A Perspective of Monocional Antibodies: Past, Present, and Future

Frank H. DeLand

In 1975, the development of the technique to pro- duce monoclonal antibodies revolutionized the ap- proach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater com- plexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has

not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to at tain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming. �9 1989 by W.B. Saunders Company.

T HE APPLICATION of labeled antibodies to tumor-associated antigens or their prod-

ucts for the detection and therapy of carcinoma has ignited the interests and investigative efforts of innumerable researchers. In the past several years, hundreds of reports have appeared in the literature, seeking the ultimate labeled antibody for medical application. This extensive effort, occurring over a short period of time, has ob- scured the slowly developing scientific data base that preceded the explosion of knowledge regard- ing antibodies, antigens, and their relationships.

Much of the earlier observations in immunol- ogy were empirical and accurate, but lacked a scientifically based mechanism. Two hundred years ago, Jenner demonstrated the principle of immunity by inoculating patients against small pox I with cow pox. He did not know that cow pox antibodies were produced and that they pro- tected the patient by reacting with the small pox antigen, yet he intuitively realized that the cow pox produced something that "fought" the cause of small pox. In the 1860s, after Pasteur discov- ered that attenuated antigens of a microorgan- ism would produce immunity to the active microorganism, 2 researchers pursued similar in- vestigations to produce antisera against a num- ber of infectious diseases.

Surprisingly, the first attempt to produce an antiserum to cancer occurred nearly one hundred

From the Health Science Center, Veterans Administration Medical Center, Syracuse, NY.

Address reprint requests to Frank H. DeLand, MD, Health Science Center, Veterans Administration Medical Center, 800 Irving Ave, Syracuse, N Y 13210.

�9 1989 by W.B. Saunders Company. 0001-2998/89/1903-0001505.00/0

years ago. Hericourt and Richet 3'4 prepared an antiserum to extracts of osteogenic sarcoma in animals. Their results in one patient were suffi- ciently encouraging that they subsequently treated fifty more patients with osteogenic sar- coma. The level of scientific research of these two researchers is attested to in their use of control patients who were administered normal serum that produced no clinical improvement. The methods of Hericourt and Richet were quite similar to those first used in the middle of this century, albeit less sophisticated.

The onset of the modern era of cancer antibod- ies began with an important discovery in 1929 by Witebsky, 5 when he described the antigenic char- acteristics of neoplasms. In 1948, Pressman and Keighly 6 demonstrated that antibodies to rat kidney developed in rabbits could be labeled with a radionuclide, and that the labeled antibody would localize in the rat kidney. Following this report, many studies of the detection and therapy of neoplasms by means of radiolabeled antibodies to tumors were performed.

To many, the era of monoclonal antibodies began with the far-reaching work of KShler and Milstein 7 in 1975. However, several pertinent events preceded their development of the hibri- doma technique. In 1846, Dalrymple 8 reported his discovery of human multiple myeloma and in 1848, Bence Jones 9 published his discovery of a unique protein in the urine of patients with multiple myeloma. Over 100 years later, Burnet 1~ proposed the clonal selection theory of acquired immunity. Shortly thereafter, myeloma proteins were described as immunoglobulins that were naturally occurring monoclonal antibodies. T M

Other reports followed, describing the additional

158 Seminars in Nuclear Medicine, Vol XlX, No 3 (July), 1989: pp 158-165

PERSPECTIVE OF MONOCLONAL ANTIBODIES 159

steps that lead to the lymphocyte hibridoma technique of K6hler and Milstein.

Following the report of Pressman and Keighly in 1948, 5 efforts were directed toward producing an antibody to a tumor-specific antigen. Al- though numerous researchers have defined anti- bodies to various tumors, these are tumor- associated antigens, not tumor-specific. 13'14 Antigens from neoplastic cells, either surface or intracytoplasmic, are not limited solely to malig- nancy, but can also be found in normal cells of more than one tissue. The encouraging aspect is that these antigens occur in higher concentration on the malignant cells than on normal cells, providing a relative quantitative basis for differ- entiating malignant cells. Even with the most meticulous purifications of antigens and the anti- bodies to these antigens, the final product is not limited to a single antibody, but rather is a polyclonal antibody. Development of the hibri- doma technique has been an important step in producing isolated antibodies to specific antigens 1 l; nevertheless, the presence of the same antigens in normal cells remains a problem) s

A major impetus to the studies of radiolabeled antibodies for in vivo scintillation imaging and subsequently to therapy, was the isolation by Gold and Freedman in 1965 of carcinoembryonic antigen (CEA), an antigen found in colonic cancer. 16 Subsequently, a number of studies reported on the use of antibodies to CEA; some were discouraging, 17'18 others encouraging) 9 The success of the latter was dependent upon com- puter processing by reducing interfering background. 2~

Until the development of monoclonal antibod- ies, the tumor markers were predominately iden- tified by names, eg, alpha-fetoprotein (an oncofe- tal antigen), human chorionic gonadotorapin (ectopic hormones), acid phosphatase (enzymes), and melanoma (tumor-associated antigens). Monoclonal antibodies have provided opportuni- ties in cancer immunology research not previ- ously possible. Hundreds of antibodies identified by letters and numbers have been developed since the report of K6hler and Milstein. 7 The possibil- ity of detecting new antibodies to neoplasms appears unlimited. For example, monoclonal an- tibodies specific for different epitopes on the same antigen are available. 21

The successful application of antibody tech- nique to diagnostic or therapeutic approaches to cancer will depend partially on antigen and antibody selection. Since tumor-associated anti- gens can be found on the cells of normal organs, it is necessary to select those antigens that occur in appreciably greater abundance on the tumor cells than on the normal cells. Previous experi- ence with polyclonal antibodies provides evi- dence of discrepancy in the concentration of antigens between normal and abnormal cells. However, it is necessary to select the most promising antibodies from the various antigen epitopes by cloning of the hibridomas. Certain problems must be evaluated in the selection of an antibody for clinical studies. For example, al- though the reaction of administered antibody with circulating antigen has not presented a serious problem for external imaging proce- dures, 19 the presence of cross-reactive antigens on circulating blood elements and cells of the bone marrow limits the quantity of antibody that can be administered. On the other hand, if there are significant sites of antigen epitopes on the blood cells, high-affinity antibodies can be consid- ered for therapy if the dosage is carefully ti- trated.

A significant problem in selecting antibodies within the same tumor is that the heterogeneity of neoplastic cells has been demonstrated by immunological means. 2a'24 In a study of mam- mary carcinomas from 39 patients, Schlom and Weeks 25 documented ten antigenic phenotypes with four monoclonal antibodies to breast epithe- lium. The variation in cellular phenotypes within the same tumor mass can be documented by the immunoperoxidase method of staining as well as the fluorescence-activated cell-sorter analysis. The variations may include focal staining of cells, diffuse cytoplasmic staining, and membrane api- cal staining. Schlom and Weeks found that the antibody was most reactive with the cell surface epitope during the S phase of the cell cycle. Since the cells within a tumor are in the different phases of the cell cycle, this finding might possi- bly explain some of the variations seen in the antibody concentration within a tumor and in microscopic slide sections stained with the immu- noperoxidase method.

Detection or therapy of metastatic lesions can present a problem of specificity. Poste et a126

160 FRANK H. DELAND

established tumors in mice from an uncloned melanoma line that had widely differing meta- static properties, including nonmetastatic clones. Compared with the original neoplasm, some of the metastases had identical metastatic proper- ties, while others had clones with different meta- static properties. Interestingly, the range of clonal diversity in heterogenous metastases was substan- tially less than in the parent line. Thus, it is possible to select an antibody to an epitope in the primary tumor that would not be present in the metastatic lesion. In a different approach, Fogel et a127 demonstrated that immunoselection might be involved in the production of metastases.

Other factors to consider in the selection of antibodies include the type of immunoglobulin, the effect of radionuclide labeling, and the use of antibody fragments. IgG and IgG2a appear to offer some advantages over the other classes of immunoglobulin. Purification by protein A and by hydroxylapatite chromatography are quite efficient for IgG and IgG2a, respectively, and IgG2a appears to have the added advantage of killing tumor cells by immunoglobulins. 2s

The objective of radionuclide labeling of anti- bodies is to maintain immunoreactivity. Measure- ment of radioactivity includes both labeling and subsequent purification. 29 For the most consis- tent results, immunoreactivity should measure at least 75% after labeling and purification. From the perspective of in vivo distribution, labeling with radioactive iodine would be preferable, but the deiodination of the antibody is a disadvan- tage. The iodine may attach to other circulating proteins or be sequestered by the thyroid gland, thereby decreasing the concentration of labeled antibody presented to the cellular epitopes. On the other hand, the indium label to antibodies is more stable in vivo, but selective concentration of the compound in the liver obscures detection of metastases. 3~

Whole immunoglobulins to tumor-associated antigens have certain inherent disadvantages that may not be present in fragments of IgG. Whole immunoglobulin is composed of two heavy and two light immunoreactive chains connected by disulfide bridges to a nonspecific arm, usually expressed pictorially as a "Y." The immunoreac- tive portion is the upper arms of the Y and the nonspecific portion, termed the Fc portion, is the base. Pepsin enzymatic cleavage occurs on the Fc

side of the disulfide links, separating the Fc from the arms. This fragment is termed F(ab')2 be- cause the combining sites are retained on the two segments that are still attached to each other. If papain enzymatic cleavage is used, the cleavage produces individual separation of each arm of the Y from the Fc portion.

The Fc portion of whole antibodies demon- strates nonspecific binding to a variety of cells that carry the appropriate receptor. For example, this phenomenon has been observed repeatedly in the thyroid gland. In some cases, even though a patient may have received adequate iodine block- ade, the sequestration of radioactivity in the thyroid (presumably the labeled antibody) may be quite intense, in other patients with the same blockade, radioactivity concentration may be minimal. Since there are Fc receptors in many organs, a fraction of whole immunoglobulin is fixed to cells that do not contribute to the definition of the objective lesion and, in fact, may obscure the regions in question.

The process of presenting the labeled immuno- globulin to the desired receptor requires escape of IgG from the vascular pool to the extracellular space. Logically, it would seem that the larger the molecular weight of a compound, the less the opportunity to permeate the vascular wall. Frag- ments of immunoglobulin, either F(ab')2 or Fab, are appreciably smaller than whole IgG. This physical difference in size cannot be directly interpreted to mean that migration of fragments through the vascular wall will occur, however, there is indirect evidence that antibody localiza- tion in tumors is greater with fragments than with whole immunoglobin. 31 Physical evidence of differences in vascular permeability between whole IgG and fragments is still to be defined in light of the observation of O'Connor and Bale 32 that the permeability of the neovascularity in neoplasms is greater than in normal vessel walls. In immunosuppressed mice with grafted human colon carcinomas, the tumor-to-tissue ratio of radioactivity between F(ab')2 and the whole antibody ranged as high as 6:1. 31 The tumor- to-blood ratio was 13:1, indicating faster elimina- tion of the fragment from the vascular pool. Although tumor-to-tissue ratios may be greater for fragments than for the whole antibody, the absolute quantity of radioactivity found in the tumors is still greater for the whole antibody. For

PERSPECTIVE OF MONOCLONAL ANTIBODIES 161

gamma camera imaging, the absolute difference in tumor radioactivity between fragments and whole antibody presents the problem of choosing between (1) decreased count density and poorer image resolution when using fragments or (2) using computer processing to improve the tumor- to-nontumor ratio when using whole antibody.

Several methods have been proposed to reduce the background of nontarget radiolabeled anti- bodies and to improve resolution. In one method, a second antibody to the primary antibody, entrapped in liposomes, is administered 24 hours after the primary antibody. 33 Within two hours, a significant reduction of the primary antibody in the circulation is observed. The disadvantage of this method is that the liposomes are sequestered by the liver, masking hepatic lesions. In another technique, second antibodies (without liposomes) are used; a moderate degree of success has been achieved with this method. 34'35 According to these authors, the nontarget primary antibody is decreased and the second antibody is not seques- tered by the liver. Within four hours after admin- istration of the second antibody, the blood level of radioactivity decreases by a factor of four. At 48 hours after administration of the first anti- body and 24 hours after the second, there is little change in the level of radioactivity in the tumor.

Polyclonal antibodies, produced in animals, were used extensively before the development of monoclonal antibodies. These tumor markers were usually classified based on organ function, oncofetal antigens, ectopic hormones, enzymes, and tissue-associated antigens. 36 The oncofetal and tissue-associated antigens are polyclonal, reacting to the cells of more than one organ. For example, carcinoembryonic antigen can be found in the respiratory and gastrointestinal tract, epidermis, cervix, vagina, urinary bladder, etc. 37'3s With the advent of monoclonal antibodies, a different nomenclature was necessary, since the different classes of polyclonal antigens are com- posed of many epitopes. The current method of designating monoclonal antibodies is organ ori- ented, ie, breast, colon, etc. A particular mono- clonal antibody may be described as oriented to breast, yet it is still not specific for breast alone. For example, 10-3D-2 is cross-reactive with lung, colon, placenta, and melanoma39; HMFG 2 with lung, uterus, and ovary4~ and 3.15.C3 with colon. 41 One of the more promising antibodies to

B72.3 antigen has a strong affinity for neoplastic cells when compared with normal adult cells. 42'43 Although this antibody reacted with 50% of the breast carcinomas we evaluated, it also reacted with >80% of colon carcinomas. Six additional antibodies to B72.3 have been developed. 44 All six are superior to the original one in terms of the percent of dose per gram of tumor and the tumor-to-nontumor ratios.

Muraro et a145 have also described a group of monoclonal antibodies to five colon carcinomas. The antibodies are divided into five distinct types based on their reaction to the surface of carci- noma cells. There was no cross-reactivity with benign or dysplastic colon lesions or with adult normal tissues; however, cross-reactivity was ob- served with breast carcinoma.

Although monoclonal antibodies to the epitopes of a number of organs have been developed, including colon, breast, lung, cervix, ovary, pan- creas, prostate, etc, cross-reactivity with cells of other organs occurs. One can only conclude that a truly tumor-specific antigen and its antibody will be a rare entity. Schlom and Weeks 25 have presented an excellent summary on this subject.

The development of antibodies to different epitopes on the same cancer tumor suggests using a mixture of such antibodies to increase tumor concentration, ie, a "cocktail." Unfortunately, this technique may not be useful. Sharkey et al, 46 evaluated the tumor localization of four mono- clonal antibodies prepared from the GW-39 human colon carcinoma. They found that two of the monoclonal antibodies demonstrated greater tumor concentration than the goat polyclonal antibody and two demonstrated less. Although statistical differences were found, tumor-to- nontumor values were not distinguished by a factor > 1.5, possibly indicating that the findings were not biologically significant. Mixtures of the two antibodies that showed greater tumor concen- tration than the whole antibody did not provide better imaging than that of a single monoclonal antibody.

A promising aspect of radioimmunoimaging is immunolymphoscintigraphy. The diagnostic method is similar to that of colloid lymphoscintig- raphy, eg, the administration of the radiolabeled antibody subcutaneously in the lymphatic drain- age field of the lymph nodes under examination. The sequestration of labeled antibody to the

162 FRANK H. DELAND

metastatic tumor in the lymph nodes has been well-documented, 47-49 as has the specificity of antibody-antigen reaction in lymph nodes. 5~ Of possible clinical significance is the sequestration of labeled antibody in lymph nodes outside of the lymphatic drainage of the tumor. In an early study of patients with breast cancer, sequestra- tion of labeled antibody was imaged in the contralateral axilla. In three of these cases, no palpable lymph nodes were present in the con- tralateral axilla. 47 In a similar series of cases reported by Thompson et al, 49 two patients with uptake of radioactivity in the contralateral axilla were observed. Although aspiration of the lymph nodes was negative for malignancy, in one pa- tient the contralateral nodes later became palpa- ble and were found positive by aspiration for malignant cells. The interpretation was that the lymph nodes were sequestering shedded antigen from a breast lesion located on the opposite side in the absence of metastatic tumor. Subse- quently, this phenomenon was demonstrated in a patient with labial carcinoma. 51 A number of lymph nodes on both sides of the mons pubis containing radioactivity were devoid of tumor and to obtain absolute proof, antigen was ex- tracted from these nodes. There have been no in-depth investigations of the clinical implica- tions of sequestered antigen in lymph nodes that may or may not be in the lymphatic drainage of the tumor. Although such information may possi- bly be important in the choice and type of therapy, this is yet to be investigated.

With the development of antibodies to tumor- associated antigens, the possibilities of using this modality for cancer therapy were immediately evident. However, the results to date can be best described as less than desired. Before the use of radiolabeled antibodies, immunotherapy had been investigated and also found wanting. Antitumor antibodies labeled with 13~I have been most frequently used because the gamma emission permits imaging and the beta emission may be advantageous for cellular lysis. Beierwaltes and Khazaeli 52 found that the uptake of 131I metaiodo- benzylguanidine (MIBG) in carcinoma of the adrenal medulla had approximately the same biological half-life as that of 131I in well- differentiated thyroid carcinoma. The percent dose per gram uptake in the adrenal was also

similar to that in thyroid therapy and elimination of the cancer was not attained. They also found that organ uptake decreased when using MIBG labeled with monoclonal antibodies or fragments.

DeNardo et a153 have postulated that the radioisotope label for an antibody to be used in therapy should have a physical half-life of one to three days, a major energy deposition from 0.5 to 30.0 cell diameters, and a simple decay scheme to ground state. Among the radionuclides men- tioned by DeNardo et al is 67CU, which releases abundant beta particles for therapy and appropri- ate gamma emissions (93 and 184 keV) for imaging. The biodistribution of 67Cu-labeled mon- oclonal antibody to human B-cell lymphoma was encouraging: Tumor uptake was 14.7% of in- jected dose per gram on day 3 and remained at this level through day 5. In normal tissues, radioactivity decreased from day 1.54

Neutron-capture therapy with antibodies la- beled with l~ is an interesting approach to cancer radioimmunotherapy. 55 When l~ ab- sorbs thermal neutrons, an alpha particle as well as recoiling 7Li ions result, with an average total kinetic energy of 2.4 MeV and a tissue range <10/~. Currently, labeling methods are under investigation to conjugate high quantities of boron without affecting the immunoreactivity.

Etlinger et a156 have approached therapy of hepatomas with combined modalities of radiola- beled antibodies, chemotherapy, and external radiation. They reported remissions in 11 of 28 patients with hepatomas. Remissions were ob- served in four of six patients with antibody therapy, and in four of five patients with inopera- ble pulmonary carcinoma, remission occurred with antibody and external radiation therapy. 57 There are a number of reports of cancer therapy with radiolabeled antibodies that may produce remission, but a paucity of cures.

Nonradionuclide conjugates with monoclonal antibodies have been investigated. Smyth et a158 evaluated the combination therapy of N-acetyl- melphalan conjugated to the monoclonal anti- body, anti-Ly-2.1, administered with recombi- nant human necrosis tumor factor, a(rTNF-a). They observed that 30% of the tumors in mice showed partial or complete regression. The possi- bility of also conjugating the antibody with an

PERSPECTIVE OF MONOCLONAL ANTIBODIES 163

appropr ia t e rad ionuc l ide could increase the tu-

mor regression.

Signif icant advances have been made in the

development of improved ant ibodies, methods of

g rea te r an t ibody product ion, procedures for the

selection of app rop r i a t e ant ibodies , selection of

radionucl ides , methods of nuc l ide-an t ibody con-

jugat ion , and imaging techniques, such as tomog-

raphy. Inves t igat ions re inforce two conclusions

previously noted. A tumor-specif ic an t ibody prob-

ab ly does not exist, and even with the purest of

ant ibodies, the r ange of tumor concentra t ion of

the an t ibody is still subopt imal for imaging and

therapy. I t appears tha t biologic response modi-

fiers may be requi red to develop the cl inical ly

successful appl ica t ions of ant ibodies. For exam-

ple, ant igen expression on the surface of mal ig-

nant cells m a y be enhanced by recombinan t

interferon. Gene t i ca l ly engineered monoclonal

ant ibodies may provide improved ant igen-ant i -

body binding for de tec t ion and therapy. For both

tumor detec t ion and therapy , immunohis tochem- istry could be a rewarding tool. A ba t te ry of ant ibodies could be quan t i t a t ed agains t the pa- t ient ' s tumor and a cocktai l made from those ant ibodies with the greates t affinities. Tumor detect ion can also be enhanced by selecting a radionucl ide label with a low rad ia t ion dose and an appropr i a t e energy for cur ren t imaging equip- ment. Wi th a radionucl ide label such as 99rnTc, a

la rger dose of labeled an t ibody can be adminis- tered, thereby improving the da t a statistics. Tu- mor - to -non tumor rat ios can be improved by the use of an t ibody f ragments . Final ly , emission tomography, pa r t i cu la r ly with a radionucl ide energy appropr i a t e for tomograph ic instruments , can improve the t a rge t - to -non ta rge t ra t io by a factor of two or three, c o m p a r e d with p l ana r imaging. Unt i l sophis t icated biologic modifiers a re developed or genet ic engineer ing techniques made avai lable , opt imal results , pa r t i cu la r ly in imaging, requi re the use of every s t ra tegy avail- able.

REFERENCES

1. Baron J: Edward Jenner, in The Encyclopaedia Britan- nica. vo125 (ed 11). New York, Britannica, 1911, pp 319-320

2. Pasteur L: Sur les maladies virulentes, et en particulier sur la maladie appel6e vulgairment cholera des poules. C R Acad Sci 90:239-248, 1880

3. Hericourt J, Richer C: Traitement d'un cas de carci- nome par la serotherapie. C R Acad Sci 120:940-950, 1895

4. Hericourt J, Richet C: De la serotherapie dans le traitement du cancer. C R Acad Sci 121:567-569, 1895

5. Witebsky E: Desponsibilet~lt und Spezifitat Alkohal- 18slicher Strukturen von Organen und bosartigen Geschwul- sten. Z Immunit~itsforsch Ext Ther 62:35-73, 1929

6. Pressman D, Keighley G: The zone of activity of antibodies as determined by the use of radioactive tracers; the zone of activity of nephrotoxic anti-kidney serum. J Immunol 59:141-146, 1948

7. K~hler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497, 1975

8. Dalrymple J: On the microscopical characteristic of mollities ossium. Dub J Med Sci 2:85-95, 1846

9. Bence Jones H: On a new substance occurring in the urine of a patient "mollities ossium." Phil Trans Royal Soc London 138:55-62, 1848

10. Burnet FM: The Clonal Selection Theory of Acquired Immunity. Nashville, TN, Vanderbilt, 1959

11. Kritzman J, Kunkel HG, McCarthy J, et al: Studies of a WaldenstrSm-type macroglobin with rheumatoid factor properties. J Lab Clin Med 57:905-917, 1961

12. WaldenstrOm J, Winblad S, Hullen J, et al: The occurrence of serological (antibody) reagin or similar gamma-

globulins in conditions with monoclonal hypergammaglobu- linemia, such as myeloma macroglobulinemia, etc. Acta Med Scand 176:619-631, 1964

13. Goldenberg DM: Oncofetal and other tumor-associ- ated antigens of the human digestive system. Curr Top Pathol 63:289-242, 1976

14. Gold DV, Goldenberg DM: Antigens associated with human solid tumors, in Sell S (ed): Cancer Markers: Develop- mental and Diagnostic Significance. New York, Humana, 1979, pp 329-369

15. Steplewski Z: Monoclonal antibodies to human tumor antigens. Transplant Proc 12:384-387, 1980

16. Gold P, Freedman SO: Demonstration of tumor- specific antigens in human colonic carcinomata by immuno- logican tolerance and absorption techniques. J Exp Med 121:439-462, 1965

17. Reif AE, Curtis LE, Duftield R, et al: Trial of radio-labelled antibody localization in metastases of a patient with tumor containing carcinoembryonic antigen (CEA). J Surg Oncol 6:133-138, 1974

18. Mach JP, Carrel S, Merenda C, et al: In vivo localiza- tion of anti-CEA antibody to colon carcinoma. Can the results obtained in the nude mice model be extrapolated to the patient situation? Eur J Cancer 1:118-120, 1978 (suppl)

19. Goldenberg DM, DeLand F, Kim E, et al: Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning. New Engl J Med 298:1384-1386, 1978

20. DeLand FH, Kim EE, Simmons G, et al: Imaging approach in radioimmunodetection. Cancer 40:3046-3049, 1980

164 FRANK H. DELAND

21. Primus FJ, Newell KD, Blue A, et al: Immunological heterogeneity of carcinoembryonic antigen: Antigenic deter- minants on carcinoembryonic antigen distinguished by mono- clonal antibodies. Cancer Res 43:686-692, 1983

22. KerbelRS: Implications of immunological heterogene- ity of tumors. Nature 280:358-360, 1979

23. Prehn RT: Analysis of antigenic heterogeneity within individual 3-methylcbolan three-induced mouse sarcomas. J Natl Cancer Inst 45:1039-1045, 1970

24. Poste G, Doll J, Fidler I J: Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal populations of B16 melanoma cells. Proc Natl Acad Sci USA 78:6226, 1981

25. Schlom J, Weeks MO: Potential clinical utility of monoclonal antibodies in the management of human carcino- mas, in Devita VT, Hellman S, Rosenberg SA, (eds): Important Advances in Ontology 1985. Philadelphia, Lippin- cott, 1985, pp 170-192

26. Poste G, Doll J, Brown AE, et al: Comparison of the metastatic properties of B 16 melanoma clones isolated from cultured cell lines, subcutaneous tumors, and individual lung metastases. Cancer Res 42:2770-2778, 1982

27. Fogel M, Gorelek E, Segal S, et al: Differences in cell surface antigens of tumor metastases and those of the local tumor. J Natl Cancer Inst 62:585-589, 1979

28. Herlyn D, Koprowski H: IgG2a monoclonal antibodies inhibit human tumor growth through interaction and effector cells. Proc Natl Acad Sci USA 79:4761-4765, 1982

29. Kennel S, Foote L, Lankford P, et al: Direct binding of radioiodinated monoclonal antibody to tumor cells: Signifi- cance of antibody purity and affinity for drug targeting or tumor imaging. Hibridoma 2:297-310, 1983

30. Halpern S, Stern P, Hagan P, et al: Radiolabeling of monoclonal antitumor antibodies: Comparison of 1-125 and In-111 ante-CEA with Ga-67 in nude mouse-human colon tumor model. Clin Nucl Med 6:453, 1981

31. Herlyn D, Powe J, AlaviA, et al: Radioimmunodetec- tion of human tumor xenografts by monoclonal antibodies. Cancer Res 43:2731-2735, 1983

32. O'Connor SW, Bale WF: Accessibility of circulating immunoglobulin G to the extravascular compartment of solid rat tumors. Cancer Res 44:3719-3723, 1984

33. Begent RHJ, Green A J, Bagshawe KD, et al: Liposo- mally entrapped second antibody improves tumor imaging with radiolabeled (first) antitumor antibody. Lancet 2: 739-743, 1982

34. Sharkey RM, Primus FJ, Goldenberg DM: Second antibody clearance of radiolabeled antibody in cancer radio- immunodetection. Proc Natl Acad Sci USA 81:2843-2846, 1984

35. Goodwin D, Meares C, Diamanti C, et al: Use of specific antibody for rapid clearance of circulating blood background from radiolabeled tumor imaging proteins. Eur J Nucl Med 9:209-215, 1984

36. Heberman RB, Mcintire KR (eds): Immunodiagnosis of Cancer. New York, Dekker, 1975, p 75

37. Harvey SR, Girota RN, Nemoto T, et al: Immuno- chemical studies on carcinoembryonic antigen-reactive gl3/co- proteins from carcinomas of the colon and breast separated by concanavalin A affinity chromatography. Cancer Res 36:3486-3494, 1976

38. DeYoung N J, Ashman LK: Physiochemieal and im- muno-chemical properties of carcinoembryonic antigen (CEA) from different tumor sources. Aust J Biol Sci 56: 321-331, 1978

39. Soule HR, Linder E, Edgington TS: Membrane 126- kilo dalton phosphoglycoprotein associated with human carci- noma identified by a hybridoma antibody to mammary carcinoma cells. Proc Natl Acad Sci USA 80:1332-1336, 1983

40. Epenetos AA, Britton KE, Mather J, et al: Targeting of iodine 123-labeled tumor-associated monoclonal antibod- ies to ovarian, breast, and gastrointestinal tumors. Lancet 2:999-1004, 1982

41. Taylor-Papadimitriou J, Peterson JA, Arklie J, et al: Monoclonal antibodies to epithelium-specific components of the human milk fat globulin membrane: Production and reaction with cells in culture. Int J Cancer 28:17-21, 1981

42. Nuti M, Teramoto YA, Mariani-Constantini R, et al: A monoclonal antibody (B72.3) defines patterns of distribu- tion of a novel tumor-associated antigen in human mammary carcinoma cell populations. Int J Cancer 29:539-545, 1982

43. Colcher D, Horan Hand P, Nuti M, et al: A spectrum of moneclonal antibodies reactive with mammary tumor cells. Proc Natl Acad Sci USA 78:3199-3203, 1981

44. Colcher D, Minelli MF, Roselli M, et al: Radioimmu- nolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies. Cancer Res 48: 4597-4603, 1988

45. Muraro D, Wunderlich A, Thor J, et al: Definition by monoclonal antibodies of a repertoire of epitopes on carcino- embryonic antigen differentially expressed in human colon carcinomas versus normal adult tissues. Cancer Res 45: 5769-5780, 1985

46. Sharkey RM, Primus F J, Shochat D, et al: Compari- son of tumor targeting of mouse monoclonal and goat polyclonal antibodies to carcinoembryonic antigen in the GW-39 human tumor-hamster host model. Cancer Res 48:1823-1828, 1988

47. DeLand FH, Kim EE, Corgan RL, et al: Axillary lymphoscintigraphy by radioimmunodetection of carcinoem- bryonic antigen in breast cancer. J Nucl Med 20:1243-1250, 1979

48. Weinstein JN, Stellar MA, Keenan AM, et al: Mono- clonal antibodies in the lymphatics. Selective delivery to lymph node metastases of solid tumors. Science 223:423-426, 1983

49. Thompson CH, Lichtenstein M, Stacker SA, et al: Immunoscientigraphy for detection of lymph node me- tastases from breast cancer. Lancet 2:1245-1247, 1984

50. Weinstein JN, Parker R J, Holton OD, et al: Lym- phatic delivery of monoclonal antibodies: Potential for detec- tion and treatment lymph node metastases. Cancer Invest 3:89-95, 1985

51. DeLand FH, Goldenberg DM: In vivo radioimmuno- logical lymphoscintigraphy in cancer. J Can Assoc Radiol 33:4-9, 1982

52. Beierwaltes WH, Khazaeli MB: Radioimmunother- apy of cancer: Historical perspectives and prospects for the future, in Burchiel SW, Rhodes BA (eds): Radioimmunoim- aging and Radioimmunotherapy. Amsterdam, Elsevier, 1983, pp 419-439

PERSPECTIVE OF MONOCLONAL ANTIBODIES 165

53. DeNardo S J, DeNardo GL: Tumor therapy with radioactive-labeled antibodies, in Moloy P J, Nicholson GL (eds): New Approaches in Biology, Diagnosis, and Treat- ment. New York, Praeger, 1983, pp 282-292

54. Deshpande SU, DeNardo S J, Meares CF, r al: Copper-67-1abeled monoclonal antibody Lym-1, a potential radiopharmaceutical for cancer therapy: Labeling and biodis- tribution in RAJ1 tumored mice. J Nucl Med 29:217-225, 1988

55. Goldenberg DM, Sharkey RM, Primus F J, et al: Neutron-capture therapy of human cancer: In vivo results on

tumor localization of boron-10-labeled antibodies to carcino- embryonic antigen in the GW-39 tumor model system. Proc Natl Acad Sci USA 81:560-563, 1984

56. Etlinger DS, Order SE, Wharam MD, et al: Phase I-II study of isotopic immunoglobulin therapy for primary liver cancer. Cancer Treat Rep 66:289-297, 1982

57. Order SE: Radioimmunoglobulin therapy of cancer. Compr Ther 10:9-18, 1984

58. Smyth M J, Pietersz GA, McKenzie IFC: Increased antitumor effect of immunoconjugates and tumor necrosis factor in rive. Cancer Res 48:3607-3612, 1988