Critical Reviews in Oncology/Hematology, 1992; 131211-282 0 1992 Elsevier Science Publishers B.V. All rigths reserved. 1040-8428/92/$5.00

271

ONCHEM 00038

Monoclonal

antibody therapy of cancer

Albert F. LoBuglio and Mansoor N. Saleh University

of Alabama at Birmingham, Comprehensive Cancer Center, Birmingham, AL, USA (Accepted 2 September 1992)

Contents I.

Introduction

II.

Types of monoclonal antibody

III.

Intact versus antibody fragments

IV.

Murine, human and genetically engineered antibodies

V.

Antibody conjugates - toxins, drugs, radionuclides

VI.

Clinically approved antibodies

VII.

Phase I and II cancer therapy trials ............................... A. Antibody alone .......................................... B. Antibody-radionuclide trials ................................. C. Antibody-toxin trials ...................................... D. Antibody drug trials .......................................

275 275 211 278 279

VIII.

Future efforts

279

Acknowledgement

. . . . . . . . . . . . . .._................................___...

.

272

.

.

.

.,.....,........,.._.....,.......__...,._._....__....,__._.

References................................................................

I. Introduction

Inherent or acquired resistance of tumor cells to conventional chemotherapeutic drugs [ 1,2] and the toxicity associated with these reagents [3] has led to a search for new options for the treatment of malignant diseases. By virtue of their unique antigen specificity, monoclonal antibodies (MoAb) targeted at tumor-associated antigens have emerged as a potentially important tool in the battle against cancer. The accomplishments of Kohler and Milstein [4] paved the way for the large-scale production of monoclonal antibodies of defined specificity.

Correspondence to: Mansoor N. Saleh, M.D., University of Alabama at Birmingham, Comprehensive Cancer Center, L.B. Wallace Tumor Institute-263. Birmingham, AL 35294-3300, USA.

271

. .

272 272 213 275

279 280

Over the last 10 years, monoclonal antibodies have moved from the laboratory bench to the forefront of innovative applications in clinical oncology [5,6]. A variety of antigens are suited as targets for monoclonal antibody therapy. These include oncofetal antigens that are highly expressed on the surface of various tumor types but present to a much lesser degree on normal human tissue (e.g., carcinoembryonic antigen) [7] and differentiation antigens expressed during normal cellular ontogeny and encountered as tumor markers during malignant transformation (e.g., lymphocyte surface antigens) [8]. In addition, advances in molecular biology have recently defined a number of cell surface receptors that are products of cellular oncogenes and play a crucial role in tumor cell growth. Such receptors (e.g., PDGF receptor, EGF receptor) present themselves for antibody targeting [9].

272

The clinical use of murine monoclonal antibodies is associated with a number of drawbacks, including immunogenicity of the foreign protein, short circulating time of the antibody and the inability to fully harness the effector and complement repertoire of the human immune system. Human monoclonal antibodies could potentially overcome some of these drawbacks [lo], but the production of human monoclonal reagents using hybridoma technology has met with a number of technical obstacles. Genetic engineering technology has allowed the production of monoclonal antibodies which retain the murine antigen binding sites with varying degrees of human immunoglobulin structure, i.e., chimeric [11] or ‘humanized’ monoclonal antibodies [ 121. In order to enhance their anti-tumor effects, monoclonal antibodies armed with radionuclides, toxins or cytotoxic drugs are also being studied [ 13,141. Although recent advances have served to partially overcome the problem of immunogenicity and short circulation time, the more complex issues of antibody transport and delivery to the tumor cell, optimal harnessing of the anti-tumor potential of the human immune system and the phenomenon of tumor heterogeneity remain to be successfully addressed. Ongoing clinical trials have led to a much better appreciation of these processes and their relevance to the clinical application of monoclonal antibodies. This chapter will serve as a brief overview of the current status of monoclonal antibody therapy in cancer. The following sections will include a discussion of the different types of therapeutic monoclonal antibodies currently undergoing clinical testing and will focus upon the merits and limitations of the various reagents. The various clinical trials that form the basis for currently ongoing efforts will be reviewed. The final section will focus on providing a brief synopsis of future strategies in the use of monoclonal reagents. II. Types of monoclonal antibody

The promise of monoclonal antibodies in the treatment of cancer lies in their unique ability to recognize and specifically bind to the target antigens expressed on tumor cells. The variable region of the immunoglobulin determines its antigen binding characteristics while the constant region conveys its capacity to interact with effector cells and complement arms of the immune system [15]. The constant region with its larger size and carbohydrate residues is also the most immunogenic component of the molecule and, furthermore, determines its pharmacokinetic characteristics. Depending on the therapeutic strategy, the antibody molecule can be modified, altered or conjugated with various cyto-

toxic moieties. The following section will briefly overview the various types of antibody molecules in clinical trials. III. Intact versus antibody fragments

Intact murine monoclonal antibodies generally have a circulating half-life of 16 to 48 hours when administered to human subjects [16,17]. This is much shorter than the 21 day half-life of human IgG (all subclasses except IgG3 which has a 5 day half-life) [18]. Murine IgG2a appears to be most efficient in mediating antibody dependent cellular cytotoxicity (ADCC), whereas the IgG3 isotype mediates potent complement-dependent cytolysis (CDC) [15]. For human antibodies, the IgGl and IgG3 subclasses are most active in mediating ADCC and are upstaged only by IgM in their ability to mediate CDC [19]. In most studies, intact murine antibodies have been found to be immunogenic in man with a majority of the antibody response directed at the constant region and a smaller component directed at the antigen binding domain (anti-idiotype) [l&17]. Fragments of monoclonal antibody can be prepared by enzymatic digestion that cleave off the Fc portion and yield either a bivalent antigen binding fragment (Fab’,) or a monovalent fragment (Fab) [ 151. Furthermore, single chain antigen binding proteins (SCA) consisting of the variable region of the light and heavy chain linked by a peptide can be produced by gene cloning. In general, these monoclonal antibody fragments retain the antigen binding specificity of the intact molecule but have a much more rapid plasma clearance and significantly reduced immunogenicity [20]. Because of their smaller size (25-100 kDa), these fragments have the advantage of a more rapid equilibrium with the extravascular space and easier distribution in tumor sites. These properties make radiolabeled antibody fragments well suited for radioimmunodetection [21]. However, antibody fragments may have reduced affinity compared to intact antibody. This combined with their rapid clearance results in a lower percent injected dose deposited in tumor sites [14]. Furthermore, antibody fragments cannot stimulate immune effector function because of the loss of the Fc component. Therapeutically, they are therefore best suited as immunoconjugates carrying cytotoxic moieties to the target cell [13,14]. IV. Murine, human and genetically engineered antibodies

Until recently, murine monoclonal antibodies were the mainstay of therapeutic efforts; however, their clinical potential was restricted by their short plasma half-

273

life necessitating repetitive administrations in order to maintain a continuous blood level [16,17]. Most studies employing intact murine monoclonal antibodies are associated with the development of human anti-mouse antibody (HAMA) in a majority of the patients unless the patient is immunocompromised. The development of HAMA frequently results in binding and rapid clearance of the infused murine antibody. Despite theoretical concerns, adverse effects (e.g., anaphylaxis, serum sickness) following repetitive administration are uncommon and in all cases reported to date readily reversible [22,23]. Human monoclonal antibodies would appear to offer considerable advantages compared to murine reagents. Techniques to produce human monoclonal antibodies have generally involved immortalizing immune B-cells with Epstein-Barr virus followed by fusion with an appropriate murine-myeloma or human-lymphoblastoid cell line [24]. The development of stable antibody producing human hybridomas has been hindered by numerous technical problems, including the inherent instability of human-mouse heterohybridomas and the paucity of fusion-efficient human myeloma cell lines. Furthermore, in the absence of immunizing human subjects with tumor cells or their products, there is no readily available source of hyperimmunized human cells. Given the current restraints only a few human monoclonal antibodies have been available for clinical testing [ 10.25,26]. They would appear to have low immunogenicity and have better pharmacokinetic characteristics than murine reagents [25]. The limitations of murine reagents and the difficulties in developing human monoclonai antibodies have led numerous investigators to utilize the tools of genetic engineering to construct antibodies that closely resemble human immunoglobulin while retaining the antigen binding characteristics of rodent monoclonal antibodies. The genes of the murine variable region can be combined with the constant region genes of the human immunoglobulin to yield a ‘chimeric’ construct. The resulting genetic construct can be transfected into appropriate recipient cells for large scale production of the antibody [ 111.Chimeric antibodies maintain the antigen specificity of the murine antibody while displaying the effector properties of the human Fc component [27]. In the clinical setting, chimeric antibodies circulate longer than their murine counterparts though not as long as normal human immunoglobulin (3-10 days) [28]. Human-mouse chimeric antibodies are generally less immunogenic than murine immunoglobulins and the immune response is primarily to the murine variable region (anti-idiotypic response) [17,28]. To further reduce the immunogenicity of the antibody, investigators

have combined the murine antigen-binding complementarity-determining-regions (CDR) with human V-region framework determinants to yield molecules that very closely resemble human immunoglobulin (‘humanized’ antibodies) [ 121. Little information currently exists for the in vivo behavior of such humanized antibodies. It is apparent that advances in technology have revolutionized the development and production of monoclonal antibodies to a degree where it may soon be feasible to ‘custom-make’ monoclonal reagents to suit specific requirements (Fig. 1). V. Antibody conjugates - toxins, drugs, radionuclides

The effectiveness of native (unmodified) monoclonal antibodies depends upon their ability to bind to the tumor and stimulate the effector arm of the human immunologic repertoire. Tumor cell killing is primarily mediated by antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). Although it has been postulated that anti-tumor responses may also be achieved by the monoclonal antibody stimulating the immune network (Jernes idiotypic network hypothesis) [29], this has not been conclusively demonstrated in humans. There is evidence that monoclonal antibodies may directly mediate tumor cell killing by inducing apoptosis [30,31]. Whether unmodified monoclonal antibodies directed at specific tumor associated antigens can mediate cell death via apoptosis remains to be demonstrated. An appealing strategy to expand the application of monoclonal antibodies and enhance their efficacy is to couple the antibodies to cytotoxic reagents. The role of the monoclonal antibody in this strategy would be to act

Murine Constant Region Murine Variable Region EEI RI!

Fig.

Human Constant Region Human Variable Region

1. Schematic diagram of various types of monoclonal

antibodies

214

as the ‘guided missile’ with the conjugate functioning as the ‘lethal warhead’. Such a strategy would confer specificity and reduce toxicity associated with using the toxic reagent alone. A wide range of plant and bacterial toxins can be coupled to monoclonal antibodies (immunotoxins) thereby targeting the cytotoxic potential of these substances to tumor cells expressing the target antigen [32,33]. In general, the toxins are internalized (receptor mediated endocytosis) and delivered to the acidic endosomes from where they cross into the cytosol, either directly or following passage through the trans-Golgi. Such plant and animal toxins kill cells by inhibiting protein synthesis at the ribosomal level [34]. Toxicity associated with immunotoxin conjugates may be due to non-specific binding of free toxin to the RES and hepatocytes (due to oligosaccharide residues) or binding of the immunotoxin to non-tumor sites (due to cross reactive antigens). Sterically blocked or deglycosylated toxins appeared to be associated with decreased RES uptake, reduced toxicity and longer plasma circulating times [32,33]. An additional limitation associated with immunotoxin therapy is the marked immunogenicity of the toxin molecule [33,35]. Similar to immunotoxins, drug-antibody conjugates may potentially offer advantage over the administration of free drug. Chemotherapeutic agents linked to MoAbs may enable delivery of drug directly to the tumor with reduced overall toxicity. Chemotherapeutic agents are not as efficient as toxins in their ability to kill cells. Direct linkage of multiple drug molecules to antibody without loss of immunoreactivity still remains a major challenge [36]. A number of limitations encountered by immunotoxins and drug-immunoconjugates can be successfully overcome by conjugating radionuclides to monoclonal antibodies (radioimmunoconjugates) [37]. The principle advantage of radioimmunotherapy is the ability, given the appropriate radionuclide, to deliver radiation to a distance of several cell diameters and thereby overcome the obstacle of tumor antigen heterogeneity. Furthermore, radiolabeled antibodies need not be internalized and their cytotoxic property is independent of the effector arm of the immune system. For radioimmunotherapy to be effective, the link between the antibody and radionuclide must be stable in vivo, the labeling procedure must not alter the biodistribution and binding characteristics of the antibody, and the radiolabeled antibody should achieve a high tumor:normal tissue localization ratio [37]. Tumor heterogeneity is less of an obstacle to the effectiveness of radioimmunoconjugates as long as the nuclide has a reasonable distribution to a tumor site and is able to deliver the required radiation

energy over an appropriate distance. The selection of the appropriate radionuclide is thus crucial and must take into account the physical half-life of the nuclide, its emission and energy spectrum, distance of radiation delivery, labeling efficiency, and availa bility of the radioactive compound [6] (Table 1). B-Emitting isotopes deliver energy over several cell diameters. The relative low energy of some of the nuclides may, however, result in inefficient tumor cell kill accompanied by toxicity to normal tissue. a-Emitters release high energy over a short range [40-80 pm] and thus require internalization to damage single cell nuclei. Individual characteristics of the radioimmunoconjugate such as dehalogenation of radiolabeled Iodine and the uptake of Yttrium by bone have to be considered when selecting radioimmunoconjugates for study. An important consideration in the use of radioimmunoconjugates is the stability of the chelated conjugate and the maintenance of immunoreactivity of the radiolabeled antibody. Toxicity associated with radioimmunoconjugates has generally been bone marrow suppression resulting from irradiation of hematopoietic cells via blood pool activity, freely circulating nuclide or marrow localization of conjugate [38]. With improvement in chelation techniques, utilization of second generation radioactive isotopes and strat-

TABLE 1 Potential radioisotopes for radioimmunotherapy Isotope

Half-life

Comment

/?-Emitters

Iodine13’

8d

Rheniumss

17h

RheniumiR6

3.5 d

Copper67

2.5 d

Yttriumgo

2.5 d

Scandium“’

3.4 d

High y-energy permits imaging. Dehalogenation in vivo. High energy B-emission. Chemistry similar to %Tc. Emission suitable for imaging and therapy. Emission suitable for imaging and therapy. Chemistry similar to “‘In. Bone uptake. High energy B-emission

a-Emitters

Asthenium2”

7h

Bismuth’” Lead”*

Ih 10 h

High energy, short range “‘I chemistry High energy, short range High energy, short range

Auger electron emitters

Iodinelz5

60 d

Bromine”

56 h

Short range electrons, dehalogenation in vivo

Fissionable n&ides

Boron-10

20 d

Very high energy. Inert until activated by external neutron source.

275

kemic/lymphoma cells. The target antigens were differentiation surface markers expressed by the malignant cells and to a certain degree also present on normal lymphoid cells [8]. The CD5 antigen is expressed by normal and malignant T-cells as well as by B-CLL cells and has been the target of antibody studies involving patients with T-ALL, T-cell lymphoma, B-CLL and cutaneous T-cell lymphoma (CTCL). Review of the literature reveals that collectively nearly 60 patients have been treated with the anti-CD5 antibody TlOl or Leu 1 [4144]. In most cases, there was a prompt but transient reduction of circulating leukemic cells but little or no change in marrow and lymph node disease. This antibody caused dramatic surface antigen down-modulation and most responses were partial and short-lived. A number of investigators have attempted to treat B-cell lymphoma with anti-idiotypic antibodies directed at the surface idiotype specifically expressed by the malignant clone [45,46]. The therapeutic strategy has proven moderately successful in nodular non-Hodgkin’s lymphoma with response rates of 25% [46]. Levy et al. reported a unique case of a complete remission that lasted for over six years employing this strategy [45]. It is now apparent that there exists a repertoire of restricted idiotypes which are shared among different B-cell lymphomas. It is thus possible to select an anti-idiotypic antibody from an existing panel and phase I/II studies using this strategy are currently ongoing [49]. The ability of native (unconjugated) monoclonal antibodies to induce durable responses in patients who have previously failed multiple chemotherapeutic modalities is exemplified by the CAMPATH- H antibodies developed by Waldmann’s group at Cambridge, UK [47.48]. The

egies to protect the bone marrow or enhance recovery (using cytokines), radioimmunotherapy may prove to be a potent tool in cancer therapy. VI. Clinically approved antibodies

To date, only one monoclonal antibody has been approved by the FDA for clinical use. The murine monoclonal antibody 0KT3 is directed against the T-cell receptor complex (CD3) ubiquitously expressed on mature T-cells. Clinical trials of the OKT3 antibody in recipients of cadaveric renal transplants demonstrated superior reversal of acute rejection when compared to conventional immunosuppressive therapy. Kidney survival rates at 1 year were significantly higher for the group treated with 0KT3 compared to the control group receiving standard immunosuppressive therapy [39]. Therapy with OKT-3, however, has been associated with a significant increase in opportunistic infections especially CMV [39]. Some cases of B-cell neoplasms have also been linked to the immunosuppression induced by such antibodies. A small number of therapeutic monoclonal antibodies currently await final FDA approval for clinical use. VII. Phase I and II cancer therapy trials

VIZ-A. Antibody alone The earliest trials using unlabeled monoclonal antibodies were conducted in patients with hematologic malignancies [4&48] (Table 2). Murine antibodies were directed against cell surface antigens expressed by leu-

TABLE

2

Therapeutic monoclonal antibodies Antibody alone (lymphomalleukemia)

Ref

MoAb

Ag

Comments

ALL

J5

CALLA

Down

modulation

of Ag

40

T-lymphoma

TlOl

CDS

Down

modulation

of antigen

41

CTCL

TlOl

CDS

Transient regression HAMA responses

B-CLL

TlOl

CD5

Transient reduction in lymphocyte No HAMA responses

B-lymphoma

Anti-Id

surface-idiotype

response

> 6 months.

Leukemia/ Lymphoma

CAMPATH-1M CAMPATH-2G

pan-lymphocyte/ monocyte

Variable

responses

Lymphoma

CAMPATH-IH (CDR-graft)

pan-lymphocyte/ monocyte

Sustained patients

Tumor

type

of cutaneous

lesions.

count.

1 CR > 6 years

43.44

45,46 41

in patients

CR in two heavily

42

pre-treated

48

216

MoAb CAMPATH-1H is a CDR grafted antibody directed at the CDw52 antigen expressed on B-cells, Tcells and monocytes. The antigen is highly expressed on lymphocytes (5 x lO*/cell), does not modulate and mediates potent ADCC and CDC in vitro. These observations support the concept that an important pre-requisite for clinical responses is that significant amounts of antibody be deposited onto the tumor surface and that the antibody be capable of effective complement and ADCC activity in vivo. This may be more readily achieved in lymphomas and leukemias which have better vascular access and exposure to cellular and humoral effector systems. Studies using unlabeled monoclonal antibodies in solid tumors have focused mainly on colorectal carcinoma [22,50-541 and malignant melanoma [55-631 (Table 3). In colorectal cancer, the most experience has been gained with the monoclonal antibody 17-1A directed at a 38 kDa glycoprotein surface antigen expressed by gastrointestinal and other adenocarcinomas. The murine monoclonal antibody 17-1A is an IgGla antibody that mediates ADCC in vitro [50]. Clinical trials demonstrated that patients tolerated the murine antibody at doses of up to 12 g with minimal nausea and diarrhea [22,50,51]. The antibody was immunogenic in man and a majority of patients developed an immune response within 8 to 10 days of therapy [15]. Repetitive therapy was well tolerated except for rare instances of anaphylactic reactions which were readily reversible [22]. With high doses of antibody (200-400 mg), the presence of HAMA did not alter the pharmacokinetics of the administered antibody [16]. Collectively, over 100

patients have been treated with this antibody with an overall response rate of approximately 5%. Addition of y-interferon to this regime did not result in enhanced anti-tumor effects [52]. The chimeric form of this monoclonal antibody (chl7-1A) has been studied and has a five- to six-fold longer half-life than murine 17- 1A and reduced immunogenicity [28,53]. A variety of monoclonal antibodies have been used for the treatment of malignant melanoma and strategies have included both systemic administration as well as intra-lesional therapy. Although tumor localization was demonstrated using the “‘In-labeled monoclonal antibody 96.5, no clinical responses were observed following therapeutic administration of the unlabeled antibody [56]. The disialoganglioside antigens GD2 and GD3 are present on tumors of neuroectodermal origin (including melanoma and neuroblastoma) and a number of trials using antibodies directed against these antigens have been reported [57-631. Administration of the murine anti-GD3 antibody R24 (IgG3) was associated with an inflammatory response at the site of cutaneous tumor deposits and partial responses were observed in 4 of 21 patients [57]. Administration of the murine antiGD2 monoclonal antibody 3F8 (IgG3) to patients with melanoma or neuroblastoma resulted in 2/17 patients achieving partial response [60,61]. Side effects included severe abdominal pain and hypertension. Recently, we have treated 12 melanoma patients with the murine anti-GD2 antibody, 14G2a (IgG2a) [62]. Patients received four doses of the antibody over an 8-day period and experienced dose-limiting abdominal/pelvic pain and occasionally delayed extremity pain. Two of the

TABLE 3 Therapeutic monoclonal antibodies Antibody alone (solid tumors)

Tumor type

MoAb

Ag

Comments

[Ref.]

Cl

17-1A

Approximately 5% overall response in -100 patients

22,50,51,52

ch 17-1A less immunogenic and circulates longer than murine 17-1A Antibody binding restricted to normal and malignant GI-epithelium

2853

D612

38 kDa glycoprotein (pan-carcinoma Ag) 38 kDa glycoprotein (pan-carcinoma Ag) 48 kDa glycoprotein

9.2.27 96.5 R24 ME36.1 human L7

HMW >400-500 kDa p 97 sialoglycoprotein CD3 GD2/GD3 CD2

55 56 51 58 59

3F8

CD2

14G2a chimeric 14.18

CD2 CD2

Tumor localization, no responses No responses Inflammation at tumor sites, some responses 1 CR in 13 patients Intralesional administration, responses at injected and some uninjected sites Some responses. Infusion-related abdominal pain and hypertension Neurotoxicity at higher doses Less immunogenic and longer half-life than 14G2a. No neurotoxicity

chimeric 17-l A

Melanoma

54

60,61 62 63

217

patients developed reversible sensory and motor neuropathy. The occurrence of neurotoxicity appeared to correlate with the dose of the antibody and presence of high levels of circulating human anti-14G2a antibody [62]. Toxicity relating to cross-reactive deposition in normal tissues, though rare, remains a concern. Morton reported responses following intralesional injection of human monoclonal L7 (IgM) anti-GD2 antibody in 16 of 21 patients. Response was documented in the injected as well as some distant lesions [59]. In general, cumulative experience with unlabeled monoclonal antibodies confirm that the antibodies are well tolerated. A majority of patients develop an immune response to the foreign protein which limits the duration of therapy. Treatment in the presence of HAMA, in most cases, is not associated with serious side effects. The anti-tumor responses to unlabeled monoclonal antibodies have been modest at best. Tumor biopsy following antibody administration has demonstrated that antibodies are deposited at tumor sites in a dose-dependent fashion [63]. Antibody deposition, however, has generally not been accompanied by tumor shrinkage. The experience with unlabeled antibodies suggest that a number of factors are important for achieving in vivo anti-tumor effect. These include delivery of adequate amounts of antibody to the entire tumor, affinity of the antibody and the ability to effectively activate effector mechanisms. Studies aimed at

addressing these issues are likely to improve the clinical utility of unlabeled antibodies. VII-B. Antibody-radionuclide trials Early work by Order and others demonstrated the clinical utility of radiolabeled polyclonal antibodies in the treatment of neoplastic disorders 1641.Monoclonal antibodies have largely replaced polyclonal reagents in the clinical arena and a number of radioimmunotherapy trials have shown objective responses, especially in the treatment of radiosensitive tumors such as lymphomas [65-681 (Table 4). Anti-tumor activity has been observed with the ‘311-labeled MoAb Lym-1 [66] as well as with the ‘3’I-labeled LL2 (EPB-2) MoAb in B-cell lymphoma [68]. A currently ongoing phase I study employs high doses (up to 600 mCi) of the pan anti-B cell MoAb MB-l followed by autologous bone marrow rescue [67]. Only patients demonstrating favorable uptake of the tracer dose receive the therapeutic dose. Using this strategy, complete responses have been achieved in a majority of the 12 previously treated patients with some surviving beyond 3 years [67]. Administration of the 1311-labeled anti-melanoma MoAb 96.5 was associated with limited clinical benefit even though the antibody demonstrated tumor localization in imaging studies [69]. Intraperitoneal administration of ‘3’I-labeled HMGFl (an antibody directed at an epithelial tumor associated antigen) resulted in favora-

TABLE 4 Therapeutic monoclonal antibodies Radioimmunoconjugate

Tumor type

MoAb

Ag

Radioisotope

Comments

CTCL

TIOI

CD5

“‘I

Myelosuppression

B-lymphoma

Lym- 1

Burkitt-cell Ag

“‘I

Very short circulating time. Responses in previously treated patients

66

B-lymphoma

MB-1

CD37

“‘I

Radioimmunotherapy responses

67

B-lymphoma

LL2 (EPB-2)

B-cell

“‘I

Responses even at low doses. Myelosuppression -50 mCi total dose

Hodgkin’s disease

polyclonal anti-ferritin IG

Ferritin

9’Y

Sixty-two percent response rate including 30% CR in refractory patients. Higher CR rate than with “‘I-anti- ferritin

63

Melanoma

96.5 (Fab) 8.2 (Fab)

P97

No responses at doses up to 500 mCi

69

Ovarian

HMF Gl/MHF AUAI, Hl7E2

70.71

GI

chimeric B72.3

G2

[Ref.]

Epithelial glycoprotein

WI

Intraperitoneal >I40 mCi

TAG-72

‘,‘I

Myelosuppression

at doses z 140 mCi

65

followed by autologous BMT, major

administration,

at 36 mCi/m’

effective at doses

at

68

38

278

ble responses in 9 of 24 patients, including one complete remission lasting >3 years [70,71]. In a recently completed phase I trial at the University of Alabama at Birmingham, patients with metastatic colon cancer received the i3’I-labeled chimeric MoAb ch B72.3 (antiTAG-72) [38]. Twenty-four patients received a total of 69 radiolabeled infusions, including one patient who received five separate treatments. Bone marrow toxicity was dose-limiting and occurred at 36 mCi/m’. Dose fractionation appears to alter the toxicity profile only slightly. One patient achieved an objective anti-tumor response. Follow-up trials using second generation MoAbs with higher antigen binding affinities are currently ongoing. A majority of these phase I studies have employed the p-emitter Iodine-131 as the radionuclide because of the availability and ease of radiolabeling. A number of studies using Yittrium-91 (a high energy p-emitter with a short half-life of 2.5 days) are currently underway in patients with B-cell lymphoma. Order’s group recently reported very encouraging results in the treatment of refractory Hodgkin’s disease using ‘lY-labeled polyclonal anti-ferritin Ig. Complete remissions were observed in 30% of evaluable patients, a higher rate than previously observed with 13’1-antiferritin [72]. VII-C. Antibody-toxin

trials

Antibody-toxin conjugates (immunoconjugates) theoretically combine the tumor cell specificity of the monoclonal antibody and the cytotoxic potential of plant or bacterial toxins. Much of the clinical experience with such conjugates stems from studies using monoclonal

antibodies linked to ricin A-chain or genetically modified pseudomonas exotoxin (PE40) [73-831 (Table 5). Administration of immunoconjugates containing native PE have been associated with significant toxicity. For example, only small doses of unmodified PE-conjugated anti-Tat MoAb could be administered to patients without causing liver damage [13]. The modified toxin (PE40) has been successfully linked to the intact as well as single chain fragment of anti-Tat MoAb. The antiTat single chain-PE40 immunotoxin demonstrated specific anti-tumor activity against IL-2R expressing cells in vitro [78]. Initial testing of the immunotoxin in animal models has been very encouraging and demonstrates a better therapeutic window than immunoconjugates employing unmodified PE. Early phase I trials are currently underway using the modified immunotoxin (Pastan, personal communication). The ricin A-chain immunotoxins have been tested in patients with CLL (TlOl-immunotoxin). Despite saturation of the CD5 antigen on leukemic cells, no clinical responses were observed [73-751. In phase I and II studies of patients with metastatic melanoma treated with ricin A-chain conjugated to Xomazyme-me1 (antiMAA), only modest responses were observed [79,80]. Therapeutic efforts using this strategy have been hampered by the short half-life of the immunotoxin, a capillary leak/hypoalbuminemia syndrome linked to the ricin A-chain and the prompt development of antibody to the murine reagent and the ricin molecule [32,33,80]. In an effort to reduce non-specific uptake of the ricin immunotoxin, modified form of the ricin molecule have been employed. Nadler treated 22 patients with a continuous infusion of anti-CD19 conjugated to ricin with the galactose binding sites blocked (blocked ricin-im-

TABLE 5 Therapeutic monoclonal antibodies Immunotoxins

Tumor type

MoAb

Ag

Toxin

Comments

[Ref.]

CLL

TlOl

CDS

Ricin A

Rapid fall in circulating leukemic cells lasting ~24 h

13,14,75

Lymphoma

-

CD19

blocked-Ricin

Transient hepatocellular toxicity. Some responses

76

Lymphoma

RFB4

CD22

dg-Ricin A

50% response rate in CD22’ tumors. Some unanticipated toxicity

77

Melanoma

Xomazyme-me1

MAA

Ricin A

Short circulation, immunotoxin highly immunogenic

79,80

Breast

260-F6

55 kDa

Ricin A

Therapy associated with neurotoxicity

81,82

Colon

XMMCO-791

72 kDa glycoprotein

Ricin A

Some partial responses

83

MAA, melanoma associated antigen. PE40, pseudomonas exotoxin A.

279 TABLE 6 Future Strategies 1. Genetically engineered antibodies

Develop chimeric and humanized antibodies with high antigen binding affinity.

Combinatorial library

Generate library of human V-region repertoire and identify heavy/light chain combinations that code for tumor binding proteins.

2. Human monoclonal antibodies

Use SCID mice reconstituted with human lymphocytes (SCID-hu mice) to serve as surrogates in immunization procedures with the aim to develop human anti-tumor antibodies.

3. Second generation radioimmunoconjugates

Develop radioimmunoconjugates using newer isotopes coupled to MoAb utilizing recently developed linker chemistry.

4. RadioimmunotherReversal of radioimmunotherapy-induced myelosuppression with autologous bone apyiautologous bone marrow rescue marrow rescue. 5. More effective and safer toxins

Use of recombinant DNA technology to produce modified toxins that retain cytocidal potential but have reduced non-specific binding/immunogenicity.

6. Improved drug conjugates

Site-specific linkage of drug to MoAb to produce stable drug-antibody complex while at the same time allow release of intact drug in cytosol. Development of immunoconjugate carrying multiple non-cross-reacting drugs.

7. Anti-idiotype vaccines

Development of ‘internal image’ anti-idiotypic antibodies that mimic the tumor antigen. Use these reagents as vaccines.

8. Bi-functional antibodies

Develop antibodies with two distinct antigen binding activities with the aim of directing cytotoxic cells to the tumor target.

munoconjugate) and observed reduced toxicity [76]. Nine of 22 patients had a partial or mixed response and one complete remission was observed in this group of patients with refractory B-cell lymphoma. In a phase I trial, Vitetta treated 15 patients with the MoAb RFB4 (anti-CD22) conjugated to deglycosylated ricin A-chain (dg ricin immunoconjugate) [77]. A third of these heavily pre-treated lymphoma patients demonstrated a clinical response. A response rate of 50% was observed in patients with CD22 positive tumors. In general, most responses have been of modest duration. Toxicity associated with the clinical administration of immunotoxin conjugates have been related to the nonspecific binding of the toxin molecule or due to the antigen specific binding of the immunotoxin to nontumor target [32,33]. The ricin A-chain has been implicated in the capillary leakhypoalbuminemia syndrome while pseudomonas exotoxin A has produced hepato-

toxicity [32]. In a recent phase I trial, several patients with refractory breast cancer treated with a recombinant ricin A-chain 260F6 MoAb immunoconjugate developed debilitating neuropathy within two to three weeks following therapy [8 1,821. Subsequent studies demonstrated binding of the antibody to human peripheral nerve myelin [82]. Gene cloning has enabled generation of recombinant immunotoxin molecules [13,78]. Development of individual recombinant components of the toxin molecules should allow a better understanding of the function of these individual components. Such information may be useful in the development of less toxic and more effective immunotoxin reagents. VII-D. Antibody drug trials

Delivery of chemotherapeutic agents will known antitumor activity to the target cells can be achived by conjugating such drugs with monoclonal antibodies [36]. A variety of chemoimmunoconjugates using agents such as doxorubicin, methotrexate, chlorambucil, vinca alkaloids and mitomycin have been explored in vitro and in animal models. Oldham treated 23 patients with a variety of malignancies using a cocktail of several monoclonal antibodies conjugated to adriamycin [36]. The MoAbs were selected based upon their reactivity to the patient’s tumor in vitro. Modest responses were seen in some patients. Dissociation of the MoAb-drug conjugate resulted in circulating free drug and dose-limiting myelosuppression. Tumor biopsies in several patients demonstrated antigen modulation following therapy. Thus, although in vitro and xenograft studies have demonstrated superior tumor killing by antibody drug conjugates compared to free drug, effective clinical application of this strategy remains to be achieved.

VIII. Future efforts The observations of the past decade have been formative in providing important insights into the strengths and limitations of currently available monoclonal reagents. Table 6 outlines some of the strategies aimed at enhancing the clinical potential of monoclonal antibody. Advances in tumor immunology, molecular biology and protein chemistry have enabled the development of novel reagents and ushered in a new era in the biologic therapy of cancer. Although a lot still remains to be unraveled, recent advances offer hope that many of the current limitations can be overcome. Acknowledgement

280

The authors wish to gratefully acknowledge Sharon Garrison for her help and expertise in preparing the manuscript. Biographies Mumoor N. Sale/r received an M.D. degree from the University of Heidelberg, Heidelberg, West Germany. His fellowship in Hematology/Oncology was at the University of Alabama at Birmingham, Birmingham, AL, where he is currently an Associate Professor, Division of Hematology/Oncology. Albert F. LoBuglio received an M.D. degree from Georgetown University Medical School, Washington DC. His fellowship in Hematology/Oncology was at Harvard University and Thorndike Memorial Laboratory, Boston City Hospital, Boston, MA. He is currently Professor of Medicine, Division of Hematology/ Oncology andDirector of the Comprehensive Cancer Center at the University of Alabama at Birmingham. Reviewer This manuscript was reviewed by Ira Pastan, M.D., National Cancer Institute, NIH, Bethesda, MD 20892, USA. References 1 Nicolson GL. Tumor cell instability, diversification and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res 47:1473-1487, 1987. 2 Dexter DL, Leith JT. Tumor heterogeneity and drug resistance. J Clin Oncol 4:244257, 1986. 3 Chabner BA, Myers CE. Clinical pharmacology of cancer chemotherapy. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer Principles and Practice of Oncology, 2nd Ed. J.B. Lippincott, 1992; 257-329. 4 Koehler G, Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256:495496, 1975. 5 Oldham RK, Smalley RV. Bioiogicals and biologic response modifiers. In: DeVita VT, Hellman S, Rosenberg SA, eds. Biologic Therapy of Cancer, 2nd Edn. J.B. Lippincott 1991; 2223-2238. 6 Dimaggio JJ, Scheinberg DA, Houghton AN. Monoclonal antibody therapy of cancer. In: Pinedo HM, Chabner BA, Longo DL, eds. Cancer Chemotherapy and Biological Response Modifiers, Annual 11, Chapter 12, Amsterdam: Elsevier Science Publishers, 1990; 177-203. I Muraro R, Wunderlich D, Thor A, Lundy J, Noguchi P, Cunningham R, Schlom J. Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryogenic antigen differentially expressed in human colon carcinoma versus normal adult tissues. Cancer Res 45:5169-5780, 1985. 8 Freedman A, Nadler L. Cell surface markers in hematologic malignancies. Semin Oncol 14:193-212, 1987. 9 Drebin JA, Link VC, Weinberg RA, Greene MI. Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen. Proc Nat1 Acad Sci USA 83:91299133, 1986. Camick JW, Bourla JM. Prospectus for the therapeutic use of human monoclonal antibodies. J Biol Response Mod 5:3799393, 1986. Morrison SL. Transfectomas provide novel chimeric antibodies. Science 229: 1202-l 207, 1985. Jones PT, Dear PH, Foots J, Neuberber MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522-525, 1986. Waldmann TA. Monoclonal antibodies in diagnosis and therapy.

Science 252:165771662, 1991. 14 Schlom J. Monoclonal antibodies: They’re more and less than you think. In: Broder S, eds. Molecular Foundations of Oncoloy. Baltimore: Williams & Wilkins, 1990; 95-134. 15 Roitt I, Brostoff J, Make D. Immunology, 3rd Edn. London, UK: Gower Medical Publishing, 1991. 16 Khazaeli MB, Saleh MN, Wheeler RH, Huster WJ, Holden H, Carrano R, LoBuglio AF. Phase I trial of multiple large doses of murine monoclonal antibody C017-IA. II. Pharmacokinetics and immune response. J. Nat1 Cancer Inst 80:937-942, 1988. 17 Khazaeli MB, Saleh M, Liu T, Reisfeld RA, LoBuglio AF. Murine and chimeric antibodies to GD2 antigen in melanoma patients: pharmacokinetics and immune response. In: Chapman and Hall, eds. Monoclonal Antibodies: Applications in Clinical Oncology. 1991; in press. 18 Waldmann TA, Strober W. Metabolism of immunoglobulin. Prog Allergy 113:l. 1969. 19 Yasmeen D, Ellerson JR, Dorrington KJ, Painter RH. The structure and function of immunoglobulin domains. IV. The distribution of some effector functions among the Cy2 and Cy3 homology regions of human immunoglobulin Gl. J Immunol 116:518-526, 1976. 20 Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M. Single-chain antigen-binding proteins. Science 242:423426, 1988. 21 Colcher D, Bird R, Roselli M, Hardman KD, Johnson S, Pope S, Dodd SW, Pantoliano MW, Milenic DE, Schlom J. In vivo targeting of a recombinant single-chain antigen-binding proteins, J Nat1 Cancer Inst 82:1191-l 197, 1990. 22 LoBuglio AF, Saleh MN, Lee J, Khazaeli MB, Carrano R, Holden H, Wheeler RH. Phase I trial of multiple large doses of murine monoclonal antibody C017-IA. I. Clinical aspects. J Nat1 Cancer Inst 80:932-936, 1988. 23 Dillman RO, Beauregard JC, Halpern SE, Clutter M. Toxicities and side effects associated with intravenous infusions of murine monoclonal antibodies. J Biol Resp Mod 5:73-83, 1986. 24 Dorfman NA. The optimal technological approach to the development of human hybridomas. J Biol Resp Mod 4:213-239, 1985. 25 Khazaeli MB, Wheeler R, Rogers K, Teng N, Ziegler E, Haynes A, Saleh MN, Hardin JM, Bolmer S, Comett J, Berger H, LoBuglio AF. Initial evaluation of a human IgM monoclonal antibody (HA-IA) in man. J Biol Resp Mod 9:178-184, 1990. 26 Ziegler EJ, Fisher CJ Jr, Sprung CL, Straube RC, Sadoff JC, Foulke GE, Wortel CH, Fink MP, Dellinger RP, Teng NNH. Allen IE, Berger HJ. Knatterud GL, LoBuglio AF, Smith CR and the HA-1A Sepsis Study Group. Treatment of gram-negative bacteremia and septic shock with HA-IA human monoclonal antibody against endotoxin: a randomized, double-blind, placebo-controlled trial. N Engl J Med 324:429436, 1991. 27 Shaw DR, Khazaeli MB, LoBuglio AF. Mouse/human chimeric antibodies to a tumor-associated antigen: biologic activity of the four human IgG subclasses. J Nat1 Cancer Inst 80:1553-1559, 1988. 28 LoBuglio AF, Wheeler RH, Trang J, Haynes A, Rogers K, Harvey EB, Sun L, Ghrayeb J, Khazaeli MB. Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc Nat1 Acad Sci USA 86:4220-4224, 1989. 29 Jerne NK. Towards a network theory of the immune system. Ann Immunol 125C:373, 1974. 30 Trauth BC, Klas C, Peters AMJ, Matzku S, Moller P, Falk W. Debatin K-M, Krammer PH. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301&305, 1989. 31 Wyllie AH. The biology of cell death in tumours. Anticancer Res 5:131-136. 1985. 32 Frankel AE, Houston LL, Issell BF. Prospects for immunotoxin therapy in cancer. Annu Rev Med 37:125-142, 1986. 33 Hertler AA, Frankel AE. Immunotoxins: a clinical review of their use in the treatment of malignancies. J Clin Oncol 12:1932-1942, 1989.

281 34 Olsnes S, Sandvig K. How protein toxins enter and kill cells. In: Frankel AE (ed). Immunotoxins. Boston: Kluwer Academic, 1988; 39-73. 35 Oeltmann TN, Frankel AE. Advances in immunotoxins. FASEB J 523342337, 1991. 36 Oldham RK. Custom-tailored drug immunoconjugates in cancer therapy. Mol Biother 3:148-162, 1991. 37 Goldenberg DM. Future role of radiolabeled monoclonal antibodies in oncological diagnosis and therapy. Semin Nuclear Med 19:3392-3396. 1989. 38 Meredith RF, Khazaeli MB, Plott WE, Saleh MN, Liu TP, Allen LF, Russell CD, Orr RA, Colcher D, Schlom J, Shochat D, Wheeler RH, Lo Buglio AF. Phase I trial of iodine-131-chimeric B72.3 (human IgG4) in metastatic colorectal cancer. J Nucl Med 33:23329, 1992. 39 Ortho Multicenter Transplant Study Group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. N Engl J Med 313:337-342. 1985. 40 Ritz J, Pesando JM, Sallan SE. Clavell LA, Notis-McConarty J, Rosenthal P, Schlossman SF. Serotherapy of acute lymphoblastic leukemia with monoclonal antibody. Blood 58:141-152, 1981. 41 Miller RA, Oseroff AR, Stratte PT, Levy R. Monoclonal antibody therapeutic trials in seven patients with T cell lymphoma. Blood 62:888995, 1983. 42 Dillman RO. Beauregard J, Shawler DL et al. Continuous infusion of T-101 monoclonal antibody in chronic lymphocytic leukemia and cutaneous T-cell lymphoma. J Biol Resp Mod 5:394410. 1986. 43 Dillman RO, Shawler DL, Dillman JB, Royston I. Therapy of chronic lymphocytic leukemia and cutaneous T-cell lymphoma with TlOl monoclonal antibody. J Clin Oncol 2:881-891. 1984. 44 Foon KA, Schroff RW, Bunn PA, Mayer D, Abrams PG, Fer M, Ochs J, Bottino GC, Sherwin SA. Carlo DJ. Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia. Blood 64:108551094, 1984. 45 Miller RA, Maloney DG, Wamke R, Levy R. Treatment of a B cell lymphoma with monoclonal anti-idiotypic antibody. N Engl J Med 306:517--522, 1982. 46 Brown SL, Miller RA, Horning SJ, Czerwinski D, Hart SM. McElderry R, Basham T, Wamke RA, Merigan TC, Levy R. Treatment of B-cell lymphomas with anti-idiotype antibodies alone and in combination with alpha-interferon. Blood 73:651661, 1989. 47 Dyer MJS, Hale G, Hayhoe FGJ, Waldmann H. Effects of CAMPATH-l antibodies in vivo in patients with lymphoid malignancies: influence of antibody isotype. Blood 73(6):1431-1439, 1989. 48 Hale G, Dyer MJ, Clark MR. Phillips JM, Marcus R, Riechmann L, Winter G, Waldmann H. Remission induction in nonHodgkin’s lymphoma with reshaped human monoclonal antibody CAMPATH 1-H. Lancet ii:13941395, 1988. 49 Miller RA, Hart S, Samoszuk M, Coulter C, Brown S, Czerwinski D, Kelkenberg J. Royston I, Levy R. Shared idiotypes expressed by human B-cell lymphomas. N Engl J Med 321:851&857, 1989. 50 Sears HF. Atkinson B, Mattis J. Ernst C, Herlyn D, Steplewski Z, Hayry P, Koprowski H. Phase I clinical trial of monoclonal antibody in treatment of gastrointestinal tumours. Lancet i:762-765, 1982. 51 Sears HF. Herlyn D, Steplewski Z. Koprowski H. Phase II clinical trial of a murine antibody cytotoxic for gastrointestinal adenocarcinoma. Cancer Res 45:591C-5913, 1985. 52 Saleh MN. LoBuglio AF, Wheeler RH, Rogers KJ, Haynes A, Lee JY, Khazaeli MB. A phase II trial of murine monoclonal antibody 17-1A and interferon-y: Clinical and immunologic data. Cancer Immunol Immunother 32:185-190, 1990. 53 Meredith RF, LoBuglio AF, Plott WE, Orr RA, Brezovich IA. Russell CD, Harvey EB, Yester MV, Wagner AJ, Spencer SA, Wheeler RH, Saleh MN, Rogers KJ. Polansky A, Salter MM, Khazaeli MB. Pharmacokinetics, immune response and biodistribution of iodine-l 3 1-labeled chimeric mouseihuman IgGl ,k 17- 1A monoclonal antibody. J Nucl Med 32:1162~1168, 1991.

54 Saleh M, Khazaeli M, Wheeler R, Allen L, Liu T. Schlom J, LoBuglio A. A phase I trial of the murine monoclonal antibody (MoAb) D612 in patients with metastatic colorectal cancer. Proceedings ASCO 11:253, 1992. Abstract No. 821. 55 Oldham RK, Foon KA, Morgan AC, Woodhouse CS, Schroff RW, Abrams PG, Fer M, Schoenberger CS, Farrell M, Kimball E. Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration. J Clin Oncol 2:1235-1244. 1984. 56 Goodman GE, Beaumier P, Hellstrom I, Femyhough B, Hellstrom KE. Pilot trial of murine monoclonal antibodies in patients with advanced melanoma. J Clin Oncol 3:340-352, 1985. 57 Vadhan-Raj S, Cordon-Cardo C. Carswell E, Mintzer D, Dantis L, Duteau C, Templeton MA, Oettgen HF, Old LJ, Houghton AN. Phase I trial of a mouse monoclonal antibody against G,, ganglioside in patients with melanoma: Induction of inflammatory responses at tumor sites. J Clin Oncol 6:1636-1648, 1988. 58 Lictin A, Iliopoulous D, Guerry D, Elder D, Heryln D, Steplewski Z. Therapy of melanoma with an anti-melanoma ganglioside monoclonal antibody: a possible mechanism of a complete response. Proc Am Sot Clin Oncol 7:247, 1988. Abstract No. 958. 59 Irie RF, Morton DL. Regression of cutaneous metastatic melanoma by intralesional injection with human monoclonal antibody to ganghoside Go,. Proc Nat1 Acad Sci USA 83:86948698, 1986. 60 Cheung NK, Lazarus H, Miraldi FD, Abramowsky CR, Kalhck S, Saarinen UM, Spitzer T, Strandjord SE, Coccia PF, Berger NA. Ganghoside G,,-specific monoclonal antibody 3F8: a phase I study in patients with neuroblastoma and malignant melanoma. J Clin Oncol 5:143&1440. 1987. 61 Cheung N-KV, Lazarus H, Miraldi FD, Berger NA, Abramowsky CR, Saarinen UM, Spitzer T. Strandjord SE, Coccia PF. Reassessment of patient response to monoclonal antibody 3F8. J Clin Oncol 10:671-674, 1992. 62 Saleh MN, Khazaeli MB, Wheeler RH, Liu TP, Urist M, Miller DM, Lawson S, Dixon P, Russell CH, LoBuglio AF. A phase I trial of the murine monoclonal anti-GD2 antibody 14G2a in metastatic melanoma. Cancer Res. Cancer Res. 52:43424347, 1992. 63 Saleh MN, Khazaeli MB, Wheeler RH, Allen L, Tilden AB, Grizzle W. Reisfeld RA, Yu AL. Gillies SD, LoBuglio AF. A Phase I trial of the chimeric anti-G,, monoclonal antibody ch14.18 in patients with malignant melanoma. Hum Antibodies Hybridomas 3:19-24, 1992. 64 Lenhard RE Jr, Order SE, Spunberg JJ, Asbell SO, Leibel SA. Isotopic immunoglobulin: a new systemic therapy for advanced Hodgkin’s disease. J Clin Oncol 3: 12961300, 1985. 65 Rosen ST, Zimmer AM, Goldman-Leikin R, Gordon LI, Kazikiewicz JM, Kaplan EH, Variakojis D, Marder RJ, Dykewicz MS, Piergies A. Radioimmunodetection and radioimmunotherapy of cutaneous T cell lymphomas using a “‘I-labeled monoclonal antibody: an Illinois Cancer Council Study. J Clin Oncol 5:562-573. 1987. 66 DeNardo SJ, DeNardo GL. O’Grady LF et al. Pilot studies of radioimmunotherapy of B-cell lymphoma and leukemia using “‘I Lym-I monoclonal antibody. Antibody Immunoconj Radiopharm 1:17-34, 1988. 67 Press OW, Eary JF, Badger CC et al. Treatment of refractory non-Hodgkin’s lymphomas with radiolabeled MB-l (anti CD-37) antibody. J Clin Oncol 7:102771038. 1989. 68 Goldenberg DM, Horowitz JA, Sharkey RM, Hall TC, Murthy S, Goldenber H, Lee RE, Stein R. Siegel JA, Izon DO. Targeting, dosimetry and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody. J Clin Oncol9:548564, 1991. 69 Larson SM, Carrasquillo JA, Krohn KA, Brown JP, McGuffin RW, Ferens JM, Graham MM, Hill LD, Beaumier PL, Hellstrom KE. Localization of “‘I-labeled p97 specific Fab fragments in human melanoma as a basis for radiotherapy. J Clin Invest 72:2101-2114, 1983.

282 70 Epenetos AA, Munro AJ, Stewart S, Rampling R, Lambert HE, McKenzie CG, Soutter P, Rahemtulla A, Hooker G, Sivolapenko GB. Antibody-guided irradiation of advanced ovarian cancer with intraperitoneally administered radiolabeled monoclonal antibodies. J Clin Oncol 5:1890-1899, 1987. 71 Epenetos AA. Clinical results with regional antibody-guided irradiation. Cancer Drug Delivery 2:233, 1985. 72 Vriesendorp HM, Herpst JM, Germack MA, Klein JL, Leichner PK et al. Phase I/II studies of yttrium-labeled antiferritin treatment for end-stage Hodgkin’s disease, including Radiation Therapy Oncology Group 87-01. J CIin Oncol 9918-928, 1991. 73 Laurent G, Pris J, Farcet J-P. Effects of therapy with TlOl ricin A-chain immunotoxin in two leukemia patients. Blood 67: 16801687, 1986. 74 Blakey DC, Thorpe PE. An overview of therapy with immunotoxins containing ricin or its A chain. Antibody Immunoconj Radiopharm 1:1-16, 1988. 75 Hertler AA, Schlossman DM, Borowitz MJ, Laurent G, Jansen FK, Schmidt C, Frankel AE. A phase I study of TlOl-ricin A chain immunotoxin in refractory chronic lymphocytic leukemia. J Biol Response Mod 7:97-l 13, 1988. 76 Grossbard ML, Freedman AS, Ritz J, Coral F, Goldmacher VS, Eliseo L, Spector N, Dear K, Lambert JM, BlBttler WA. Serotherapy of B-cell neoplasms with anti-B4 blocked ricin: a phase I trial of daily bolus infusion. Blood 79576585, 1992. 77 Vitetta ES, Stone M, Amlot P, Fay J, May R, Till M, Newman J, Clark P, Collins R, Cunningham D. Phase I immunotoxin trial in patients with B-cell lymphoma. Cancer Res 51:40524058, 1991.

78 Chaudhary VK, Queen C, Junghans RP, Waldmann TA, FitzGerald DJ, Pastan I. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonus exotoxin. Nature 339~394-397, 1989. 79 Spitler L, de1 Rio M, Khentigan A, Wedel NI, Brophy NA, Miller LL, Harkonen WS, Rosendorf LL, Lee HM, Mischak RP. Therapy of patients with malignant melanoma using a monoclonal antimelanoma antibody-ricin A chain immunotoxin. Cancer Res 47:1717-1723, 1987. 80 Herder AA, Spitler LE, Frankel AE. Humoral immune response to a ricin A chain immunotoxin in patients with metastatic melanoma. Cancer Drug Deliv 4245-253, 1987. 81 Weiner LM, O’Dwyer J, Kitson J, Comis RL, Frankel AE, Bauer RJ, Konrad MS, Groves ES. Phase I evaluation of an anti-breast carcinoma monoclonal antibody 260F6-recombinant ricin A chain immunoconjugate. Cancer Res 49:4062-4067, 1989. 82 Gould BJ, Borowitz MJ, Groves ES, Carter PW, Anthony D, Weiner LM, Frankel AE. Phase I study of an anti-breast cancer immunotoxin by continuous infusion: report of a targeted toxic effect not predicted by animal studies. J Nat1 Cancer Inst 8 1:775781, 1989. 83 Byers VS, Rodvien R, Grant K, Durrant LG, Hudson KH, Baldwin RW, Scannon PJ. Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer. Cancer Res 49:6153-6160, 1989.