Human monoclonal antibodies as cellular and molecular probes: a review

Molecular and Cellular Probes (1987) 1, 3 3-54

REVIEW

Human monoclonal antibodies as cellular and molecular probes : a review

M . J . O'Hare and C. Y . Yiu* Institute of Cancer Research, Royal Cancer Hospital, Sutton, Surrey SM2 5PX and *Department of Surgery, University College London, University Street, London WC1E 611, UK (Received 12 January 1987, Accepted 22 January 1987)

KEYWORDS : human-antibodies, hybridomas, EBV transformants, monoclonals, cancer.

BACKGROUND The use of rodent monoclonals in human immunotherapy is generally deemed undesirable because of anaphylaxis and clinical effects of immune complex formation . Most human monoclonal antibodies have, therefore, been prepared with the ultimate aim of therapeutic and diagnostic administration to patients . Human monoclonals also provide a unique means of probing human immune responses in, for example, autoimmune disease and cancer . There is also a possibility that specificities might occur in man which are not readily available in xenogeneic immunizations, notably in relation to polymorphic antigens . To date, however, the number of human monoclonal antibodies with 'useful' specificities has barely reached a hundred, as compared with thousands of mouse antibodies . The reasons are essentially twofold . The methods of producing human monoclonals have been significantly less efficient than their murine counterparts, and many studies have relied on natural immunization processes . The search for human monoclonals reacting specifically with human cellular and in particular membrane-associated antigens has, therefore, been laborious .

TECHNIQUES Human monoclonals generated in the laboratory date from 1977' and human hybridoma systems have been widely available for the past 5 years . It is now possible, therefore, to assess which methods are proving the most productive . Human monoclonal antibodies can be made in one of two ways . Lymphocytes 0890-8508/87/010033+22 $03 .00/0

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© 1987 Academic Press Inc . (London) Ltd

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M. J . O'Hare and C. Y . Yiu

Table 1 .

Methods of human B-cell immortalization*

Method Lymphocyte x human fusion : Epstein-Barr virus transformation : Lymphocyte x mouse fusion : EBV X human fusion : EBV X mouse fusion : Lymphocytes x mouse/human' fusion : EBV X mouse/human' fusion

34 27 20 8 4 4 3

Based on reports published 1980-6 . Using mouse x human heterohybridomas as parental line .

can be transformed with Epstein-Barr virus or they can be immortalized by an adaptation of the hybridoma technology developed by Kohler & Milstein' for making murine monoclonals . In the latter case a suitable human B-cell derived line is directly hybridized with fresh human lymphocytes . Attempts to improve these systems have led to the construction of both human-human and mouse-human hybrids as fusion partners, and to fusions between EBV-transformed lymphocytes and myeloma, lymphoma or lymphoblastoid lines . Alternatively, rodent myeloma cell lines can themselves be used directly as the fusion partner for human lymphocyte immortalization . All of these strategies have been employed in the past 5 years . A numerical comparison of published papers (Table 1) indicates that the direct human x human fusion systems are preferred at present, although they are run a close second by EBV transformation . Current trends suggest that the combined technique of hybridizing EBV-activated cells may prove a reliable means of generating human monoclonals with pre-defined specificities, obviating the problems of stability, loss of antibody secretion and clonal senescence by EBV-transformed cultures .' The choice of fusion partner is more problematical . The long-term karyotypic stability of heterohybridomas (mouse x human) remains contentious . All human lines that have been used extensively for fusion stem from relatively few lymphoblastoid lines as the myeloma-derived SKO-007 4 has not been widely used . They include the HMy2 line of O'Hare' as well as other lymphoblastoid lines, including UC729-6 6 and GM4672, the GM1500-derived line . 7 Comparative aspects of their behaviour as fusion partners have been documented .'

SPECIFICITIES The practical problems involved in making human monoclonal antibodies have been extensively discussed elsewhere . 9 ' 10 We propose here to review their specificities, with particular emphasis on recognition of cellular antigens, i .e ., their value as cellular and/or molecular probes .



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Human monoclonals

Anti-viral monoclonals Human anti-viral antibodies could provide a means of passive immunization against infections, and thus substitute for conventional serotherapy in potentially fatal primary infections, such as varicella-zoster virus (VZV) and cytomegalovirus (CMV) in immunocompromised individuals . The first human monoclonals produced by hybridoma technology were to viral antigens . Croce et al .' derived two hybrids which reacted with measles virus nucleocapsid polypeptide in a fusion of peripheral lymphocytes from a patient with subacute sclerosing panencephalitis (SSPE) . Nowinski et al ." using heterohybridoma technology prepared a stable cell line which secreted IgM reacting with the terminal disaccharide of the Forssman antigen, a highly immunogenic glycolipid which is a constituent of the influenza virion . One advantage when raising anti-viral monoclonals is that successful methods of in vitro immunostimulation with viral proteins have been used to enrich cultures for reactive cells prior to immortalization 12 when specific antibody-producing cell lines can be significantly increased .", " Active immunization in vivo is also possible, and has been successful where fusions with non-immunized donors having high natural antibody titres had failed ." Several groups have now produced human anti-viral monoclonals . Some react with capsid polypeptide nuclear antigens and others detect membrane-associated viral antigens in infected cells . Most show considerable specificity . They are summarized in Table 2 . In addition to measles and Forssman antigen, monoclonals

Table 2.

Human monoclonal anti-viral antibodies

Antibody

Class

Antigen

D3 C5 Ri-BM Various C10 C15 C28

IgM IgM

Measles nucleocapsid polypeptide Measles nucleocapsid polypeptide HSV glycoprotein D HSV Influenza type A NP

C29 C75 UT17 H1-C4

IgG1,A IgG3,K IgM IgM IgG1,K IgG1,2 IgG,K IgG,A IgG1 IgG1,2. IgG,2 IgG,K IgG,K IgG,K IgG,2. IgG1,K

1-A2 2-1D5 312 .A C1 C23 Hu-HBsAG-1 JHB-2,3,4,10 TAKA-SP-8 FUKA-SP-3 TAKA-B-1 SAKA-B-2 0.5 a

IgG1,K NS IgG1,K IgG1,K IgG1,2

Influenza A Influenza A H3 Influenza B/Sing Influenza A H3 Influenza A H3 Forssman glycolipid Varicella-zoster virus Varicella-zoster virus CMV nuclear antigen CMV (non-neutralizing) CMV (neutralizing) Hepatitis B surface Hepatitis B surface EBV membrane antigen p93 EBV capsid antigen p120 EBV capsid antigen EBV capsid antigen HTLV-1 env-glycoprotein

Reference 7 7 20 13 12 15 15 15 15 21 11 17 17 18 14 14 19 22 16 16 16 16 23



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M. J . O'Hare and C. Y. Yiu

against herpes simplex, various influenza serotypes, varicella zoster, cytomegalovirus, hepatitis B, Epstein-Barr virus and HTLV-1 have been obtained . Although in many instances comparable murine monoclonals are available16 there is some evidence that the human monoclonals may react with antigens and epitopes not readily detected by mouse antibodies . 17 Some, but not by any means all, of these anti-viral antibodies have been shown to be protective in animal model systems ." Others have been shown to neutralize virus infection in Vitro 14,16,17 and those which react only with nuclear antigens in infected cells or which do not neutralize the virus for others reasons may, nevertheless, be of diagnostic value . 18,19

Anti-bacterial monoclonals Relatively few human monoclonals to specific bacterial antigens or toxins have been reported to date other than those reacting with tetanus toxin . Nevertheless, serotherapy offers a significant therapeutic modality in certain acute bacterial infections that are antibiotic and/or drug resistant, such as infant Haemophilus influenzae infection and pneumococcal bacteraemia and meningitis .

Antibodies to capsule antigens and endotoxins Two antibodies (16M3C8 and 16M4F2) to the capsular polysaccharide (PRP, polyribosylribitolphosphate) of Haemophilus influenzae type b, were reported by Gigliotti et al. 24 Both protected infant rats against H . influenzae infection, as did the antibody prepared by Hunter et al . 25 These may have prophylactic value in man, especially in infants who do not respond to active immunization with PRP . An antibody against group A streptococcal carbohydrate was generated by Steinitz et al. 26 More recently an IgM,K monoclonal reacting with pneumococcus capsular polysaccharide type B was identified in an EBV-transformed cell line (NAD) from a patient who had been treated with the polyvalent anti-pneumococcal vaccine Pneumovax; 27 like several monoclonal IgMs this also bound protein A, a reaction seen with only a minority of human serum IgMs . An IgA-secreting line (NAD-Sel) with an identical specificity from the same source has also been obtained ; 28 this possesses bactericidal activity, and specifically opsonizes type 8 pneumococci . An IgM monoclonal (BA-G2) to PPD (purified protein derivative of tuberculin) has also been reported, 29 and several antibodies reactive with Mycobacterium leprae antigens . 3o Human monoclonals to Gram-negative endotoxin (LPS) have been sought by two groups . 31,32 In one case the J5 mutant of Escherichia coli, which has a core oligosaccharide common to all Gram-negative bacteria, was used in an in vivo immunogen, and an IgM-secreting hybrid (A6(H4CS)) which protected mice against bacteraemia was obtained ." Several antibodies binding to various strains have recently been reported by Bogard et al . 32 Pseudomonas aeruginosa is an infection frequently encountered in immunocompromised burns and cancer patients . Sawada et al ." have identified a human



Human monoclonals

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monoclonal P3D9, which reacts with a lipopolysaccharide of P . aeruginosa type 5 . This IgG2,), antibody also protects mice against experimental infection . Chlamydia trachomatis is a small DNA/RNA-containing coccus with a complicated life-cycle which can cause blindness . Using lymphocytes from patients with chlamydial salpingitis, Rosen et al." established two cell lines (EN and SALP) which secrete IgG1 directed against a common genus-specific chlamydial surface antigen . These cross-reacted with only Streptococcus pyogenes amongst a wide range of other bacteria tested . The antigen detected is probably the 10 kD lipoproteinpolysaccharide complex against which mouse monoclonals have been raised . The utility of the human antibodies is diagnostic .

Antibodies to bacterial exotoxins Tetanus toxoid has been used frequently as an 'experimental' immunogen to test both the efficacy of in vitro selection and sensitization protocols and various immortalization techniques, including fusion of EBV lines with various myelomalymphoblastoid partners . 35 Consequently there are a relatively large number of antiTT human monoclonals, whose properties are summarized in Table 3 . Conjugation to tetanus toxoid may also provide a means of enhancing the immunogenicity of small polypeptides (such as bombesin) for in vitro immunostimulation procedures . 31 In some, but not all, instances a protective effect on toxin administration has been demonstrated .4°-43 Other anti-toxin human monoclonals include one against diphtheria toxin (16M3F10) reported by Gigliotti et al.," which also neutralized toxin activity .

Table 3 . toxin

Human monoclonal antibodies against tetanus

Antibody

Class

3CG/4LP-B4 B6 TT-1 9F12 UN* UN* (3) Hu-TT-1t 16M3C9 3G6 UN (2) UNt a-TT1,2

IgG IgM,K IgM, IgG,x IgG,K IgG (2) and IgM (1) IgG1,x NSt lgM,K IgM and IgG IgM IgG1,x

* UN, undesignated . EBV line subsequently xenohybridized . 47 $ Selected by FACS . NS, not stated .

Reference 37 38 39 40 41 42 43 24 44 36 45 46



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M . J . O'Hare and C . Y . Yiu

Anti-protozoal monoclonals Human monoclonals reacting with parasitic protozoans are also few in number at present . Malaria (Plasmodium sp .) and its life-cycle offers an opportunity of interfering with merozoite re-invasion by which the parasite spreads in the erythrocyte population . Schmidt-Ullrich et al ." have produced anti-plasmodial antibodies reacting with the Pf195 schizont/merozoite antigen of P . falciparum . Several lines, predominantly producing IgG1 or 2, were described, one of which, KJ7 2C11D (IgG2), significantly inhibited parasite growth in vitro . Berzins et al ." and Udomsangpetch et al." describe two antibodies, G2 (IgM) and B6 (IgG) which stain schizonts, trophozoites and merozoites, and react with Pf155, a surface protein which binds to human erythrocyte glycophorin . Both inhibited merozoite reinvasion . These and other" 52 human anti-malarial antibodies will be valuable in identifying and characterizing antigenic merozoite peptides, mapping the relevant portion of the plasmodial genome, and possibly in treating severe cases of drugresistant malaria .

Anti-human monoclonals Erythrocyte, blood group and histocompatibility antigens

Interest in human monoclonals as a resource was stimulated in part by the belief that mice might not prove an efficient source of monoclonals against human polymorphisms . Many murine anti-H LA antibodies react with monomorphic framework antigens, as distinct from type-specific polymorphisms . Specific human alloantibodies in the form of immune sera are important clinical diagnostic reagents for blood typing in cases of transfusion and transplantation, and for prophylaxis of Rhesus haemolytic disease of the newborn . Limited availability of such sera has, however, been a restraint on their use . An antibody to the influenza virus-associated Forssman antigen" has already been mentioned ; it reacts with a disaccharide (aGaINAc-ßGaINAc) also found, in a modified form, in blood group Ab glycolipid . The reaction of the antibody (H1-C4) with the latter was not recorded, although it lysed sheep, but not human red blood cells . Human antibodies to SRBC were also obtained by Strike et al . 53 in a study of in vitro immunization ; we ourselves were unsuccessful in generating such antibodies in our first study with HMy2, although many hybrids were prepared ." A human anti-A monoclonal has been derived by Foung et al ." using splenic lymphocytes from a type O patient with haemolytic anaemia, providing a reagent which reacted as well as or better than commercial antisera .S6 Hirohashi et al ." identified a human IgM antibody NCC-1004 which reacts with blood group i antigen, agglutinating cord blood erythrocytes and binding to lacto-norhexaosylceramide plus sialyosyl-lacto-norhexaosylceramide . The i antigen is the precursor of ABH I antigen and is developmentally regulated, appearing in increased amounts in both embryos and tumours. A number of human monoclonals have been raised against Rhesus D antigen because of its importance in haemolytic disease of the newborn (Rh incompatibility), where post-partum administration of Rh(D) immunoglobulin to RH-negative



39

Human monoclonals

Table 4 .

Human monoclonal antibodies to rhesus antigens

Antibody

Class

Specificity

Ks-P* R* UCH-D4 D4-B2 EC10- cl , Hs DS DZ REY-1t UN§

IgG1 IgM lgG1,x lgG3,2 IgG3, NS IgG1,ic Igd1 IgG3

Rh (D) Rh (D) Rh (D) Rho (D) Rho (D) Rh (D) Rh (D) Rh (G) Rh (Du)

IgM

Rh (Du)

GAD-2 MAD-2

Reference 58 59 60 61 61 62 63 64 65 65

Uncloned . Derived from same fusion as D4-B2 . EBV-transformed line which eventually senesced . a UN, undesignated .

sensitized women has dramatically decreased maternal isoimmunization and resulting Rh disease . They are summarized in Table 4 . The majority react with Rh(D), although a human monoclonal reacting with Rh(G), an antigen strongly associated with D or C, has recently been identified by Foung et al. 64 The immediate clinical utility of anti-Rh human monoclonals has led to some debate as to their safety, vis-à-vis potential EBV contamination . This has been cogently discussed by Crawford et al . 66 who concluded that the hazard is actually small in comparison with conventional serotherapy, and have shown that such monoclonals can be readily purified from all detectable DNA . 67 A monoclonal human IgM apparently reacting with an HLA framework antigen was reported by Hulette et al . 68

Autoimmune antigens Autoimmune diseases provide a potentially fertile source of human monoclonal antibodies directed against cellular antigens . Furthermore, a potential cure for autoimmune disease can be envisaged with the aid of monoclonal antibodies . Thus, anti-autoantibodies could provide a means of interrupting the vicious circle of tissue destruction and endogenous antibody production . Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) have both been used as source of monoclonal autoantibodies, as well as autoimmune thyroiditis, insulin-dependent diabetes and Graves' disease .

Rheumatoid arthritis In rheumatoid arthritis (and some other autoimmune diseases) patients develop antibodies which bind, like complement, to circulating immune complexes (rheumatoid factors) . An IgM,), rheumatoid factor-secreting cell line (RF-AN) was estab-



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M. J . O'Hare and C . Y . Yiu

lished by Steinitz and his colleagues 69 '70 as a reagent for the universal detection and quantitation of immune complexes . Its advantages over conventional methods are that it preferentially binds IgG in immune complexes, and being an IgM does not bind non-specifically via Fc receptors . Anti-RF-secreting lines were also obtained by Normasell & Lane. 71 Other human lines producing RF have, however, proved unstable72 although the same fusions yielded lines which reacted with a variety of cellular constituents, including proteoglycans, vimentin, cardiolipin and DNA . 73

Systemic lupus erythematosus Most anti-DNA human monoclonals have been generated from lymphocytes obtained from patients with SLE, whose serum contains autoantibodies that react with nuclear, cytoplasmic and cell-surface antigens . Human autoantibody-secreting cell lines have been derived from such patients by several groups, notably by Shoenfeld and his colleagues . All have been IgMs . Shoenfeld et al ." reported 16 lines from an individual with thrombocytopenic purpura, of which seven reacted with single-stranded DNA (denatured DNA) . A further six reacted with platelets by the same antibody binding site responsible for DNA-binding, but with a platelet epitope not involving DNA, protein or sialic acid .75 Further lupus fusions yielded a total of 30 hybrids, 76 and analysis of their specificity showed that a majority reacted with ssDNA, native double-stranded DNA as well as left-handed double-stranded DNA (z-DNA) and synthetic polynucleotides, although with individually distinct patterns of binding . A third of them also reacted with the glycolipid cardiolipin . Despite the apparently disparate nature of these antigens, the human 'anti-DNA' antibodies appeared to be reacting with appropriately spaced phosphodiester groups, which it has been speculated may constitute an important group of autoantigens in SLE . Two of the autoantibodies described by this group have subsequently been shown to possess lymphocytotoxic activity.77 This poses the question of how the same antibody binds to both DNA and lymphocyte membranes, and to Raji cells . 78 Diesters or simply repeated negatively charged groups are candidates although the possibility of polyfunctional binding sites on the antibodies 79 cannot be excluded . Over half of the same antibodies reacted with cellular cytoskeletal elements, corresponding to the intermediate filament protein vimentin . 80 IgM-secreting anti-DNA cell lines have also been derived by Littman et al ." and Sasaki et al ." from SLE patients with similar properties to those prepared by Shoenfeld . Rauch et al." also obtained similar antibodies from rheumatoid arthritis patients as well as SLE, and even from normal individuals . Antibodies to DNA were also produced by Winger et al." by a rosetting precursor enrichment technique from lymphocytes from normal individuals ; they have also been obtained from cancer patients . 85 Thus the specific pathogenicity of anti-DNA autoantibodies may not bear any relationship to their nucleic acid antigen-binding characteristics . Although anti-DNA antibodies are seen in patients with RA they are not thought to be of major pathogenic significance in this disease . Furthermore, the presence of anti-DNA activity in SLE does not always correlate with disease activity . A further study with anti-idiotypic antisera has indicated that although there is a substantial degree of idiotypic sharing among SLE patients" human monoclonal



Human monoclonals

41

anti-DNA antibodies from SLE may share a specific idiotype which is not found on RA-derived antibodies of similar antigenic specificities . 87

Insulin-dependent diabetes, Hashimoto's thyroiditis and Graves' disease The polyspecificity of lupus monoclonals is mirrored to some extent in antibodies generated from other autoimmune diseases . Serum from patients with autoimmune thyroiditis reacts with many organs, posing the question of whether it contains a multiplicity of organ-specific antibodies or a few antibodies reacting with antigens or epitopes expressed on many tissues . Similar reactions are seen in sera of insulindependent diabetic patients, who may also have anti-thyroid responses . Several antibodies to pancreatic antigens have been prepared from diabetics . Eisenbarth et al ." reported an IgM antibody (B6) reactive with pancreatic islet cells ; its extra-pancreatic reactivity was not documented . A similar human 1gM monoclonal (MC4E4) was reported by Thivolet et al .," reacting with a cell-surface islet antigen of 64 kD ; again a tissue distribution was not reported, although intermediate filament reactivity was excluded . A human monoclonal to human insulin has also been reported, 90 an IgM which cross-reacts with other species but which does not bind isolated A- and B-chains of the hormone . Many of the antibodies from diabetic patients have also reacted with thyroid antigens as well as the pancreas and other tissues . Thus, Satoh et al." hybridized lymphocytes from patients with insulin-dependent diabetes and with anti-thyroid microsomal antibodies . Nine autoantibody-secreting hybridomas were isolated, representing 6% of the total generated . Two reacted with nuclear antigens and the remaining seven IgMs (MOR-hl-7), stained defined populations of cells in more than one organ, including anterior pituitary, thyroid, gastric mucosa and pancreas . From differences in staining patterns and sensitivity of antigens to fixation it was inferred that they did not all recognize the same antigenic determinant . Several stained cultured cells in a manner which indicated binding to intermediate filaments . The same group have also raised a series of similar multiple organ-reactive human monoclonals by EBV transformation of lymphocytes from similar patients, using a rat insulinoma cell line WIN F5) as a rapid screen . All were IgMs which also reacted with human pancreatic islet and thyroid acinar cytoplasmic antigens" ," with varying degrees of individual tissue specificity . One antibody (E10-1-19) reacted with thyroid colloid . A similar anti-thyroglobulin monoclonal (HML 3 . 21) has been described by Foster et al.," together with an anti-TSH receptor antibody (HML 3 . 22) both from a patient with diabetes ; neither of these IgG antibodies reacted with pancreatic tissues . One of the multiple organ-reactive hybridoma antibodies produced by Notkins and his colleagues, MOR-h1, has been extensively s tudied . i t recognizes a 35 kD intracytoplasmic protein in pituitary, thyroid, stomach and pancreas and binds to growth hormone (hGH, 21 kD) .95 Anti-idiotypic mouse monoclonal antibodies which inhibit ligand-binding to this human antibody have been generated ." One paratope-directed antibody (4E6) has been used in turn to raise a rabbit anti-antiidiotype IgG antiserum which has the same staining pattern as the original human IgM monoclonal ." More specific anti-thyroid monoclonals have also been prepared from Graves'



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M. J . O'Hare and C . Y . Yiu

disease patients (thyrotoxicosis with exophthalmos) . Graves' disease serum immunoglobulin inhibits TSH-binding and itself acts as an intrinsic thyroid-stimulating factor . Two IgG human monoclonals (208F7 and 206H3) made by Valente et al ." using the heterohybridoma technique, also stimulated thyroid function as measured by increased rat and human thyroid cell cyclic AMP levels in vitro, and thyroidal iodine release in mice in vivo . Both interacted with receptor-associated gangliosides . Two other antibodies (129HB and 122G3) were initially identified (like 208F7 and 206H3) by their capacity to bind to bovine thyroid membrane preparations . They did not, however, stimulate thyroid function but inhibited thyrotropin (TSH) action and bound to the high-affinity glycoprotein receptor component rather than gangliosides . They thus appeared to be representative of the blocking antibodies also found in Graves' disease serum . One of the blocking antibodies and both of the stimulating ones enhanced thymidine incorporation by thyroid cells . In a recent investigation another thyroid-stimulating human monoclonal (307H6) uniquely stimulated collagen biosynthesis in cultured fibroblasts . 99 The pluritopic nature of GD antibodies was thus unequivocally demonstrated, as well as a possible mechanism for the orbital pathology via shared thyroid/orbital antigens . There is also evidence for such antigens from other human monoclonal studies 100 in which anti-thyroglobulin antibodies cross-reacted with orbital connective tissue membranes .

Other autoimmune diseases The CREST variant of scleroderma is a form of progressive systemic sclerosis with circulating antibodies to nuclear antigens, particularly centromeres . Someya & Kondo 101 recently isolated human monoclonal antibodies from such a patient . A chromosomal protein fraction was used as the screen and one IgM antibody (7D9B) was identified which reacted with a nuclear antigen . It did not, however, stain centromeres and immunoblotting identified a 33 kD protein antigen . Myasthenia gravis is an autoimmune disease in which autoantibodies to acetylcholine receptor impair neuromuscular transmission . A human monoclonal to AchR with myasthenic properties was derived by Kamo et al.' 02 by EBV transformation of cells from an MG patient .

Cancer-associated antigens Serologically detectable anti-tumour responses in man have been the subject of intensive study for many years, notably by the Sloan-Kettering group of Lloyd Old . These workers have also carried out an extensive study of monoclonal responses in cancer patients, using hybridoma tech no Iogy, 103,1°4 The most notable result from these studies was quantitative . Over 4350 Igsecreting hybrids have been made from 335 fusions 104 of which 305 (8%) reacted with either cell-surface or intracytoplasmic antigens . Most of these antigens had an intracytoplasmic location and were widely distributed . Intermediate filaments were prominent as reactive antigens, as with many antibodies from autoimmune patients (see above) .



Human monoclonals

43

Although in some cases intermediate filament reactivity may be due to nonidiotypic binding to vimentin 105 most such antibodies identified by Cote et al."' reacted with cytokeratins, and binding to other cytoplasmic elements as well as nuclear staining were demonstrated . Many reacted only with tumour cells . It is generally presumed that such antibodies reflect immune responses to 'normal' intracytoplasmic antigens liberated from necrotic tumour cells ; this may, however, be an overly simplistic interpretation . 106 In their first studies the Sloan-Kettering group also identified two human monoclonals which reacted with tumour-associated cell-surface antigens . These were Ri37, an IgG 107 and Ma4, an IgM . 103 . Ma4 recognizes a neutral glycolipid which is expressed in nearly half the cultured malignant cells tested, including breast, colon, lung, renal and bladder cancer, as well as gliomas and melanomas, but not on any normal cells tested . Ri37 antigen is apparently a protein found on lung, bladder and melanoma lines, as well as B lymphocytes but not on lines from normal solid tissues (or EBV-transformed lines) . In their recent paper a further four membrane-reactive antibodies have been characterized by screening them on a large panel of cell lines . The total of six contrasts with nearly 300 monoclonals reactive with intracytoplas104 mic antigens ; it corresponds to approximately 0 . 4% of all hybrids generated . The other membrane-reactive antibodies of Cote et al . 104 were all IgMs and two (Sp909, Kr73) reacted with most tumour and normal cells tested . Ev248 detected an antigen on most epithelial, but not neuroectodermally-derived tumours or normal cells, while Gr169 antigen is expressed by subsets of both epithelial and neuroectodermal cells . Collectively, therefore, the membrane-reactive antibodies ranged from broadly reactive (Sp909) to highly restricted (Ma4, Ri37) monoclonals . No panel of cell lines can, however, be representative of all normal tissues, and their definition as true tumour antigens awaits an immunocytochemical investigation of tissue distribution . Other studies confirm the difficulty of generating antibodies to tumour-associated antigens . Our own relatively limited study with breast cancer lymphocytes 54 yielded no strongly reactive monoclonals . Sikora et al."' obtained seven hybrids reacting with glioma cells from glioma lymphocytes ; these also had limited crossreactivity with a small panel of lung and colon lines . Further studies yielded over 150 hybrids from a wide variety of cancer patients, 109 with weak anti-tumour binding in 12 (8%) but with no evidence of specificity to individual tumours, although the antibodies did not bind to normal fibroblasts . Similar results were obtained by Glassy et al .' There is some evidence that melanomas may be more than usually immunogenic and monoclonals from melanoma patients have been prepared by several groups . Thus Warenius et al . 110 obtained weak binding by several monoclonals but without extensive examination of their specificity . Irie et al."' used melanoma lymphocytes to prepare two IgM,K-secreting lymphoblastoid lines (L55 and L72) which reacted with human OFA-I tumour antigens . L55 (anti-OFA-I-1) reacted with a wide variety of tumours while L72 (anti-OFA-I-2) reacted only with melanomas, gliomas and neuroblastomas (plus fetal brain) . OFA-1-2 was identified as the acidic sphingoglycolipid ganglioside GM2, and the L72 antibody has been used to develop an ELIZA assay for endogenous anti-GD2 activity . 112 This human monoclonal has been shown to suppress human melanoma growth in a xenograft model system, when injected directly into the tumour, apparently by a complement-mediated lytic effect ."',"'



44

M. J . O'Hare and C. Y. Yiu

Anti-melanoma human monoclonals cross-reacting with several other tumour types, notably colon and prostate, were prepared by Kan-Mitchell et al ."' The two antibodies, 2-139-1 (IgG1) and 6-26-3 (IgG2), respectively, reacted with cytoplasmic but not membrane antigens in all 25 melanoma samples tested . The antigen was not apparently cytoskeletal and normal tissues showed little staining although tumour binding was weak . Attempts to generate human monoclonals specific for individual carcinomas have included colon, lung, breast and prostate. Colon-reactive antibodies have been derived by two groups, Haspel et al ."' and Borup-Christensen et al .' 17 Haspel used peripheral lymphocytes from autologous tumour-immunized patients and isolated 36 colon-reacting monoclonals of which 25 (all IgMs) reacted with cellsurface antigens . Some reacted weakly with normal colon, others did not, and none was to carcinoembryonic antigen (CEA) . While other normal tissues did not react, their reactivity to other carcinomas was not documented . Unusually for tumourreactive monoclonals, antibodies that stained tumours did so relatively homogeneously, although not all tumours stained with each antibody . Fusions from nonimmunized patients were apparently unsuccessful . The IgM antibody (D4213) Borup-Christensen et al ."' binds to autologous and allogeneic colon cancer, but only weakly to normal tissue ; it did, however, cross-react with three melanoma lines tested . Cole et al.' 18 prepared lines from non-immunized lung carcinoma patients with disappointing results . A number of initially positive lines were obtained (-80/0), but most were not tumour-cell specific, reacting with lymphoblastoid cells from the same patient, and no stable reactive lines were obtained . Human anti-breast antibodies are few in number . An early example is the IgM MBE6 heterohybridoma derived by Schlom et al."' This reacted with most breast carcinomas tested but only weakly with benign breast disease, and very weakly or not at all with normal mammary epithelium . This pattern closely resembles many mouse monoclonals directed against membrane glycoproteins expressed on breast tumour cells . An IgM reactive with prostatic carcinoma (MHG7) has been described by Lowe et al . 120 which also fails to react with normal cells . It is possible that this and many of the other anti-carcinoma human monoclonals described above are reacting with carbohydrate epitopes in membrane proteins whose glycosylation is aberrant in the tumours . A human monoclonal with some specificity for leukaemias has been obtained by Olsson et al ."' from a patient with acute myeloid leukaemia . The antibody (aml18,IgG,K) bound to AML cells, promyeloid cells and weakly to ALL and erythroleukaemia cell lines but not to normal bone marrow . Forty per cent of leukaemia samples analysed were positive with heterogenous staining, including some ALLs . The aml-18 reacted with a 58 kD tumour antigen, and unlike most mouse monoclonals did not stain normal peripheral mononuclear cells . In a further study two more human antibodies, aml-19 and cml-20, were obtained, with somewhat similar autologous and allogeneic reactivities to ami-18 . 122 Both were cytotoxic IgGs ; they reacted with intracytoplasmic components in normal marrow cells but with membrane antigens only in tumour cells . Neither reacted with ALL or CLL cells but they did stain 70% of AML samples .



Human monoclonals

45

CONCLUSIONS AND FUTURE TRENDS Although many human monoclonal antibodies have not been fully characterized at present there is little evidence that they readily afford a unique set of reagents . In general the specificity of human monoclonals as they emerge is broadly matching that of existing mouse antibodies ."' This is true, for example, with anti-viral antibodies, 16 anti-bacterial monoclonals, 34, polyspecific organ-reactive antibodies, 124 anti-DNA antibodies 125 and anti-thyroid antibodies .126 It may also turn out to be the case with carcinoma-associated membrane glycoproteins, the accessible epitopes of which are often heterogeneously expressed . 127 In the context of immunotherapy human monoclonals should, nevertheless, have distinct advantages as their correct species-specific carbohydrate side chains and Fc receptor sequences should ensure a longer half-life in vivo, compared with murine monoclonals . Host immune responses are not likely to be a problem when immunotherapy is used in response to an acute life-threatening infection, intoxication or envenomation where the use of conventional serum globulin preparations may be increasingly hampered by the risk of AIDS contamination and its effect on blood donation . Even if human-specific reactivities are identified, their long-term administration in vivo can pose further problems . Many existing antibodies are IgMs, and are thus unsuited to direct administration for tumour localization, and sometimes not even for serotherapy . For example, IgM does not prevent haemolytic disease, and may even enhance sensitization to Rh antigen . The immunogenicity of human monoclonals is difficult to assess because few have as yet been administered therapeutically . Sikora et al. 128 used two antibodies for radiolocalization of glioma and bronchial carcinomas and also administered one monoclonal via a transcutaneous abdominal chamber . 129 Anti-idiotypic responses were not, however, measured . Human immune responses to multiple injections of mouse monoclonal IgG have been documented 130-132 with both general and antiidiotypic responses occurring in varying degrees even in immunocompromised patients . Anti-idiotypic responses need not, however, be invariably deleterious . Koprowski and his colleagues have reported that the best clinical effects in gastrointestinal cancers were obtained in patients in whom the most vigorous anti-mouse Ig responses were detected . 133 It was inferred that these perturbed host immune networks with the eventual result that the endogenous host anti-tumour responses were enhanced . This phenomenon was not, however, observed in the melanoma patients treated by Schroff et al."' One possible solution to the anti-idiotype response with its potential abrogation of antigen binding is treatment with different monoclonals, either sequentially, as mixtures, or possibly as bispecific antibodies ."' Thus, while patient responses to human variable region domains must be anticipated it is possible that they can be minimized in this manner, or by administration of large tolerising doses, which seems to work with mouse antibodies . 135 The potential utility of human monoclonals is not, however, restricted solely to in vivo administration . Such antibodies facilitate vaccination strategies based on antiidiotypic reagents . Anti-idiotypic antibodies as pseudoantigens may be of considerable importance as potential vaccines if appropriate quantities of viral or bacterial



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M . J . O'Hare and C . Y . Yiu

and protozoal antigens cannot be obtained . The causes of immunodeficiencies can

be probed' 16 and human antibodies with tumour-associated specificities may also be useful for the in vitro 'arming' of lymphokine-activated killer cells prior to the reinfusion of the latter in vivo . In the case of cancer results have, however, frankly been disappointing to date, with few high-affinity membrane-reactive antibodies when lymph node lymphocytes are used directly for fusion . Our own attempts with colonic antigens have yielded only about a dozen reactive antibodies from over 1500 hybridomas, a yield

which agrees with other large-scale studies on non-immunized patients . 104 In vivo immunostimulation has been used to generate antibodies to 'artificial' antigens such

as keyhole limpet haemocyanin 137 as well as tetanus toxin 138 and some success has been claimed with autologous tumour vaccination .' 16 In vitro methods have, however, proved more difficult to develop, although antibodies to prostatic acid phosphatase have been obtained by in vitro immuniza-

tion of human spleen cells . 139 Pre-selection of lymphocytes by rosetting or panning techniques is possible if the donor has a high titre of reactive antibody, as occurs

when synthetic haptens such as NNP and TNP are employed . 140,141 With less immunogenic antigens clonal expansion in vitro is desirable, although antiphosphorylcholine antibodies have been obtained directly . 142 Bone marrow lymphocytes, suppressor and helper thymus-derived lymphocytes, macrophage/monocytes and a variety of B-cell growth factors have all been found to play a role in specific in vitro immunostimulation . These procedures are, however, complicated by concurrent activation of non-specific B-cell proliferation . One solution to this problem is to use fluorescence-activated cell sorting to separate reactive lymphocytes prior to fusion, or reactive hybridomas subsequent

to fusion as has been done with mouse hybridomas . 143 Casali et a1.45 have recently used FACS to separate lymphocytes reactive to tetanus toxoid or thyroglobulin by virtue of their surface Ig, prior to immortalization by EBV . This is obviously a powerful technique, particularly if combined with in vitro immunostimulation

methods, and/or antigen-directed fusion techniques . 144 If a purified antigen is not available as a marker then anti-idiotypic antibodies might serve the same purpose, and enable the human equivalents of any murine monoclonal to be identified via the agency of a murine anti-idiotype . The chimaeric antibody is an alternative approach to monoclonals with predefined specificities based on murine reactivities within an essentially human antibody molecule . Genetic engineering techniques have been used to combine the variable region exon sequences of a mouse immunoglobulin gene with the constant regions of a human Ig gene, and transfect this recombinant gene into a mouse myeloma cell, where it is expressed and a corresponding chimaeric protein synthesized .

Details of the technology are described by Morrison 145 and more recently Jones et al."' who restricted the 'graft' solely to the hypervariable complementaritydetermining region . Such techniques have been used to transfer the variable region from murine monoclonal B6 .2 to human constant region light and heavy chain

genes . 147 The resultant chimaeric antibody recognized human carcinoma cell membranes, just like the original B6 .2 mouse monoclonal . These techniques become more attractive the less likely it seems that novel specificities will result from human B-cell immortalization . They may also be used for isotype 'switching' if appropriate classes of human antibody are not available .



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47

It seems likely, therefore, that the quest''for therapeutically efficient human or 'humanoid' monoclonal antibodies will continue . Future progress in developing human monoclonals as specific cellular and molecular probes will probably result from the use of these biological and molecular techniques for predefining the specificity of the antibodies .

ACKNOWLEDGEMENTS One of us (C .Y .Y .) is grateful to the Wellcome Trust for support ; we are also grateful to Drs C . Dean and M. G . Ormerod for helpful discussion concerning anti-idiotypic antibodies and fluorescence-activated cell sorting .

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