneous erythema, fluid retention, eo-sinophilia, anemia, and moderate to severe hepatic and renaldysfunction (111). A limited number of patients develop neuropsychiatrie complications heralded by confusion. This may

last days to weeks. Most other IL-2 toxicities resolve withinhours to days following discontinuation of IL-2. IL-2-activatedlymphocytes adhere to endothelial cells in a dose-dependentmanner. These cells are cytotoxic in vitro to endothelial cellswhich may explain some of the systemic toxicity (112). Increased expression of HLA-DR antigens occurs in tumor, endothelial cells, and in perivascular T-cells in patients receiving IL-2 (104). Preliminary data suggest that this may be greater inpatients whose lesions respond to IL-2 treatment (109). Themechanisms by which IL-2 mediates antitumor effects are notknown but histolÃ³gica! examination of tumors before and aftertreatment shows infiltration with large activated T-cells consistent with a cell-mediated immune response (113).

IL-2 is known to stimulate all subsets of T-cells. A strategywas designed to decrease T-suppressor cells by injecting low-dose cyclophosphamide prior to IL-2 administration (114). Sixof 24 patients with melanoma responded and all respondingpatients had LAK cell activation in vivo. Other strategies toimprove IL-2 activity include combinations with interferons,tumor necrosis factor, or monoclonal antibodies. One preliminary study reported 6 of 21 (29%) persons with advanced renalcell carcinoma responding to the combination of IL-2 and ÃŸ-interferon (115).

Tumor Necrosis Factor

TNF was first identified in mice primed with BCG andchallenged with endotoxin. Serum from these mice causedtumor necrosis when transferred into tumor-bearing animals(116). This factor is produced by monocytes and is called TNF-a. Recent data indicate that cachectin, responsible for wastingin chronic parasitic diseases, is identical to TNF-a (117). Arelated cytotoxic protein, called lymphotoxin or TNF-/3, isproduced by lymphocytes (118, 119). The genes for encodingboth TNF-a and TNF-/ÃŽhave been molecularly cloned andsequenced (120-124). Both molecules have cytostatic and cytotoxic effects in vitro against a variety of human tumors (125-127). Antitumor effects have been demonstrated in syngeneicmurine tumor models and a human tumor xenograft model innude mice (128). Tumor necrosis factor has recently beenstudied in Phase I trials (129-132). Toxicity was over a broadrange of doses; antitumor activity was minimal. Some serioustoxicities such as hypertension are not dose related. Someinvestigators have found synergistic antitumor activities in vitroand in vivo when TNF is combined with interferon or cyclophosphamide (133-136). TNF is currently being studied withother biologicals.

Adoptive Cellular Therapy

A variety of immune cells have antitumor activity includingT-cells, NK cells, killer cells, monocytes/macrophages, andpolymorphonuclear leukocytes. B-cells mediate their antitumoreffect by producing antibodies that combine with killer cells,monocytes, or polymorphonuclear cells and mediate antibody-dependent cellular cytotoxicity. Monocytes make TNF andperoxides which are toxic to tumor cells. Polymorphonuclearcells also generate peroxides and contain enzymes that are toxicto tumor cells. NK cells, killer cells, and cytotoxic T-cells allmake lymphokines, such as natural killer cytotoxic factor, thatkill or inhibit the growth of tumor cells.

The different cells that kill tumor cells recognize their targetsin distinct ways. Cytotoxic T-cells recognize the tumor-associated antigen together with self-antigens. Killer cells, monocytes,

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and polymorphonuclear cells mediate antibody-dependent cellular toxicity by binding of their Fc receptor to the Fc portionof the antibody attached to the target tumor cell.

The most extensively studied adoptive cellular therapy is theLAK cell described by Grimm et al. (137). They demonstratedthat incubation of normal mouse splenocytes or human peripheral mononuclear cells with IL-2 in vitro for 3-5 days resultedin generation of cells capable of lysing a spectrum of fresh andcultured tumor cells in vitro. LAK cells are distinguished fromNK cells because of their IL-2 dependence. LAK cells are non-major histocompatibility complex restricted, and lack matureT-cell markers (CD3) although they have other T-cell markerssuch as CD2. They also express the Fc and C3bi receptors (138).No efficacy or major toxicity was seen in cancer patients treatedwith LAK cells alone. Later studies demonstrated that lymphocytes obtained by repetitive leukapheresis, cultured with IL-2for 3-4 days, and reinfused with IL-2 resulted in both partialand complete responses in patients with disseminated solidtumors (101). Several investigators have reported responses inpatients with melanoma, renal cell carcinoma, colorectal carcinoma, and non-Hodgkin's lymphoma treated with IL-2 and

LAK cells (111, 139-145) (Table 3).There are critical questions regarding the mechanism by

which IL-2 in combination with LAK cells mediates theirantitumor effect. In addition, the relative roles of IL-2 and LAKcells remain to be clarified. It has not been demonstrated thatthe LAK cells themselves actually infiltrate the tumor orwhether they are necessary in addition to the IL-2. Currenttrials are addressing these issues. Most of the toxicity associatedwith this therapy is thought to be related to increase in capillarypermeability directly or indirectly related to IL-2. Recent studies using lower doses of IL-2 given by continuous infusion (3-6 milliunits/m2 daily) in combination with LAK cells have

reported efficacy (139). Toxicity is decreased with minimal fluidretention; few patients required intensive care. The in vivogeneration of LAK cells appears to be greater following continuous infusion compared to bolus therapy (146). Trials are underway to determine the efficacy of low dose continuous infusionaltherapy. Other approaches to LAK cell therapy with IL-2include La., i.p., and intrapleural infusions (147-151); clinicalresponses have been observed. However, i.p. fibrosis has beena problem following i.p. therapy (147). Other strategies toimprove LAK therapy include in vitro incubation with IL-4 inaddition to IL-2 which may synergize the generation of LAKcells (152) (Table 4).

Another approach to adoptive immunotherapy is to expandtumor-specific T-lymphocytes that have infiltrated the tumorwith IL-2 in vitro. These cells are referred to as tumor-infiltrating lymphocytes and are reported to be 50 to 100 times moreeffective than LAK cells in lysis of autologous tumor cells fromtumor-bearing mice (102). IL-2-activated TIL have been isolated from human melanoma, renal and head and neck tumors

Table 3 Clinical responses to interleukin 2 with and without lymphokine-activated killer cells

Table 4 How can we improve 1L-2/LAK therapy?

IL-2 + LAKTotal

Ã©valuable CR PR % Refs.

Renal cellcancerMelanomaColorectal

cancerLungNon-Hodgkin's

lymphomaHodgkin'sdiseaseGlioblastoma

multiforme"13183261044692111003216212303122121050750111,

139-143111,139,141,143,

144111,139111,139111,

139,141138,141145

Â°Injected through an Ommaya reservoir.

Optimize dose of IL-2.Optimal method of delivery (continuous r.v.bolus).Enhance activation of LAK (i.e., IL-4).Regional therapy (i.e., i.p., i.a.).Combination therapy with chemotherapy agents (i.e., cyclophosphamide).Combinations with other biologicals (i.e.. a-interferon, monoclonal antibodies).Improved killer cells (i.e., tumor-infiltrating lymphocytes).

(153-156). TIL may expand better and have higher antitumorcytotoxicity than LAK cells and may be a potentially betteradoptive immunotherapy modality. TIL appear to be heterogeneous and have demonstrated both major histocompatibility-restricted (156) and nonrestricted activity (154,155). TIL demonstrated strong cytotoxicity when tested against several allo-geneic fresh tumor cells and cell lines. The actual cytotoxiccell(s) within the TIL population is controversial; althoughmost TIL are reported to be cytolytic CDS-positive T-lymphocytes (154), the antitumor effector cells were reported to belarge granular lymphocytes by others (155). Clinical trials withTIL are under way; promising initial results have been reported(157, 158). In one recent trial (158) 60% of 15 melanomapatients previously not treated with IL-2 responded to therapywith cyclophosphamide, TIL, and IL-2. Interestingly, 2 of 5patients (40%) who had failed prior IL-2 therapy responded tothis combined therapy.

A phase I clinical trial of 7-interferon-activated autologousmonocytes was performed in patients with colon carcinomalimited to the peritoneal cavity (159). These investigators usedcountercurrent centrifugal elutriation to isolate pure preparations of peripheral blood monocytes. These cells were activatedwith 7-interferon and administrated to patients who had undergone an attempted curative resection of residual i.p. coloncancer. The therapy was well tolerated but efficacy is unknown.

Human Growth Factors

Hematopoietic cells are derived from self-renewing pluripo-tent stem cells. Pluripotent stem cells are able to differentiateinto committed progenitor cells which eventually give rise todiscrete cell lineages. A variety of in vitro assays are used toexamine stem cells and their progeny. The least differentiated,pluripotent stem cell that can be identified in culture is theCPU (colony-forming unit)-blast (160). The CFU-GEMM(granulocytes, erythrocytes, megakaryocytes and monocytes)assay detects a progenitor cell with limited self-renewal but thecapacity to generate all of the above cell types (161). Morecommitted colony-forming stem cells have been identified.

These stem cells can form erythroid, megakaryocytic and mixedgranulocyte/monocyte colonies. Growth factors are essentialfor differentiation of all of the aforementioned progenitor cells.Some growth factors are specific for one type of progenitor cell,while others are pleiotropic and affect many types of progenitors (162,163). Growth factors are produced by many differentcells (164). For instance, T cells produce IL-3 and GM-CSF.Monocytes produce M-CSF and G-CSF after contact with IL-3 and GM-CSF. Monocytes also produce IL-1 and TNF whichstimulate GM-CSF, G-CSF and M-CSF production by endo-thelial cells. A number of growth factors can now be producedin large quantities since the complementary DNAs have beencloned (165-169) (Table 5). In Fig. 1, the hematopoietic cellsresponsive to each of the growth factors are shown. While acomprehensive review of growth factors is beyond the scope ofthis paper, this section will focus on those that have currentclinical utility. IL-3 (multi-CSF) appears to be a pan-growth

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Table 5 Human hematopoietic growth factors

GrowthfactorGM-CSFG-CSF

M-CSFIL-3IL-1Alternate

nameCSF-alphaCSF-beta

CSF-1Multi-CSFLymphocyte

activating factorSize

of protein(kD)14-3518-22

35-40 (dimer)20-2612-19Chromosome5q23-3117qll.2-q21

5q33.15q23-312ql3Colonies

stimulated"CFU-GEMM,

CFU-GM,BFU-E, CFU-MEG

CFU-GM, BFU-ECFU-MCFU-GEMM, CFU-GM,

BFU-E, CFU-MEGSynergy with M-CSF and IL-3

Â°CFU-GM, colony-forming unit-granulocyte, monocyte; BFU-E, burst-forming unit-erythrocyte; CFU-GEMM, colony-forming unit-granulocyte, erythrocyte,monocyte, megakaryocyte; CFU-MEG, colony forming unit-megakaryocyte.

Fig. 1. Hematopoietic cells responsive to thevarious colony-stimulating factors [reproducedby permission from Dr. J. D. Griffin (164)).CPU-Blast, blast colony-forming unit; CFU-GEMM, CPU for granulocytes. erythrocytes,megakaryocytes, and monocytes; CFU-E, eryth-roid CPU; CFU-GM, granulocyte/monocyteCFU; CFU-Mega, megakaryocyte CFU; Pro,promyelocyte; Promono, promonocyte; Gran,granulocyte; Mono, monocyte.

* GM-CSFCFU-GEMM

NormoBlast

â€”¿�M-CSF â€”¿�I , I

Platelets

factor for all lineages. GM-CSF enhances the growth of allprogenitor cells beyond the CPU-blast and is an effective enhancer of granulocyte and monocyte function. /// vitro administration of G-CSF causes an increase in circulating neutrophils.M-CSF causes an increase in monocyte number and function.

IL-1 represents a family of polypeptides with a wide range ofbiological activities including augmentation of cellular immuneresponses (T-, B-, and NK. cells); proliferation of fibroblasts;

chemotaxis of monocytes, neutrophils, and lymphocytes; stimulation of prostaglandin E2, increased blood neutrophils; andneutrophil activation (170). Murine and human complementaryDNAs have been cloned (171,172). IL-1 appears to synergizewith other growth factors as well as having a survival-enhancingor maintenance effect on primitive hematopoietic cells (173,174). IL-1 is known to synergize with interferon and IL-2 inenhancing tumor killing by NK cells (175).

Administration of recombinant growth factors to non-humananimals and patients have been accomplished. While IL-3 hasnot been used in humans, injection into experimental animals

activates all types of progenitor cells and induces cell cycle entry(176-178). In non-human primates, GM-CSF has been reported to cause an increase in neutrophils, eosinophils, plateletsand lymphocytes (179). Recently, neutropenic patients withAIDS had an increase in neutrophil counts after receiving GM-CSF (180), and patients with metastatic sarcoma who weregiven recombinant GM-CSF immediately following combination chemotherapy demonstrated a significant reduction induration and degree of neutropenia (181). GM-CSF also stimulated hematopoiesis in patients with myelodysplastic syndromes and aplastic anemia with short-term hematologicalimprovement (182,183). Patients with melanoma and breastcancer receiving high dose chemotherapy and autologous bonemarrow transplantation had an accelerated granulocyte recovery following infusions with recombinant GM-CSF (184). Thetoxicity of GM-CSF has been limited to low grade fever, myalgias, phlebitis, and flushing. Patients with transitional cellsarcoma of the bladder received recombinant G-CSF after thecompletion of combination chemotherapy and had an absolute

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neutrophil count over three times higher than those not receiving G-CSF (185) without toxicity. G-CSF was also shown toreduce neutropenia caused by melphalan in patients with advanced malignancies (186). The role for M-CSF is less clearbecause it is not very effective in promoting in vitro growth ofhuman monocyte progenitor cells. However, it is a potentstimulator of monocyte cytotoxicity.

There is a potential role for growth factors to decreasemyelosuppression secondary to chemotherapy and/or radiationtherapy. It appears likely that growth factors may allow forhigher doses in situations where the major toxicity is bonemarrow suppression. The increased doses may lead to betterand more durable responses. Clearly, the toxicity to otherorgans will limit how high a dose can be given. In the settingof AML a potential problem is that the myeloid leukemia cellsmay proliferate in response to the growth factors (187). Thegrowth factors may be useful for local administration intocertain infected sites. Enhanced monocyte function by growthfactors may be clinically useful. For example, certain monoclonal antibodies are active in monocyte-mediated, antibody-dependent cellular cytotoxicity. Combination therapy with suchantibodies and M-CSF may augment tumor lysis.

Patients with pancytopenia secondary to aplastic anemia,myelodysplasia, infections, etc., may also benefit from growthfactor therapy. While aplastic anemia patients could not beinfused continuously with growth factors they may benefitduring infections or bleeding episodes. In transient pancyto-penic states growth factors may be a major benefit.

While growth factors may enhance leukemic cell growth invitro, it is possible that appropriate growth factor(s) may leadto terminal differentiation of leukemic cells. Alternatively, thestimulation of leukemic cell proliferation may be desirable ifcombined with cell-cycle specific chemotherapy. Early trailswith growth factors have demonstrated their potentially important clinical role. Further trials will determine the optimumdose schedules, toxicity and whether combinations of growthfactors are more effective than single growth factors. This is anexciting new area of cancer research which will generate important clinical data over the next few years.

Monoclonal Antibodies

The development of technology to produce monoclonal antibodies created substantial enthusiasm for using this approachin the diagnosis and treatment of cancer. Monoclonal antibodies are specific for single antigens, can be produced in largequantities from ascites fluid or by tissue culture productiontechniques with high degrees of purity (greater than 90%), andcan be efficiently coupled to isotopes, drugs, and toxins. Thespecificity of monoclonal antibodies should theoretically reducetoxicity to normal tissues unreactive with the antibody. Unlikeconventional antisera, there is little batch to batch variation,and they are of a single immunoglobulin subclass.

Critical Factors for Successful Monoclonal Antibody Therapy.Monoclonal antibodies (8) are useful for in vitro diagnosisparticularly in leukemias and lymphomas (199). In vivo applications have evolved more slowly. Several critical factors needto be addressed (Table 6). One problem is that for in vivo usethese antibodies must have minimal cross-reactivity with normal tissues. Most monoclonal antibodies cross-react with somenormal tissues. This is not unexpected since these antibodiesare directed against tumor-associated rather than tumor-specific antigens. To be effective these antibodies must not cross-react with antigens on normal tissues or at least the antigen

Table 6 Critical factors for monoclonal antibody tumor localization

Cross-reactivity with normal tissues.Distribution of surface membrane antigens.Affinity of antibody.Antigenic modulation.Circulating antigen.Whole immunoglobulin r.\. fragments.Tumor vascularization.Tumor size.Degree of tumor necrosis.

density should be less than on tumors. Ideally, the antigenshould be dense and homogeneous on the tumor cell surfaceand the antibody should bind to the antigen with high affinity.It is also important that the antigen-antibody complex notdissociate from the cell membrane although internal incorporation of the antigen-antibody complex by pinocytosis might beadvantageous when immunoconjugates with drugs, toxins, orisotopes are used. Some tumor-associated antigens are presentin the blood. These might bind the antibody and prevent tumorlocalization. Ideally, one should choose antibodies selective fornoncirculating antigens.

Another critical issue is the form of the antibody. Intactimmunoglobulin can be broken down into various fragments.Intact IgG has a half-life of approximately 24 h, F(ab')2 10 h,

and Fab 90 min. The choice of the appropriate form of antibodyvaries with the tumor antigen system and the conjugate. Forunlabeled antibody therapy, fragments of antibody would notbe useful as the Fc portion is critical for activating humaneffector systems.

For antibody to react with tumor cells, the tumor must bevascular. The number of blood vessels in the tumor decreasesproportionately as the tumor enlarges. In addition, as the tumorenlarges, the diameter of the capillaries increases leading to adecrease in the total vascular cross-sectional area of the tumor.It is not unusual for the outer, better perfused, more viableportions of tumors to concentrate more antibodies than the lessvascular, more necrotic interior portions. Other problems arearteriovenous shunts. Blood entering the tumor is then shuntedto the systemic circulation before it can traverse the capillarybed, thus preventing exchange of the antibody. In addition, thelow pH of tumors decreases capillary flexibility which furtherdecreases vascular perfusion.

Human Antiglobulin Response. One major problem in clinicaltrials using murine monoclonal antibodies is development ofhuman anti-mouse antibodies (antiglobulin response). Theseantibodies can form immune complexes and result in tissuedamage. They also alter the clearance and organ distribution ofinjected antibodies, may neutralize the antibody, or may preventit from binding to tumor cells.

Development of antiglobulin varies in different tumor systems (200). Patients with advanced chronic lymphocytic leukemia, who are typically hypogammaglobulinemic, rarely develop untÂ¡globulins (201, 202). Most patients with cutaneousT-cell lymphomas and solid tumors will develop antiglobulins(200). In some tumors like melanoma, only one-third to one-half of patients developed antiglobulins (203, 204).

The specificity of the antiglobulin is important. Most anti-globulins cross-react with almost all mouse immunoglobulinclasses. However, in several cases, one component of the anti-

Table7 Potentialapproachesto theantiglobulinproblemLarge quantities of antibody (.-400 mg) may tolerize patient.Cyclophosphamide may destroy the antiglobulin clone.Human antibodies or chimeric human-mouse antibodies may be substi

tuted for murine antibodies.Fragments of immunoglobulin may reduce antigenicity.

1626



BIOLOGICALRESPONSE MODIFIERS

Table 8 Monoclonal antibody clinical trials using unlabeled antibodies

DiseaseB-lymphomaB-lymphomaB-CLL"B-CLLCTCLT-ALLATLCALLAMLGastrointestinalMelanomaMelanomaAntibody/classAnti-idiotype/IgGl,IgG2a,

orIg2blF5/IgG2a(anti-CD20)Anti-idiotype/IgGland

IgG2bT101/IgG2a(anti-CD5)T101/IgG2a,

anti-Leu-l/IgG2a(anti-CD5)Anti-Leu-

l/IgG2a(anti-CDS)12E7/IgGl4H9/IgG2aAnti-Tac(anti-CD25)Anti-J5/IgG2a(anti-CD

10)PM/81/IgM,AML-2-23/IgG2b,

PMN29/IgM17-lA/IgG2a9.2.27/IgG2aR24/IgG3No.

ofpatients114118308243202021ToxicityFever,

chills,nausea,vomiting,headache,diarrhea,

transientdyspneaFever,myelosuppressionFever,

urticariaDyspnea,

hypotension,fever,malaise.urticariaDyspnea,

urticaria.fever,hypotensionCoagulopathyNoneFeverFever,

arthralgia.myalgiaUrticaria,bronchospasm,

mildhypotensionFever,

serumsicknessUrticaria,pruritis.fever,

wheezingEffect1

complete and5partialresponses1

partialresponseTransient

reductionincirculatingcellsTransient

reductionincirculatingcellsMinor

responses in10patientsTransient

reductionincirculatingcells1

partialresponseTransient

reductionincirculatingcellsTransient

reductionincirculatingcellsLimited

responsesNone4

partial responsesRef.221,222212201,202,213,214202,214,215216220217218206203,219223

' B-CLL, B-chronic lymphocytic leukemia; CTCL,

leukemia; AML, acute myelogenous leukemia.cutaneous T-cell lymphoma; ATL, adult T-cell leukemia/lymphoma; CALL, common acute lymphoblastic

globulin appeared specific for administered antibody. This istermed an anti-idiotype. Development of anti-idiotype antibodies clearly indicates a primary immune response. Responses toother components of administered antibodies such as the Fcfragment are rapid in onset and consist primarily of IgG suggesting a secondary immune response (200).

Several putative approaches are used to prevent developmentof anti-globulins (Table 7). Some data suggest that the use offragments will reduce this problem but this is controversial(205). In other studies, large quantities of antibodies (>400 mg)are used to "achieve tolerance" (206); these data are also

controversial (203). In one recent study, nonspecific or unrelated monoclonal antibodies were injected just prior to therelevant monoclonal antibody (207). The objective was to increase the exogenous antibody mass and thereby render thespecific antibody less immunogenic. Other approaches to prevent antiglobulins include concomitant use of immunosuppres-sive drugs, such as azothioprine or cyclophosphamide, given atan appropriate time to eradicate responsive clones of B cellsthat would otherwise produce antiglobulin (208). Other studiesuse cyclosporine to interfere with T-cell stimulation of the liceli responses. None of these approaches has been convincinglyshown to be effective. Another approach might be using humanantibodies or genetically engineering antibodies so that the Fcand common portions of the antibody are of human originwhereas the Fab portion is of murine origin (209-211). Thesechimeric antibodies offer the possibility of reduced "antigenic-ity." However, these chimeric molecules would not prevent

anti-allotypic or -idiotypic responses.Clinical Trial with Unlabeled Antibodies. Clinical trials with

unlabeled monoclonal antibodies, i.e., not bound to drugs,toxins, or isotopes, have resulted in only minor responses (Table8). This was not surprising because most murine antibodies donot activate human effector systems or have direct cytotoxic

effects. The most impressive clinical responses with unlabeledantibodies have been with anti-idiotypic antibodies in B-ccll

lymphoma patients (221, 222) and in melanoma patientstreated with an antibody that activates human complement andeffector cells (223).

In some studies monoclonal antibodies to regulatory molecule receptors have been studied (224, 225). Preclinical trialsindicate responses in mice treated with monoclonal antibodydirected to epidermal growth factor (224) and transferrin receptors (225). A trial in patients with adult T-cell leukemia treatedwith the anti-Tac antibody (anti-IL-2 receptor, or CD25 antigen) demonstrated a transient response in one patient; a secondpatient had a 6-month remission with regression of skin lesionsand hematological abnormalities (220).

One novel therapeutic approach involves the use of monoclonal anti-idiotype antibodies in B-cell malignancies. Unlikethe aforementioned antibodies in which the antigen is a tumor-associated antigen; anti-idiotype antibodies are directed againsta tumor-specific antigen; the idiotype of the cell surface im-munoglobulin present on the malignant B-cells. Since B-celllymphomas and leukemias are donai, all of the malignant cellsshould express the same idiotype. Since each patients' lymphoma idiotype is unique; anti-idiotype antibodies must be"tailor-made" for each patient.

The largest experience with anti-idiotype therapy is that ofMiller et al. (224). Their initial report of this therapy was in apatient with rapidly progressive lymphoma resistant to chemotherapy and Interferon. Following eight continuous 6-h i.v.infusions spaced over the period of 1 month, the patient entereda complete clinical remission that was sustained for 6 yearswithout further treatment.3 The mechanism responsible for this

is not clear. Because the antitumor response continued long

3 R. Levy, personal communication.

1627




after the period of passive antibody administration, evidence ofan anti-anti-idiotype antibody response by the patient was investigated; none was detected. It remains possible that indirectmechanisms may be involved. The immune system is regulatedby networks of interactions between idiotypes and anti-idiotypes(226). The anti-idiotype could have triggered these interactionsleading to an antiproliferative response against the tumor.

These investigators treated 10 additional patients with individually tailored anti-idiotype antibodies of different subclasses(222). Some were treated with multiple antibodies with differentisotype or epitope specificity. Tumor responses were reportedin 50% of patients; no complete responses were observed andmost lasted for less than 6 months. Problems included anti-globulin responses and free idiotype antibody in the serumwhich blocked the murine antibody and lead to fever, anthral-gias, dyspnea, and headaches. A more serious problem wasemergence of idiotype variants within tumors during treatment(227). These investigators are currently generating monoclonalanti-idiotype antibodies that identify "shared idiotypes". Shared

anti-idiotype antibodies react with the tumor cells from morethan one patient. Panels of "shared idiotype" antibodies would

lead to a much broader application of this novel approach toB-leukemia/lymphoma therapy.

Combination therapy with other biological response modifiers might increase the antitumor effect of monoclonal antibodies. For instance, M-CSF stimulates monocyte tumoricidalactivity and phagocytosis (164). Antibodies that act throughmonocyte-dependent cellular cytotoxicity might have a greatereffect when given with M-CSF or 7-interferon (228). IL-2activates human killer cells (98-100) and can enhance theactivity of antibodies that mediate killer cell cytotoxicity (229).IL-2 and a-interferon have been shown to synergistically enhance the effect of anti-idiotype antibodies in a transplantablemurine B-cell lymphoma (230, 231). A combination of IL-2and LAK cells incubated with an appropriate monoclonal antibody in vitro is particularly appealing. Combination trialssuch as those described are under way with several antibodiesand biologicals.

At the turn of the century, Ehrlich coined the term "magicbullet" (232). He envisioned antibodies (at the time polyclonal

antisera) delivering toxic agents directly to tumor cells to eradicate the tumor cells. This concept was repopularized with theintroduction of the monoclonal antibody technology. A varietyof potentially toxic molecules have been linked to monoclonalantibodies. Most of this work has been focused on the use ofisotopes, toxins, and drugs coupled to antibodies. Some advantages of these immunoconjugates are reviewed in Table 9.Radioisotopes have the advantage that the radiation field canradiate beyond the antibody-binding cell. The extent of theradiation field depends on the type of isotope used (discussedbelow). The disadvantage is that some normal tissues may beradiated. Another advantage of radioisotopes is that it is notessential that they be internalized. Toxins and drugs require

Table 9 Advantages of different immunoconjugates

RadioisotopesLarge variety with widely varied characteristics.Most isotopes radiate beyond a single cell.Most isotopes will not require internalization.

ToxinsExtremely potent.Well denned chemistry.

DrugsProven "track record" in cancer therapy.

Toxicity well defined.Maximal tolerated doses known.New and more potent drugs may be useful as immunoconjugates.

internalization to destroy the target cell.Immunoconjugates with Toxins. Several potent plant and bac

terial toxins have been coupled to antibodies. Plant toxinsinclude ricin, abrin, gelonin, pokeweed antiviral protein, andsaporin. The most commonly studied bacterial toxins are diphtheria toxin and Pseudomonas exotoxin. All of these immuno-toxin conjugates demonstrate specific antitumor cytotoxicity invitro and in vivo (233-237). For many toxins a single molecule

may destroy a cell. Some toxins, such as ricin, abrin, anddiphtheria toxin, consist of A and B chains. The A chain iscytotoxic to the tumor cell by inhibition of protein synthesis.The B chain binds galactose which is found in high density onthe surface of most cells. Entry of the A chain into cells dependson B chain binding to the cell surface. For in vivo therapy withimmunotoxins it is necessary to isolate the A chain and cova-leni ly link it to the monoclonal antibody. Elimination of the Bchain prevents nonspecific binding. Clinical trials of this approach are under way (238,239). In one of these studies modestactivity was reported in patients with malignant melanoma(239).

Immunoconjugates with Cytotoxic Drugs. Another approachto generating immunoconjugates is by attaching cytotoxic drugssuch as doxorubicin, daunorubicin, methotrexate, vinblastine,or melphalan to monoclonal antibodies (233, 240). The advantage of using drugs is that their antitumor activity and toxicityare well defined. Conjugation to antibodies allows direct targeting of the drug to tumor cells and should reduce toxicity tonormal tissues. Some drugs may be more active against a tumorwhen delivered to the tumor cell by a monoclonal antibody. Inaddition, some potent drugs not used clinically because ofexcessive toxicity may be usable when incorporated into immunoconjugates. The disadvantage of drugs is that they are notas potent as toxins and cannot kill cells other than the singlecell the antibody binds.

Antibody-coated liposomes containing chemotherapeuticagents provide an alternate method of selective drug delivery.Antibodies are either conjugated to the liposome or linked viaprotein A (241). Antibody-labeled liposomes containing chemo

therapy drugs are demonstrated to increase cytotoxicity againstmurine and human tumor cell lines (242, 243). A correlationbetween growth inhibition and liposome internalization wasdemonstrated in one study. These data suggest that this approach may not be useful with antibodies that do not modulate(244).

Immunoconjugates with Isotopes. The major limitation ofdiagnostic imaging techniques is the lack of specificity. Theattractive feature of radiolabeled monoclonal antibodies is thepotential to specifically localize to tumor cells. Similarly, conventional radiation therapy is not specific for tumor cells. Thefield that can be radiated is also limited. Radiolabeled antibodies should be able to radiate tumor deposits throughout thebody with minimal radiation to normal tissues (reviewed inRefs. 245 and 246).

Diagnostics. Several radionuclides are available that can belinked to monoclonal antibodies for diagnostic purposes. Thechoice is governed by radionuclide energy, half-life, technicalrequirements for conjugation, conjugate stability, safety, andcost. It is preferable to use radionuclides with energies between100 and 200 keV which is the optimal energy for the gammacameras currently in use (Table 10). The radionuclides shouldhave minimal paniculate radiation to maximize their safety.They should have an adequate half-life to permit tumor localization and background clearance but sufficiently short to minimize radiation exposure of the patient and medical staff.

1628




99mTc, '"In, and 123Ihave suitable properties for diagnosticimaging. I31I is most often used because of its availability andlow cost. However, 13IIis not ideal because of high 7-ray energythat degrades images, dehalogenation in vivo; its 8-day half-lifeand paniculate ÃŸemissions limit the total dose. Currently, I23I

is too costly, is not widely available, and dehalogenates in vivo.'"In is generally conjugated to antibodies via dictation to

diethylenetriaminepentaacetic acid. Nonspecific localization inthe liver and spleen has been a problem with the '"In. 99mTc

would appear to be the optimal isotope, if the problems ofkinetics and clearance of antibody can be overcome.

The first diagnostic imaging studies were with monoclonalantibodies using I3'l-labeled anti-carcinoembryonic antigen

monoclonal antibody. Background subtraction was required inorder for the tumor to be imaged (247). In another study I23I-

labeled antibodies that reacted with the human milk fat globulewas used (248). The first systematic human study of 13ll-labeled

monoclonal antibody Fab fragments was reponed in patientswith malignant melanoma (249). Approximately 60% of theknown sites were detected by scanning and more than 90% ofpatients had at least one site detected. Tumors with diameters<1.5 cm and those with poor antigen expression accounted formost negative findings. Significant deiodination of the proteinin vivo was reported. Numerous other studies of iodinatedmonoclonal antibodies have been reported; all have similarproblems.

There have been a number of studies using '"In-labeled

antibodies. Lesions as small as 1 cm have been detected (250,251) (Fig. 2). Problems encountered with "'In have been retic-

uloendothelial uptake of the antibody which has compromisedimaging of lesions in the liver and spleen.

Several studies report improved localization of the radiola-beled antibody when unlabeled antibody is given simultaneouslywith "'In-labeled antibody (251, 252). Detection rates haveincreased 2- to 3-fold using this technique. Apparently theunlabeled antibody favorably affects the biodistribution of thelabeled antibody and improves imaging. An irrelevant antibodythat does not bind to the same site can achieve similar results.

Excellent imaging of cutaneous T-cell lymphoma patients isobserved using '"In-labeled T101 antibody (250). T101 bindsto circulating T-lymphocytes and the antigen-antibody complexmodulates internally (253). It is believed that the circulatingcells with the internalized complex traffic to the malignantlymph nodes and skin enhancing the localization at these sites.Concentrations of 0.01 to 0.03% injected dose per g wereachieved in diseased lymph nodes, which is approximately 10-fold greater than previously achieved in other systems. A problem with this study was nonspecific reticuloendothelial uptake

Table 10 Isotopes for monoclonal antibodyimagingâ€¢Y-Energy

Isotope Half-life(keV)"Te6 h14013II

8 days364'"I

13 h159'"In

3 days 173247AdvantagesAvailability,7-energy,inexpen

siveIodine

chemistry, inex

pensive,availableIodine

chemistry1-EnergyDisadvantagesRapid

kinetics,renaland gas

trointestinal uptake,complexchemistryDehalogenation,high

i-ray,ÃŸ-emissionsDehalogenation,expensive,

notavailableLeaches

offchelate.expensive,affin

ity for reticuloendothelial system

^Ã‰Ã‰yc

^^ I^^P^H^^

Front RearFig. 2. Image of a patient with cutaneous T-cell lymphoma (with the SÃ©zary

syndrome) taken 48 h after the i.v. injection of 5 mCi of '"In linked to 1 mg of

T101 antibody + 10 mg unlabeled TI01. Full body scans shows front (left) andrear (right) views demonstrating excellent localization in cutaneous disease andinvolved lymph nodes with nonspecific localization in the liver and spleen.

leading to localization within the liver and spleen. Some studiesin animals (254) report that linking the chelate to oxidizedcarbohydrate decreases reticuloendothelial uptake. If confirmedin humans this could improve results with "'In conjugates.

Utilizing "'In-labeled T101, immunolymphoscintigraphy

with s.c. injections intradigitally led to excellent images belowthe diaphragm with minimal uptake in the liver and spleen inpatients with cutaneous T-cell lymphoma (255). This studyreported efficient and specific antibody binding in lymph nodesnearest to the injection site with progressive uptake of remaining antibody in more distal nodes as proximal sites approachsaturation. In one patient, inguinal-femoral lymph node biopsycontained over 2% of the injected dose of '"In-labeled T101

antibody at 7 days. Although this technology does not allow fora total body image, it may offer advantages by improving thequality of imaging in critical regions important for stagingcertain lymphoproliferative diseases.

A diamide-dimercaptide (N2S2)chelate was developed to labelantibody with 99nTc (256). Using the N2S2 chelate, excellent

results in imaging malignant melanoma were demonstratedusing F(ab'): and Fab fragments of an anti-melanoma antibody

(257). MÃ©tastasesin skin, lymph node, lung, liver, brain, bone,and spleen were detected as early as 6 h after injection. In Fig.3 lymph node and subcutaneous lesions are demonstrated. Anumber of these subcutaneous lesions were not detected clinically prior to the scan. There was no significant uptake in thereticuloendothelial system. Biliary and renal excretions werevisualized but cleared with time. Up to 0.03% injected dose per

1629




Fig. 3. Image of a patient with malignant melanoma taken 8 h after theinjection of 10 mCi of ""Te linked to 10 mg of the Fab fragments of the NR-Ml.-05 antibody demonstrating multiple lymph node and s.c. nodules (arrows).Blood pooling in the heart and nonspecific localization in the sinuses are alsoseen.

g was observed in excised tumors. This was comparable to thatreported using "'In-labeled T101 in cutaneous T-cell lym-

phoma (250). Approximately 80% of known lesions were detected and an equal number of not previously diagnosed lesionswere found.

In summary, imaging with monoclonal antibodies is feasible.Questionable areas can be evaluated by other techniques suchas computerized axial tomography or magnetic resonance imaging. Some data indicate that tumors undetectable with presenttechniques may be visualized with isotope-labeled monoclonalantibodies.

Therapy. Radioisotopes such as 32P and 13IIhave been used

to treat malignancies such as polyeythernia vera and metastaticthyroid carcinoma for several years (Table 11). A reasonableextension of this approach is to link isotopes to antibodies.Several isotopes that can be used include Â«-emitters; low-,medium-, and high-energy /3-emitters; and radionuclides thatact by electron capture and/or internal conversion (Auger electrons).

Choice of the appropriate radioisotope for therapy is complex. Longer particle length radionuclides, such as 90Y and

l88Re, are preferred in tumors with heterogeneous antibody

binding. This should allow for destruction of nonlabeled tumorcells but might increase toxicity to normal tissues.

Shorter particle length radionuclides are preferred wherethere is uniform antibody binding. Electron capture or internalconversion decay radionuclides such as 125Iwould be optimalin such a situation; other choices include Â«-emitters such as211Astatine.

Anti-ferritin polyclonal antibody labeled with 131I(Table 12)produced responses in persons with hepatomus and Hodgkin's

disease (258, 259). Single doses as high as 150 mCi were givenwith acceptable hematological toxicity. Interestingly, iodinatedmonoclonal anti-ferritin antibody were not as effective as polyclonal antibodies. Studies in animal models demonstrate thatisotope-labeled monoclonal antibodies can be effective (264).There have been relatively few reports of human studies usingradiolabeled monoclonal antibodies. Responses have been reported (260) in patients treated with the anti-Lym-1 antibodylabeled with 131I.Although these patients were heavily pre-treated several responded. I31l-labeled T101 antibody (150-250

mCi) has been used to treat patients with cutaneous T celllymphoma (261). Responses were observed in two patients withskin and lymph node involvement but lasted only 2 to 3 months.Myelosuppression was dose limiting. Two B-cell lymphomapatients were treated with 131I-labeledMB-1 (250 and 480 mCi)

with 1 partial and 1 complete response (262). Although bothhad significant myelosuppression, no intervention was required.

A Phase I study of 131I-labeledFab in patients with malignant

melanoma was recently reported (249). Patients received up to529 mCi of I31Iin divided doses; target organ toxicity was bone

marrow associated. Despite good antibody localization in selected patients with estimated tumor doses of 1300-5500 cGy,anti-tumor effects were modest.

Therapy with 131I-labeled anti-ovarian antibodies i.p. dem

onstrated responses in 9 of 24 patients (263). Doses >140 mCiwere more effective than lower doses. Only patients with smallvolume (<2 cm) disease responded.

The first isotope other than 131Ito enter clinical trials was90y Â»OYhas a more energetic /3-particle than 131I(2.2 versus 0.6

MeV). It is bound by diethylenetriaminepentaacetic acid but ithas no imagible 7-ray. Rhenium has two different /3-emittingisotopes (186Re,188Re)that are chelated by the same N2S2 ligandused for 99mTc. Rhenium is bound irreversibly to N2S2 as is99mTc.Both isotopes have imagible 7-ray energies. Clinical trials

with rhenium are expected to commence in 1988.The next 2 years should see the first systematic studies of

targeting radiation therapy directly to metastatic carcinomawith monoclonal antibodies. Issues such as single dose versus

Table 11 Isotopesfor monoclonalantibodytherapy

Isotope'"Re""Re13IJ67Cu"Â¥'"AstatineiÂ»!Half-lifeDecaymode17

hÃŸ3.5

daysÃŸ8

daysÃŸ2.5

daysÃŸ2.5daysÃŸ7h

a2.5

days Electroncapture(Auger)l'ari

Â¡culaiemaximumenergy(MeV)2.11.10.60.4-0.62.35.97.5AdvantagesGenerator

produced,Tcchemistry"Te

chemistryIodine

chemistry, inexpensiveImagesIndium

chemistryIodine

chemistry,shortrangeIodine

chemistryDisadvantagesVery

energetic0-particleAvailability,

lowspecificactivityDehalogenation,

abundant7-emission,half-lifeAvailability,costNo

imaging, leachesoffchetateDehalogenationNo

imaging,dehalogena-tion,availability

1630




Table 12 Clinical trials with '"Â¡-labeled monoclonal antibodies

DiseaseHepatoma"Hodgkin's

diseaseB-lymphoma

andB-CLLCTCL*B-LymphomaMelanomaOvarianAntibody/classAntiferritin/het-eroantiseraAntiferritin/het-eroantiseraLym-l/IgG2aT101/IgG2a(anti-CD5)MB-1/IgGl8.2/IgGlc96.5/IgG2aHMFG1,HMFG2''AUA1,H17E2/IgGlNo.

ofpatients105381862724ToxicityThrombocytopeniaMyelosuppressionFever,

rash,myelo-suppressionDyspnea,

fever,urticaria,myelosuppressionMyelosuppressionMyelosuppression,

chills.fever,hypotensionMyelosuppression,

fever,diarrheaEffect7%

complete and41%partialresponses40%

partialresponses2

complete and 7 partialresponses2partial, and 3minorresponses1

complete and 1 partialresponseNo

responses9

partial responsesRefs.258259260261262249263

" Some patients were also treated with external beam irradiation and chemotherapy.* B-CLL, B-chronic lymphocytic leukemia; CTCL, cutaneous T-cell lymphoma.' Fab fragments were used in this study.* i.p. therapy.

fractionated dose must be addressed in these studies. Imagingof patients for dosimetry prior to the delivery of therapeuticdoses of isotopes is of critical importance. Parallel advances inin vitro purging of tumor cells from bone marrow followed byautologous bone marrow transplantation permits the use ofbone marrow-ablating doses of radiation if required. These

studies can be undertaken while further advances in tumorlocalization and blood clearance are pursued.

Bone Marrow Transplantation. An attractive therapeutic application of monoclonal antibodies is to remove tumor cellsfrom bone marrow prior to autotransplantation. Patients inclinical remission often have undetectable tumor cells in thebone marrow. Theoretically these could be detected and removed with specific antibodies. Most of the obstacles andtoxicities with monoclonal antibody infusion would be eliminated since the treatment is in vitro. Such an approach has beenreported using the anti-Bl monoclonal antibody to purge autologous bone marrow in patients with non-Hodgkin's lym

phoma (265). Several antibodies were used in patients withALL (266, 267). Early results have demonstrated that bonemarrow reconstitution occurs in almost all patients. Therapy iswell tolerated and most patients enter remission. In ALL onlyone-third of the patients remain in remission for longer than 1

year, a result not different from that with allogeneic bonemarrow transplants. The results for non-Hodgkin's lymphoma

are more promising with over 50% of the patients remaining inremission for a median duration of 22 months. However, in theabsence of controlled trials there is no evidence in lymphomaor ALL that results of these autotransplants were related to theantibody treatment.

Another transplantation approach with monoclonal antibodies is to utilize antibodies in vitro for T-cell depletion. Thisapproach has been highly successful in preventing graft-verau-

host disease but has led to higher incidences of graft rejectionand recurrent leukemia (188-190). Mechanisms underlying

these adverse consequences may be due to the lack of essentialhelper cell function and/or growth factors provided by T-cellsleading to graft rejection and loss of graft-vmiw-leukemia effect

leading to recurrent leukemia.

Tumor Vaccines

Another potential antitumor strategy is activation of hostdefenses against tumor-associated antigens. This is referred to

as active specific immunotherapy which attempts to boost the

host's immune response. The tumor-associated antigens must

be accessible on the tumor cell to serve as a site for antibody-mediated or cell-mediated destruction. Most studies of active

specific immunotherapy in humans with hematological malignancies have been disappointing. For example, early studies inALL suggested that children could be successfully immunizedwith allogeneic leukemia cells (3). These results could not bereproduced in controlled trials (268, 269). Similarly, earlyresults in acute myelogenous leukemia patients suggested prolonged survival with BCG and/or allogeneic leukemia cell (4,5, 270-272) immunization. Again, these results could not be

reproduced (7, 273).Great promise was held for BCG immunotherapy with or

without cellular vaccines for malignant melanoma (2); long-

term results were disappointing. However, a series of interestingstudies focused on melanoma patients demonstrated activeimmunity in patients with promising preliminary clinical results(Table 13) (274-280).

Clinical vaccine trials for other solid tumors are limited innumber. In one trial patients with colorectal cancer with trans-

mural extension of tumor or nodal metastasis were randomizedinto groups treated by resection alone or resection with activespecific immunotherapy with autologous tumor cells plus BCG.At 28 months follow-up recurrences were significantly fewer in

vaccinated patients and they demonstrated cellular immuneresponse to autologous tumor cells (281, 282). In a metastaticrenal cell vaccine study, 4 of 14 patients responded to vaccinetreatment with autologous tumor cells mixed with Corynebac-terium parvum (283). In another study patients with broncho-

genie carcinoma received a vaccine of allogeneic tumor cells incomplete Freund's adjuvant. A 10-year follow-up demonstrated

a statistically significant 5-year survival advantage for patients

treated with specific active immunotherapy (284).A major problem with tumor vaccines is our inability to

standardize the contents of a vaccine, particularly with referenceto the tumor-associated antigen(s) of interest. Perhaps the anti-

idiotype vaccine approach discussed below will adequately address this problem.

Anti-Idiotype Vaccines

Another novel approach to tumor vaccines based on theidiotypic network hypothesis warrants discussion (226). Tumor-associated antigens are often a part of "self and evoke a very

poor immune response in the tumor-bearing host because of T-

1631



BIOLOGICALRESPONSE MODIFIERS

Table 13 /mmunotherapy trials for patients with malignant melanoma

No. ofpatients Stage Vaccine

Activeimmunity

Clinicalresults Ref.

42

55

39

10

19

17

26

III Autologous with melanoma cells with BCGplus cyclophosphamide

II and III Polyvalent melanoma antigen

I and II Vaccinia melanoma onco-lysate

III Semiautologous melanomahybrids with C. parvum

III Autologous melanoma cell(Â±cyclophosphamide)

II Allogeneic melanoma lineswith BCG or C. parvum

Allogeneic melanoma celllines

Reduction in CD4+ CD45+T-cellsÂ°

69% stage II53% stage III64%

Positive delayed hypersensi-tivity

88% with cyclophosphamide;29% without cyclophosphamide

94% developed antibodies toalloantigens

15% developed anti-GP2 antibodies.

38% developed anti-GM2antibodies.

NR"

MR

Increasedsurvival

No responses

2 completeresponses

NR

NR

274

275

276

277

278

279

280

Â°CD4+ CD45+ T-cells are the subset that induce suppression.* NR, not reported.

cell-mediated suppression (285, 286) and tolerance to the antigen (287). Recent data indicate that a weak antigen can beturned into a strong antigen by simply changing the molecularenvironment of the haptenic structure. These changes in thehapten carrier activate T-cell help and increase the immuneresponse. In addition, altering the carrier can turn a tolerogenicantigen into an immunogenic antigen. The immune status ofcancer patient is often suppressed and they can respond to onlycertain T-dependent antigens but not to other antigen forms.These considerations suggest introduction of molecular changesinto the tumor antigens before using them as vaccines. Presently, this is impossible to accomplish because tumor antigensare not well defined and are difficult to purify.

According to the idiotypic network hypothesis, certain anti-idiotype antibodies express three dimensional shapes whichresemble the structure of external antigens. The existence ofsuch internal antigenic idiotypes comes as a statistical necessityof the diverse and "complete" network of idiotypes (288, 289).

The internal antigens are stereochemical copies of nominalantigens which produce specific immune responses similar toresponses induced by nominal antigen and also can competewith normal antigens in binding assays.

Assuming one is aiming for a tumor-specific immunotherapy,the anti-idiotype hybridoma route of making surrogate antigenscould alleviate the problem of making a large amount of purifiedantigen associated with a given tumor. Furthermore, idiotype-

based tumor antigens are free of the danger of transmittinghuman tumor viruses which theoretically may contaminate cell-based human tumor vaccines. Parenthetically, it makes nodifference in this approach whether a de novo tumor antigen isthe target for immunotherapy or whether the target is a "normal" but highly tumor-associated antigen. If a tumor-specifichybridoma is available then a second anti-hybridoma (anti-idiotypic hybridoma) can be made and selected for the structurewhich represents the tumor antigen. In Fig. 4, this approach isdiagrammed. An anti-tumor-associated antibody (Abl) is generated and is used to make a second antibody or an anti-idiotypeantibody (Ab2).

There are three different types of Ab2 antibodies a, 0, and 7.The a type of Ab2 recognizes an idiotype that is distinct fromthe binding site of Abl. The y type of Ab2 recognizes a targetidiotope that is close to the binding site so that it can interferewith the antigen binding (288). The ÃŸtype of Ab2 is the internalimage of the antigen for Ab2 (289, 290). If the ÃŸtype is usedlike an antigen, an Ab3 antibody is induced. It has an antigen-

immunize Mouse with Tumor Cell to Generote Abi

Immunize Mouse with Ab) to Generate Anti-idiotype (Ab2)

Ab2Abt

Immunize Patients with Anti-idiotype (Subcutaneous with

Adjuvant and/or Carrier)

Ab3

Ab2

Fig. 4. Idiotype tumor network. Concept of monoclonal anti-idiotypic vaccines. An Abl is generated to a tumor-associated antigen. The Abl is used toimmunize mice to generate an anti-idiotypic monoclonal Ab2. The Ab2 which isthe mirror image of the tumor-associated antigen is then used to immunizepatients to generate an Ab3 response to the tumor-associated antigen. Note thatthe Ab3 (or Abl') has a binding specificity identical to that of the Abl.

combining site (paratope) similar to that of Abl. Because ofthis paratope similarity, the Ab3 is also called the Abl' to

indicate that it might differ in its other idiotypes from the Abl.The cyclic nature of complementary binding sites and idi-

otopes serves as the basis for the idiotope vaccines. The experimental evidence that idiotypes or anti-idiotypes mimic antigensand produce antitumor immunity is quite strong (288, 291-293). Treatment of mice with injection of anti-idiotype thatappears to mimic tumor antigen structures has been reportedto induce antitumor immunity in human melanoma (294),mammary adenocarcinoma (295), and rat sarcoma (296). Recently, anti-idiotypes produced in goats that contained antibodycomponents that mimic human gastrointestinal tumor-associated antigens have been generated. Mice and rabbits immunizedwith this anti-idiotype produced antibodies (Ab3) that boundto human tumor antigen (297). Monoclonal anti-idiotypes to aleukemia/lymphoma T cell associated antigen have evoked anAb3 response in animals (298, 299). These Ab3 anti-anti-idiotypes bound to the tumor antigen and immunoprecipitatedan antigen identical to the Abl. In a clinical trial where patientswere given i.v. injections of murine antitumor antibodies, thosepatients that developed an anti-idiotype response improvedclinically and had longer remissions from their disease (300).

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Indirectly, these results appeared to be consistent with the anti-

idiotypic hypothesis. They did not demonstrate Ab3 responsesin these patients, however. In another clinical trial (301), patients with colorectal carcinoma were immunized with goatanti-idiotypic antibodies to murine monoclonal antibody CD 17-1A (Abl). All patients developed Ab3 with binding specificitieson the surfaces of tumor cells similar to the Abl specificity.The Ab3 also inhibited binding of Ab2 to Abl suggesting sharedidiotopes with Abl. Three of 30 treated patients responded. Inanother clinical trial 15 patients with disseminated melanomawere immunized with monoclonal anti-idiotypic antibodies toa high molecular weight melanoma-associated antigen (302).Seven patients developed antibodies (Ab3) which inhibited thebinding of anti-idiotypic antibody (Ab2) to the Abl antibodyand 3 patients showed tumor reduction. Current clinical trialsare ongoing to further study the effects of anti-idiotypic vaccine.

Conclusion

Biological therapy may be considered the "Fourth Modalityof Cancer Treatment" (321). The past decade of research has

demonstrated that there is indeed a role for biological agentsin cancer therapy. Over the next decade, combination therapies,new cloned molecules, and more sophisticated chemistry forimmunoconjugates should lead to major advances in cancerdiagnosis and therapy.

Acknowledgments

I am indebted to Drs. R. P. Gale, M. Goldrosen, L. Vaickus, and W.Biddle for their careful review of this manuscript, NeoRx Corporationfor the "Te scan, and Pat Alvarado for the preparation of this

manuscript.

ImmunomodulationReferences

4-

Immunomodulation is a rapidly expanding field and is applicable in a variety of clinical settings including autoimmunediseases, graft rejection, as well as the treatment of cancer.Immunomodulation with lymphokines such as interferon andIL-2 have been discussed previously, and the use of BCG,

Cornybacterium parvum, and other stimulants of the immunesystem was also briefly reviewed. There exists a growing list ofdrugs, synthetic agents, hormones, and bacterial cell products 5.

that have immunomodulatory activity.Cyclophosphamide, in relatively low doses, has an anti-sup

pressor T-cell effect. Cyclophosphamide has been used to en- 6-hance the response to IL-2 (1 14) and tumor vaccines (274, 278)and also as a single agent. In one melanoma study, cyclophos- 7.ph amide depression of suppressor T-cells was restricted to thosepatients who demonstrated increased suppressor T-cell activity g

prior to Cyclophosphamide treatment (303). Other cytotoxicagents such as mitoxantrone (304, 305) and doxorubicin (306)also have immunomodulatory activity. Corticosteroids have a 10.profound suppressive effect on both humoral and cellular immunity and are used to treat autoimmune diseases and graftrejection. They are also lympholytic and are used to treat a 12.variety of lymphoid neoplasms. Diethylstilbestrol (307) and theantiestrogen tamoxifen (308) also have immunomodulatoryactivity. Cyclosporin has been shown to have suppressor effectson both humoral and cellular arms of the immune response(309). Nonsteroidal antiinflammatory agents (310), morphine(311), marijuana (311), and antibiotics such as amikacin and 15-

cefotoxime (312) also have immunomodulatory activity. 16A variety of synthetic agents modulate the immune system

such as polyribonucleotides which augment natural killer activity and induce interferon production (313). Thymosin fractionS acts as an immunostimulant and has a significant antitumor 18.effect in animal models (314). Removal of IgG and circulatingimmune complexes by passing plasma over a protein A columnmay also modulate the immune system. Responses have beenreported in a variety of cancer patients (315). Bacterial cellproducts such as Bordetella pertussis (316), Corynebacterium 21.granulosum (317), and Listeria monocytogenes (318) and manyothers have potent immunomodulatory activity.

Another innovative approach to immunomodulation has been 23.

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ies can also be utilized systemically to alter cell populationssuch as T-cells or T-cell subsets (319, 320). 26.

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Biological Response Modifiers: The New Immunotherapy · Biological Response Modifiers: The New Immunotherapy Kenneth A. Foon1 Division of Clinical Immunology, Roswell Park Memorial