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Clinical trials of antibody therapy
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Clinical trials of antibody therapy Martin J. Glennie and Peter W.M. Johnson Much of the 25 years since Kohler
T
and Milstein first described making
he first indication that monoexisting mouse mAbs into mouse–human monoclonal antibodies (mAbs) has clonal antibodies (mAbs) might chimerized Ab, and humanized reagents have significant therapeutic where only the Ab complementarity-deterbeen spent trying to develop these potential came in 1982 when an mining regions (CDR) are of murine origin reagents to treat human disease. individual with lymphoma, Philip Karr, (reviewed in this issue). More recently, the Until recently, progress has been showed a complete response to brief treatproduction of fully human mAb has been ment with a ‘tailor-made’ mouse antimade routine, using either phage technology frustratingly slow and by 1994 only idiotype (anti-Id) mAb (Ref. 1). This notable or transgenic mice12. one mAb, anti-CD3 (OKT3), had This technology has had a major effect success, together with limited animal data, been licensed for clinical use. In the upon the immunogenicity of the mAb, its sparked widespread academic and comability to recruit effectors such as complemercial interest, followed by a period of past five years, however, the ment and cytotoxic cells, and its half-life very rapid investment in a plethora of new situation has changed dramatically, in the circulation. Although relatively munantibody-based biotechnology companies with numerous mAbs now showing dane in comparison with the questions of (Fig. 1; Ref. 2). It was anticipated that the specific antibody targeting and effector progress mAbs had brought to biomedical clinical potential, and a further mechanisms, the issue of repeated antibody research and clinical diagnostics would be seven approved for human dosing at high levels with limited toxicity repeated for therapeutics. Unfortunately, treatment. Furthermore, all was a vital one to resolve in the development this was not the case and subsequent trials of clinical applications. Chimeric, humanwith different types of anti-cancer mAb indications are that this upward ized and human mAbs still have the potenfailed to match the success of anti-Id. By the trend will continue, with a quarter tial to stimulate immune responses to the end of the 1980s enthusiasm for therapeutic of all new biological products variable (V)-domains of the Ab, although mAbs was waning. It was further eroded the frequency of this is usually fewer than by the pharmaceutical problems of mAbs currently undergoing clinical 12% of immune-competent patients (see as they were expensive to produce, needed development being antibody based. Clark, this issue, p. 397). The incidence of imspecialist expertise to administer, and were munogenicity is variable and depends upon often associated with considerable toxicity. Reports of severe toxicity following treatment with immunoconju- the nature of the target antigen, the disease process being treated and gates of Ab and toxins, and unfavourable results from various trials the schedule of administration. In the largest study of patients of anti-endotoxin mAbs in individuals with septic shock, com- treated with a second course of rituximab for lymphoma, none of the pounded the situation3. Confidence was further eroded in 1994 60 patients developed human antichimeric antibody (HACA)13. This when development of Campath 1H as a treatment for rheumatoid might relate to a degree of immunosuppression caused by prior arthritis (RA) was halted by rather harsh commercial judgement of chemotherapy, or by the lymphoma itself. It is also possible that its lack of efficacy, acute toxicity and immunosuppressive effect. mAb immmunogenicity can play some part in the therapeutic effect. These early disappointments notwithstanding, the past five years Certain studies with the mouse anti-colorectal cancer mAb, antihave seen a major change of fortune, with a total of eight mAbs now EpCAM (17-1A, Panorex; Centocor, Malvern, PA, USA), have sugapproved by the US food and drug administration (FDA) for clinical gested that a human anti-mouse antibody (HAMA) response might use. These include reagents against cancer4,5, transplant rejection6, a actually improve the outcome14. Other successful uses of rodent variety of immunological conditions such as RA and Crohn’s dis- mAbs, such as radioimaging, radioimmunotherapy and suppression ease7,8, antiviral prophylaxis9, and also an anti-thrombotic reagent10. of acute allograft rejection (OKT3), rely upon single use or multiple More than 70 mAbs are currently in commercial trials beyond Phase use within a short period of a week to 10 days, before the anti-antiI and Phase II (Table 1; Ref. 11). In this review we consider the factors body response develops. Studies of mouse anti-CD20 with iodinethat have led to this turnaround, and why it has taken more than 20 131 (Tositumomab; Coulter Pharmaceuticals, South San Francisco, years to achieve some clinical success. CA, USA) for the treatment of lymphoma have shown a high rate of HAMA responses when this is used early in the course of the illness. Patients who had received no prior therapy developed HAMA Human–mouse chimerization: a key step in clinical responses in 56% of cases, as against 12% of those who received the application radioconjugate for tumour recurrence after chemotherapy15. The development of genetic engineering has been central to the cliniThe other major benefit of human IgG, or rodent IgG genetically cal use of antibodies. This technology has allowed the conversion of engineered to express human constant regions, is their increased PII: S0167-5699(00)01669-8
0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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Table 1. A selection of naked mAb currently in clinical developmenta Indication
Target Ag
Antibody name
Product type
Sponsors
Trial status
Psoriasis
IL-8 CD11a ICAM-3 CD80 CD2 CD3
ABX-IL8 Anti-CD11a ICM3 IDEC-114 MEDI-507 SMART anti-CD3
Human Humanized (IgG1) Humanized Primatizedb Humanized Humanized (IgG)
Abgenix Genentech/Xoma ICOS Pharm IDEC Pharm/Mitsubishi Medimmune/BioTransplant Protein Design Lab
II I and II Preclinical I I I/II
RA
Complement (C5) TNF-a TNF-a TNF-a CD4
5G1.1 D2E7 CDP870 Infliximab IDEC-151
Humanized Human Humanized (Fab) Chimeric (IgG1) Primatizedb (IgG1)
II III II FDA approved (1999) II
CD4
MDX-CD4
Human (IgG)
Alexion Pharm CAT/BASF Celltech Centocor IDEC Pharm/SmithKline Beecham Medarex/Eisai/Genmab
Crohn’s disease
TNF-a TNF-a
Infliximab CDP571
Chimeric (IgG1) Humanized (IgG4)
Centocor Celltech
FDA approved (1998) II
Ulcerative colitis
a4b7
LDP-02
Humanized
LeukoSite/Genentech
II
Autoimmune disease
CD4
Humanized (IgG)
Ortho Biotech
II
CD3
OrthoClone OKT4A Smart anti-CD3
Humanized
Protein Design Lab
I/II
SLE
C5 CD40L CD40L Complement (C5)
5G1.1 Antova IDEC-131 5G1.1
Humanized Humanized (IgG) Humanized Humanized (IgG)
Alexion Pharm Biogen IDEC Pharm/Eisai Alexion Pharm
I II (on hold) II I/II
Multiple Sclerosis
VLA-4 CD40L
Antegren IDEC-131
Humanized (IgG) Humanized
Elan IDEC Pharm/Eisai
II II
Autoimmune haematological disorders
CD64 (FcgR)
MDX-33
Human
Medarex/Centeon
II
Asthma/Allergy
IL-5 IL-5 IL-4 IgE
SCH55700 SB-240563 SB-240683 rhuMab-E25
Humanized (IgG4) Humanized Humanized Humanized (IgG1)
II II II III
CD23
IDEC-152
Prim’ed
Celltech/Schering SmithKline Beecham SmithKline Beecham Genentech/Norvartis/ Tanox Biosystems IDEC Pharm
I
GVHD
CD147 CD2 CD2
ABX-CBL BTI-322 MEDI-507
Murine (IgM) Rat (IgG) Humanized
Abgenix Medimmune/BioTransplant Medimmune
III II I/II
Allograft rejection
CD3 CD3 CD25
Orthoclone/OKT3 SMART anti-CD3 Zenapax
Murine (IgG2a) Humanized Humanized (IgG1)
FDA approved (1986) I/II FDA approved (1997)
CD25 b2-integrin CD4 CD147 CD40L CD18
Simulect LDP-01 OKT4A ABX-CBL Antova Anti-LFA-1
Chimeric (IgG1) Humanized (IgG) Humanized (IgG) Murine (IgM) Humanized (IgG) Murine [F(ab´)2]
Ortho Biotech Protein Design Lab Protein Design Lab/ Hoffman-La Roche Novartis Pharm LeukoSite Ortho Biotech Abgenix Biogen Pasteur-Merieux/ Immunotech
Stroke
b2-integrin
LDP-01
Humanized (IgG)
LeukoSite
I/IIa
Glaucoma surgery
TGF-b2
CAT-152
Human
Cambridge Ab Tech
II
Anti-coagulant
Fact VII
Corsevin M
Chimeric
Centocor
I
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Table 1. A selection of naked mAb currently in clinical developmenta (cont.) Indication
Target Ag
Antibody name
Product type
Sponsors
Trial status
Complications of coronary angioplasty
Glycoprotein IIbIIIa receptor PDGFbR
ReoPro Abciximab CDP860
Chimeric (Fab)
Centocor/Lilly
FDA approved (1994)
Humanized F(ab´)2
Celltech
II
Myocardial infarction
CD18
anti-CD18
Humanized F(ab´)2
Genentech
II
Virus RSV
F protein
Synagis
Humanized (IgG1)
MedImmune
FDA approved (1998)
HIV
gp120
PRO542
CD4 fusion
II
Hep B
Hep B
Ostavir
Human
CMV
CMV
Protovir
Humanized (IgG1)
Progenics/ Genzyme transgenics Protein Design Lab/ Novartis Protein Design Lab/ Novartis
Toxic shock
TNF-a CD14
MAK-195 (SEGARD) Murine F(ab’)2 IC14 ?
Knoll Pharma/BASF ICOS Pharm
III I
VEGF
anti-VEGF
Humanized (IgG1)
Genentech
III
Ovarian
CA 125
OvaRex
Murine
Altarex
II/III
Colorectal
17-1A cell surface antigen
Panorex
Murine (IgG2a)
Glaxo Wellcome/ Centocor
Approved (1995) in Germany
Lung
anti-idiotypic GD3 epitope
BEC2
Murine (IgG) Merck KGaA
ImClone Sys
III
Head and Neck
EGFR
IMC-C225
Chimeric (IgG)
Imclone Sys
III
Breast
HER2/neu
Herceptin
Humanized (IgG1)
Genentech
FDA approved (1998) for metastizing tumours; Phase III for early tumours
Sarcoma
aVb3 integrin
Vitaxin
Humanized
Applied Molecular Evolution (formerly Ixsys)/ Medimmune
II
CLL
CD52
Campath1H/ (LDP-03)
Humanized (IgG1)
Leukosite
BLA
AML
CD33
Smart M195
Humanized (IgG)
Protein Design Lab/ Kanebo
III
NHL
CD20
Rituxan
Chimeric (IgG1)
FDA approved (1997)
CD22 HLA HLA DR
LymphoCide Smart 1D10 Oncolym (Lym-1)
Humanized (IgG) Humanized Radiolabelled murine
IDEC Pharm/Genentech Roche/Zenyaku Immunomedics Protein design Lab Techniclone
Cancer (general)
II (on hold) III (studies completed)
I/II I II/III
Adapted from Refs 10 and 50, and from individual company websites. Every effort has been made to check the current status of the antibodies listed in this table; however, we cannot guarantee the accuracy of these data. aAbbreviations: AML, Acute myeloid leukaemia; CD, cluster of differentiation; BLA, Biologics License Application; FDA, Food and Drug Administration; NHL, Non-Hodgkin’s lymphoma; RA, rheumatoid arthritis; IL-8, interleukin 8; ICAM, intercellular adhesion molecule; IgE, immunoglobulin E, TCR, T-cell receptor; FcgR, Fc receptors for IgG, LFA-1, leukocyte function-associated molecule 1; CMV, cytomegalovirus; Hep B, hepatitis B; EGFR, epidermal growth factor receptor; HLA, human leukocyte antigen; GVHD, graft-versus-host disease. bThese primatized mAbs contain variable regions from cynomolgus macaque (.90 % homologous with human consensus sequence) and human IgG1 constant domains10.
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cytokine, thereby providing clinical improvements in Crohn’s disease that were Multiple Abs show able to justify FDA approval. The placeboclinical efficacy Eli Lilly purchase controlled trials demonstrated improvements Hybritech ($350 m) in the severity as measured by a clinical Launch of multiple Molecular index17, endoscopic findings18 and the healAb-based companies, engineering ing of enterocutaneous fistulae19. In RA, studCancer trials First mAb (e.g., Hybritech) provides chimeric, show lack of ies of the antibody alone20 or in combination produced humanized and efficacy with methotrexate7 have demonstrated sighuman Ab Success of antinificant improvements in joint pain, inflamIdiotype in lymphoma mation and mobility when compared with placebo. 1975 1982 1986 1994 A similar blocking action underlies the use of anti-CD25 mAb for the suppression of FDA approve OKT3 Panorex (Germany) and FDA approve for acute allograft rejection ReoPro (FDA) approved for rejection following allotransplantation. As a six mAbs for (Ortho Biotech) colorectal cancer and acute treatment for rejection, the mAb is successclinical use coronary syndromes, ful, although its use in acute graft-versusrespectively host disease (GVHD) has had mixed results21. Immunology Today In almost all these applications the mAb Fig. 1. Key events and ‘landmarks’ in the therapeutic monoclonal antibody (mAb) industry. Abbre- appears to act by blocking either a key cytokine or a receptor–ligand interaction in the viation: FDA, US Food and Drug Administration. immune system (Fig. 2). A similar blocking half-life. Most rodent IgG have half lives of less that 20 h, whereas action probably occurs in most applications of mAbs to virus infecIgG with human constant regions will persist with a half-life of tions (Table 1). This is entirely in keeping with the primary function many days and sometimes closer to the 21 days measured for en- of Ab: defending the body by blocking and neutralizing the entry dogenous human IgG. Obviously, attainment of these long half lives and spread of pathogens. A key aspect of this process is that the can only occur once the antigen load in the patient has been exposure to antibody might only need to be brief in order to moderquenched by mAb. For example, in studies of anti-cancer mAbs, the ate the response, break the inflammatory cycle and allow the repair presence of bulky tumour deposits resulted in a shortening of the process to begin. In effect, mAbs are achieving the goal of ‘a longhalf-life for humanized antibodies to a figure much closer to that of term effect following a short-term treatment’ suggested by Waldmann the rodent types. However, once saturation of antigen has occurred, and colleagues25. the consequence of prolonged survival is a greatly increased biological dose of human mAb delivered to the target. We now understand how this biological survival is controlled16: the Fc regions of human Targeting IgG allow binding to specialized Fc receptors on endothelial cells, In contrast to their use in autoimmunity and immunosuppression, called FcRn or the Brambell receptors, which ensure that endocy- mAbs are required to perform a very different task when used in the tosed IgG is continually recycled back into the blood pool instead of treatment of cancer: cellular ablation. The original intent of tumour being broken down intracellularly. By contrast, rodent mAbs fail to targeting by mAbs was that when tumour-specific reagents could be bind to FcRn and are rapidly removed from the circulation into the made, they would opsonize the malignant cells and allow effectors intracellular degradation pathway. within the immune system to destroy them (Fig. 2), a prejudice that was supported by the early success of anti-Id mAbs in lymphoma. It is now less clear to what extent such Ab-effector mechanisms operMechanisms of action: blocking, targeting and ate in vivo and whether they ever have the capacity to deal with subsignalling stantial numbers of cancer cells. In the case of complement-mediated Blocking mAbs cytotoxicity for example, it is clear that regulatory proteins Most of the early clinical investigations with mAbs were conducted expressed by host cells are highly capable of protecting normal and in cancer patients, where they were used to target tumour cells and tumour tissues from Ab attack26. Likewise natural killer (NK) cells often to deliver an insult such as radiation or toxin. However, the possess a wide range of regulatory receptors that can switch off cymajority of unconjugated mAbs now in clinical development are for totoxicity if engaged by their ligand, major histocompatibility comautoimmunity and immunosuppression, where it appears that they plex (MHC) class I (Ref. 27). This is not to say that targeted cellular act by blocking or modulating responses. A good example is in effectors do not have a key role in the activity of some anti-cancer the use of anti-tumour necrosis factor a (TNF-a) mAb (Infliximab; mAbs, and the evidence from Fc receptor knockout mice suggests Centocor, Malvern, PA, USA) for treating RA and Crohn’s disease7,8. that in certain systems the binding of Fc regions is paramount for The mAb binds and blocks the action of this pro-inflammatory therapeutic activity28. Furthermore, it has recently been demonstrated Severe side effects halt multiple trials and Wellcome drops Campath 1
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C1q
Targeting in elegant experiments using human xenografts that loss of the activation Fc receptor results in reduced therapeutic effects from rituximab and trastuzumab against lymphoma and breast cancer models, respectively29. Conversely, the effects of these antibodies can be increased strikingly if the inhibitory Fc receptor, FcgRIIB, is disrupted by genetic knockout. These results are significant for two reasons: first, because they underline the central role of Fc receptorexpressing effectors in mAb-mediated tumour therapy; and second, because they indicate an important potential role for macrophages. Inhibitory FcgRIIB is expressed on macrophages but not NK cells, so that the enhanced effect in FcgRIIB2/2 mice must be attributed to cells other than NK cells. In the clinical setting, much less information is available regarding the importance of mAb targeting. Campath1H, which is currently involved in a Biological Licence Application for the treatment of chronic lymphatic leukaemia in the USA, is a potent recruiter of effector cells in vitro and is generally felt to operate via this mechanism in vivo30. Careful studies in patients have shown that the isotype of Campath 1H is crucial to its therapeutic success, and that its interaction with host effectors might well relate to the strong surface expression and the stability of the target antigen, CD52 (Ref. 31).
Signalling
Effector cell
Target cell
mAb crosslinking
Reactive cell (recognizing target cell) Blocking Soluble factor receptor Crosslinked receptor Target-molecule for Ab-attack Target-molecule for reactive cell
Signalling
mAb
Soluble factor
Signal
Fc receptor
Target-molecule ligand
Block Immunology Today
Fig. 2. Mechanisms operating with therapeutic monoclonal antibodies (mAbs). A number of potential mechanisms have been identified that allow mAbs to operate in vivo. Antibodies have traditionally been seen as glycoproteins that protect the body by blocking invasion by microbes. The current success of mAbs in the treatment of a range of diseases (Table 1) demonstrates that this blocking function is highly efficient and can operate at a number of levels. Thus, the ‘blocking’ section shows therapeutic mAbs preventing access of a growth factor, cytokine, or other soluble mediator by binding directly to the soluble factor itself or to the factor’s receptor. Similarly, mAbs can prevent cell–cell crosstalk by blocking receptor–ligand interaction. Once microbes have invaded, then Ab can target effector systems, such as complement and Fc receptor-bearing cells. The ‘targeting’ section shows activation of the classical pathway of complement (C1q) and recruitment of a cellular effector against a target cell. It is these mechanisms that have become accepted as the most likely processes that operate when tumour cells become coated with IgG mAbs. In vitro data suggest that such Abs work best when the targets are highly expressed and tend to remain at the cell surface when bound by mAbs (i.e., they do not internalize). The B-cell marker CD20 is an example of such a molecule and has proved an ideal target to treatment of B-cell lymphoma with mAbs (Refs 22,23). Finally, the ‘signalling’ section shows an alternative mechanism that might operate in vivo with certain mAbs, such as anti-Id and anti-CD20. In this situation, the mAb appears to crosslink the target molecule and deliver transmembrane signals that control cell division. It appears that crosslinking of neighbouring receptors is a prerequisite of such activity and that mAbs might become more active in this regard when hypercrosslinked by Fc receptor-bearing effector cells24.
With more successful anti-cancer mAbs, such as anti-Id, rituximab (anti-CD20) and trastuzumab (anti-HER2/neu), there is a growing feeling that while they are able to recruit effectors, they might also employ direct signalling mechanisms to achieve their cytotoxic effect4. The first clinical evidence for such activity came from anti-Id studies, which showed a strong positive correlation between the therapeutic efficacy of a patient’s anti-Id mAb and the ability of that mAb to trigger tyrosine phosphorylation of intracellular proteins32. Such results were consistent with in vitro studies showing that crosslinking of the B-cell receptor (BCR) with Ab results in growth arrest and apoptosis in both normal and neoplastic B cells. A number of mouse lymphoma models also support a role for BCR signalling33, for example BCL1 tumour cells emerging after long periods of anti-Id-induced dormancy still express surface Id, but often show
alterations in levels of vital signalling proteins such as Syk, HS-1 and Lyn (Ref. 34). The role of transmembrane signalling produced by other anticancer mAbs is less clear4. Both rituximab and trastuzumab carry human Fc regions and, in vitro at least, recruit complement and cellular effectors. However, this activity does not distinguish them from a range of mAb reagents that performed well during in vitro
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development, but then failed to perform in vivo. In the case of trastuzumab (anti-Her-2/neu), mAb binding has been shown to induce a number of signalling events that might play a role in controlling tumour growth35. Given that the antigen is a member of a family of growth factor receptors that provide important mitogenic signals, it is likely that the mAb could block an important ligand–receptor interaction normally responsible for promoting tumour growth. In the case of the CD20 target, crosslinking with various mAbs, including rituximab, induces a number of signalling events including increased protein tyrosine phosphorylation, activation of protein kinase C and upregulation of Myc (Ref. 4). In vitro, these changes can regulate B-cell growth and appear to depend upon the association of CD20 with an ill-defined factor that couples it to the Src-kinases Lyn, Fyn and Lck (Ref. 36). Although the function of CD20 is still unknown and CD20-knockout mice do not show a prominent phenotype37, it does appear to play a role in Ca21 transport as cells transfected with CD20 increase their resting levels of intracellular Ca21. A number of studies have shown that certain mAbs, including rituximab, can induce apoptosis in some B-cell lines. Interestingly, the level of such activity is increased when the mAb is crosslinked by a second layer of Ab or by Fc receptor-bearing effectors, such as macrophages24. Some of the most interesting data on CD20 signalling comes from work showing that when crosslinked by mAb, CD20 is rapidly reorganized in the lipid membrane38. CD20 might be one of many signalling molecules, including the kinases Lyn, Fyn and Lck, able to partition into the sphingolipid and cholesterolrich microdomains so important in antigen receptor signalling in lymphocytes.
Function of the Fc region of therapeutic mAbs Interestingly, most anti-cancer mAbs, at least in animal studies, do not function therapeutically when used as Fab9 or F(ab9)2 fragments. This lack of activity has been cited as evidence to support the role of whole mAbs in recruiting effectors, although an alternative explanation might be that Fc receptor interactions with normal cells increase the density of Ab crosslinking on the targets. In studies with anti-lymphoma mAbs it has been found that F(ab9)2 fragments from anti-Id and anti-CD40 mAb are not therapeutic, even when all efforts are made to compensate for their shorter half-life (M.J. Glennie and P.W.M. Johnson, unpublished). The importance of Fc receptors was most clearly seen in the ‘CD40-culture’ system developed by Banchereau and colleagues39. Here, normal B cells could be maintained in culture for extended periods with anti-CD40 and IL-4, but only if a feeder layer of CD32-expressing L cells was provided to present and crosslink the mAb. The ability of trimeric soluble CD40 ligand to produce the same effects is an indication that crosslinking rather than the presence of other cellular interactions is crucial in this process40. Thus, it is likely that the presence of Fc receptor-expressing effector cells, not as cytotoxic effectors but as providers of IgG crosslinking, could prove to be an important factor in immunotherapy and explain the lack of activity normally seen with F(ab9)2 fragments.
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How will antibody-based therapies be used in cancer treatment? Although current research in illnesses ranging from autoimmunity to infections suggests that the future of therapeutic, unconjugated, mAbs is on an upward trajectory, their application in cancer is more problematic. Despite the successes seen in clinical trials, with tumour regressions produced by rituximab and trastuzumab, it does not appear that these approaches will prove curative for advanced malignancies. Patients with low-grade B-cell lymphomas that respond to rituximab have a median time to recurrence of approximately one year22,23. The use of mAbs in the setting of low-volume residual disease, for example, as adjuvant therapy after surgery to remove a primary tumour, might be more likely to succeed in curing some patients. The trials currently underway to examine the effects of trastuzumab and the chimeric 17-1A antibody in breast and colorectal cancer, respectively, will be important in addressing this question14.
Conjugated mAbs in tumour therapy Although outside the scope of this review, an expanding field in antibody-based cancer therapy is the use of mAbs to direct selective cytotoxic agents. Radionuclides, toxins and prodrug-converting enzymes have all been conjugated to mAbs and are at various stages of development in clinical trials. The most promising results have been seen with radioimmunotherapy for lymphoma, where there appears to be synergy between the signalling induced by anti-CD20 and the delivery of beta-radiation, an effect also seen in mouse lymphoma models41. High response rates have been seen in both newlydiagnosed patients42 and those previously treated extensively with chemotherapy43,44. There is early evidence from a randomized trial to confirm the impression that this approach is superior to the use of ‘naked’ rituximab45; whether it will prove superior to naked antibody plus external-beam radiation is not known. The approaches of immunotoxin therapy and ADEPT (antibody-directed enzyme prodrug therapy) are at an earlier stage of clinical development, and the next few years will show whether these can achieve a useful therapeutic effect5,11.
Target selection for mAb therapy For any mAb-based cancer therapy, target selection appears crucial and to date most mAbs have not made useful therapeutics. If, as we believe, a direct cytotoxic action by mAb is important, we need to know more about the normal function of cellular targets and whether they are likely candidates for growth control or induction of apoptosis. Perhaps one of the most exciting prospects for the future is the development of a new class of anti-cancer mAb that operate not via a direct effect on the tumour, but as a result of agonistic interaction with key receptors on cells of the immune system (Fig. 3). In this way, they might be able to potentiate weak, ineffective, antitumour responses to a level that provides effective therapy. Such mAbs have so far only been evaluated in animal models, a situation that will change very rapidly. The most exciting reagents described include anti-CTLA4 (Ref. 46), anti-CD40 (Ref. 47), anti-CD137
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loads are greater and have evolved to escape immune detection, and where tumours might be less immunogenic.
T-cell-based response Tumour
Stimulatory mAb (e.g., a-CD137 a-CTLA4 a-BAT)
T cell
MHC I Tumour antigen
CTL
Tumour processing MHC II priming MHC I X-priming Stimulatory mAb (e.g., a-CD40 a-RANK a-Flt3)
APC
Co-stimulation (e.g., CD28–B7)
Cytokines (e.g., IL-12) Immunology Today
Fig. 3. Monoclonal Ab (mAb) in the potentiation of immune responses. Recent data show that mAbs, by crosslinking certain trigger molecules on cells of the immune system, can stimulate rapid, powerful, T-cell responses to unknown tumour antigens. Such reagents can perform as either agonists, in which case they appear to stimulate antigen presenting cells (APCs) or lymphocytes [e.g., anti-CD40 (Ref. 46) and anti-CD137 (Ref. 47)], or as antagonists that block the inhibitory signals delivered by certain receptors, such as CTLA-4 (Ref. 48). Interestingly, a number of these stimulatory targets happen to belong to the tumour necrosis factor (TNF) receptor superfamily and function at the level of the APC, for example, anti-CD40 mAb, or the responding lymphocytes, such as anti-CD137. In most cases the stimulatory mAbs act like a surrogate ligand and, at least in a functional sense, mimic the crosslinking activity of the natural ligand. The great potential of such reagents lies not only in their ability to augment existing ineffectual responses, but in their ability to leave the treated host immune to tumour rechallenge. Only time will tell if similar reagents will function in humans. Abbreviations: CTL, cytotoxic T lymphocyte; Ag, antigen. (Ref. 48) and anti-BAT (Ref. 49). With these, the mAb provides blocking or crosslinking function on cytotoxic lymphocytes or APC in such a way that anti-tumour T-cells and inflammatory cells residing in the immune system are stimulated and expand to a level capable of eradicating metastatic disease. In most cases, the mAb binds to its target on T cells or APC, and mimics the natural ligand of the target molecule. Thus, in the case of anti-CD40 mAb, it probably mimics the action of the CD40L on helper T cells and, by activating CD401 APC, promotes the efficiency with which tumour antigens are processed and presented to CD8 cytotoxic T cells47. Anti-CTLA4 works in a different way, by blocking the inhibitory effects of CTLA4–B7 interaction, it appears to alter the balance of T-cell costimulation so that the expansion-inducing effects of CD28 predominate. Regardless of the initiating mechanism, the result of treatment is the rapid and, in some cases, massive expansion of CD8 cytotoxic T cells that recognize and destroy the tumours, and leave the animals partially or completely immune against tumour rechallenge. The next step will come in seeing whether similar immune stimulation can be achieved in patients, where tumour
Concluding remarks Technologies now in place allow the production of chimeric or human reagents, with long half-lives, reduced chance of stimulating anti-antibody responses and efficient interaction with natural effectors. In the future it is likely that we will see an expansion of human mAbs, made either by phage technology or in human-Ab transgenic mice, to act as blocking reagents for a variety of immune and infectious diseases. This technology is already being licensed for a plethora of new targets. These reagents will be directed at a variety of cytokines, chemokines or their receptors involved in controlling the immune system, and at extracellular receptors and ligands that control cell–cell interactions. In the case of cancer, progress might be somewhat slower with ‘naked’ mAbs. Specificity appears to be the key issue, and selecting reagents that can modulate weak, existing, immune responses is an exciting way forward.
We thank Tenovus, Cardiff, the Cancer Research Campaign and the Leukaemia Research Fund for their support, and are indebted to colleagues for help and discussion of the manuscript.
Martin J. Glennie ([email protected]) is at the Tenovus Research Laboratory, and Peter W.M. Johnson ([email protected]) is at the CRC Medical Oncology Unit, The Cancer Sciences Division, Southampton University School of Medicine, General Hospital, Southampton, UK SO16 6YD. References 1 Miller, R.A. (1982) Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody. New Engl. J. Med. 306, 517–522 2 Dickman, S. (1998) Cancer therapy – antibodies stage a comeback in cancer treatment. Science 280, 1196–1197 3 Baumgartner, J.D. and Glauser, M.P. (1993) Immunotherapy of endotoxemia and septicemia. Immunobiology 187, 464–477 4 Cragg, M.S. et al. (1999) Signaling antibodies in cancer therapy. Curr. Opin. Immunol. 11, 541–547 5 Farah, R.A. et al. (1998) The development of monoclonal antibodies for the therapy of cancer. Crit. Rev. Eukaryotic Gene Expr. 8, 321–356 6 Berard, J.L. et al. (1999) A review of interleukin-2 receptor antagonists in solid organ transplantation. Pharmacotherapy 19, 1127–1137 7 Maini, R. et al. (1999) Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. Lancet 354, 1932–1939 8 Sandborn, W.J. and Hanauer, S.B. (1999) Antitumor necrosis factor therapy for inflammatory bowel disease: a review of agents, pharmacology, clinical results and safety. Inflamm. Bowel Dis. 5, 119–133 9 Saez-Llorens, X. et al. (1998) Safety and pharmacokinetics of an intramuscular humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. Pediatr. Infect. Dis. J. 17, 787–791 10 Juberlirer, S.J. (1999) Acute profound thrombocytopenia following C7E3 Fab (abciximab) therapy: case reports, review of the literature and implications for therapy. Am. J. Hematol. 61, 205–208
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11 Quan, M.P. and Carter, P. The rise of monoclonal antibodies as therapeutics. In Lung Biology in Health and Disease Anti-IgE and Allergic Disease (Jardieu, P.M. and Fick, R., eds), Marcel Dekker (in press) 12 Vaughan, T.J. et al. (1998) Human antibodies by design. Nat. Biotechnol. 16, 535–539 13 Davis, T.A. et al. (1999) Final report on the safety and efficacy of retreatment with rituximab for patients with non-Hodgkin’s lymphoma. Blood 94, 88a 14 Riethmuller, G. et al. (1998) Monoclonal antibody therapy for resected Dukes’ C colorectal cancer: seven-year outcome of a multicenter randomized trial. J. Clin. Oncol. 16, 1788–1794 15 Vose, J.M. et al. (1999) Iodine-131 Tositumomab for patients with follicular non-Hodgkin’s lymphoma (NHL): overall clinical trial experience by histology. Blood 94, 89a. 16 Ghetie, V. and Ward, E.S. (1997) FcRn: the MHC class I-related receptor that is more than an IgG transporter. Immunol. Today 18, 592–598 17 Targan, S.R. et al. (1997) A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease. New Engl. J. Med. 337, 1029–1035 18 D’haens, G. et al. (1999) Endoscopic and histological healing with infliximab anti-tumor necrosis factor antibodies in Crohn’s disease: a European multicenter trial. Gastroenterology 116, 1029–1034 19 Present, D.H. et al. (1999) Infliximab for the treatment of fistulas in patients with Crohn’s disease. New Engl. J. Med. 340, 1398–1405 20 Elliott, M.J. et al. (1994) Repeated therapy with monoclonal antibody to tumour necrosis factor alpha (cA2) in patients with rheumatoid arthritis. Lancet 344, 1125–1127 21 Cahn, J.Y. et al. (1995) Treatment of acute graft-versus-host disease with methylprednisolone and cyclosporine with or without an antiinterleukin-2 receptor monoclonal antibody – a multicenter Phase III study. Transplantation 60, 939–942 22 McLaughlin, P. et al. (1998) Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J. Clin. Oncol. 16, 2825–2833 23 Foran, J.M. et al. (2000) European Phase II study of rituximab (chimeric anti-CD20 monoclonal antibody) for patients with newly diagnosed mantle-cell lymphoma and previously treated mantle-cell lymphoma, immunocytoma, and small B-cell lymphocytic lymphoma. J. Clin. Oncol. 18, 317–324 24 Shan, D. et al. (1998) Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 91, 1644–1652 25 Waldmann, H. and Cobbold, S. (1998) How do monoclonal antibodies induce tolerance? A role for infectious tolerance. Annu. Rev. Immunol. 16, 619–644 26 Morgan, B.P. (1999) Regulation of the complement membrane attack pathway. Crit. Rev. Immunol. 19, 173–198 27 Renard, V. et al. (1997) Transduction of cytotoxic signals in natural killer cells: a general model of fine tuning between activatory and inhibitory pathways in lymphocytes. Immunol. Rev. 155, 205–221 28 Clynes, R. et al. (1998) Fc receptors are required in passive and active immunity to melanoma. Proc. Natl. Acad. Sci. U. S. A. 95, 652–656 29 Clynes, R.A. et al. (2000) Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443–446 30 Kennedy, B. et al. (1999) Campath-1H therapy in 29 patients with refractory CLL: ‘true’ complete remission is an attainable goal. Blood 94 (Suppl.), 603A 31 Dyer, M.J.S. et al. (1989) Effects of Campath-1 antibodies in vivo in patients with lymphoid malignancies – influence of antibody isotype. Blood 73, 1431–1439 32 Vuist, W.M.J. et al. (1994) Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumour expression of high levels of Bcl–2 protein. Blood 83, 899–906
33 Tutt, A.L. et al. (1998) Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors. J. Immunol. 161, 3176–3185 34 Vitetta, E.S. et al. (1997) Tumor dormancy and signaling (V): regrowth of the BCL1 tumor after dormancy is established. Blood 89, 4425–4436 35 Goldenberg, M.M. (1999) Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin. Ther. 21, 309–318 36 Popoff, I.J. et al. (1998) The association between CD20 and Src-family tyrosine kinases requires an additional factor. Mol. Immunol. 35, 207–214 37 O’Keefe, T.L. et al. (1998) Mice carrying a CD20 gene disruption. Immunogenetics 48, 125–132 38 Deans, J.P. et al. (1998) Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment. J. Biol. Chem. 273, 344–348 39 Banchereau, J. et al. (1994) The CD40 antigen and its ligand. Annu. Rev. Immunol. 12, 881–922 40 Johnson, P.W.M. et al. (1998) Soluble CD40 ligand induces proliferation rather than apoptosis in primary human low-grade lymphoma: implications for immunotherapy. Blood 92, 484a 41 Illidge, T.M. et al. (1999) Radioimmunotherapy of B-cell lymphoma: the importance of antibody-specificity in determining successful treatment of established disease. Blood 94, 233–243 42 Kaminski, M.S. et al. (1998) I-131 anti-B1 antibody for previously untreated follicular lymphoma (FL): clinical and molecular remissions. Proc ASCO, 17, 2a 43 Press, O.W. et al. (1995) Phase II trial of I-131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 346, 336–340 44 Kaminski, M. S. et al. (1996) Iodine-131-anti-B-1 radioimmunotherapy for B-cell lymphoma. J. Clin. Oncol. 14, 1974–1981 45 Witzig, T.E. et al. (1999) Prospective randomized controlled study of Zevalin (TM) (IDEC-Y2B8) radioimmunotherapy compared to rituximab immunotherapy for B-cell NHL: report of interim results. Blood 94, 631a 46 Leach, D.R. et al. (1996) Enhancement of antitumor immunity by CTLA-4 blockade. Science 271, 1734–1736 47 French, R.R. et al. (1999) CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat. Med. 5, 548–553 48 Melero, I. et al. (1997) Monoclonal antibodies against 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 3, 682–685 49 Hardy, B. et al. (1997) A lymphocyte-activating monoclonal antibody induces regression of human tumors in severe combined immunodeficient mice. Proc. Natl. Acad. Sci. U. S. A. 94, 5756–5760 50 Kim, J.H. and van de Broek, R.A. (1999) The Renaissance of Antibodies. (Industry Report), Hambrecht & Quist (San Francisco, CA, USA)
Comment If you would like to comment on any of the articles featured in Immunology Today, please contact our Editorial Office at the numbers below. All correspondence will be forwarded to the original author(s) who will then have an opportunity to reply. E-mail: [email protected] Fax: 144 (0)20 7611 4485 :
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Clinical trials of antibody therapy Martin J. Glennie and Peter W.M. Johnson Much of the 25 years since Kohler
T
and Milstein first described making
he first indication that monoexisting mouse mAbs into mouse–human monoclonal antibodies (mAbs) has clonal antibodies (mAbs) might chimerized Ab, and humanized reagents have significant therapeutic where only the Ab complementarity-deterbeen spent trying to develop these potential came in 1982 when an mining regions (CDR) are of murine origin reagents to treat human disease. individual with lymphoma, Philip Karr, (reviewed in this issue). More recently, the Until recently, progress has been showed a complete response to brief treatproduction of fully human mAb has been ment with a ‘tailor-made’ mouse antimade routine, using either phage technology frustratingly slow and by 1994 only idiotype (anti-Id) mAb (Ref. 1). This notable or transgenic mice12. one mAb, anti-CD3 (OKT3), had This technology has had a major effect success, together with limited animal data, been licensed for clinical use. In the upon the immunogenicity of the mAb, its sparked widespread academic and comability to recruit effectors such as complemercial interest, followed by a period of past five years, however, the ment and cytotoxic cells, and its half-life very rapid investment in a plethora of new situation has changed dramatically, in the circulation. Although relatively munantibody-based biotechnology companies with numerous mAbs now showing dane in comparison with the questions of (Fig. 1; Ref. 2). It was anticipated that the specific antibody targeting and effector progress mAbs had brought to biomedical clinical potential, and a further mechanisms, the issue of repeated antibody research and clinical diagnostics would be seven approved for human dosing at high levels with limited toxicity repeated for therapeutics. Unfortunately, treatment. Furthermore, all was a vital one to resolve in the development this was not the case and subsequent trials of clinical applications. Chimeric, humanwith different types of anti-cancer mAb indications are that this upward ized and human mAbs still have the potenfailed to match the success of anti-Id. By the trend will continue, with a quarter tial to stimulate immune responses to the end of the 1980s enthusiasm for therapeutic of all new biological products variable (V)-domains of the Ab, although mAbs was waning. It was further eroded the frequency of this is usually fewer than by the pharmaceutical problems of mAbs currently undergoing clinical 12% of immune-competent patients (see as they were expensive to produce, needed development being antibody based. Clark, this issue, p. 397). The incidence of imspecialist expertise to administer, and were munogenicity is variable and depends upon often associated with considerable toxicity. Reports of severe toxicity following treatment with immunoconju- the nature of the target antigen, the disease process being treated and gates of Ab and toxins, and unfavourable results from various trials the schedule of administration. In the largest study of patients of anti-endotoxin mAbs in individuals with septic shock, com- treated with a second course of rituximab for lymphoma, none of the pounded the situation3. Confidence was further eroded in 1994 60 patients developed human antichimeric antibody (HACA)13. This when development of Campath 1H as a treatment for rheumatoid might relate to a degree of immunosuppression caused by prior arthritis (RA) was halted by rather harsh commercial judgement of chemotherapy, or by the lymphoma itself. It is also possible that its lack of efficacy, acute toxicity and immunosuppressive effect. mAb immmunogenicity can play some part in the therapeutic effect. These early disappointments notwithstanding, the past five years Certain studies with the mouse anti-colorectal cancer mAb, antihave seen a major change of fortune, with a total of eight mAbs now EpCAM (17-1A, Panorex; Centocor, Malvern, PA, USA), have sugapproved by the US food and drug administration (FDA) for clinical gested that a human anti-mouse antibody (HAMA) response might use. These include reagents against cancer4,5, transplant rejection6, a actually improve the outcome14. Other successful uses of rodent variety of immunological conditions such as RA and Crohn’s dis- mAbs, such as radioimaging, radioimmunotherapy and suppression ease7,8, antiviral prophylaxis9, and also an anti-thrombotic reagent10. of acute allograft rejection (OKT3), rely upon single use or multiple More than 70 mAbs are currently in commercial trials beyond Phase use within a short period of a week to 10 days, before the anti-antiI and Phase II (Table 1; Ref. 11). In this review we consider the factors body response develops. Studies of mouse anti-CD20 with iodinethat have led to this turnaround, and why it has taken more than 20 131 (Tositumomab; Coulter Pharmaceuticals, South San Francisco, years to achieve some clinical success. CA, USA) for the treatment of lymphoma have shown a high rate of HAMA responses when this is used early in the course of the illness. Patients who had received no prior therapy developed HAMA Human–mouse chimerization: a key step in clinical responses in 56% of cases, as against 12% of those who received the application radioconjugate for tumour recurrence after chemotherapy15. The development of genetic engineering has been central to the cliniThe other major benefit of human IgG, or rodent IgG genetically cal use of antibodies. This technology has allowed the conversion of engineered to express human constant regions, is their increased PII: S0167-5699(00)01669-8
0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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Table 1. A selection of naked mAb currently in clinical developmenta Indication
Target Ag
Antibody name
Product type
Sponsors
Trial status
Psoriasis
IL-8 CD11a ICAM-3 CD80 CD2 CD3
ABX-IL8 Anti-CD11a ICM3 IDEC-114 MEDI-507 SMART anti-CD3
Human Humanized (IgG1) Humanized Primatizedb Humanized Humanized (IgG)
Abgenix Genentech/Xoma ICOS Pharm IDEC Pharm/Mitsubishi Medimmune/BioTransplant Protein Design Lab
II I and II Preclinical I I I/II
RA
Complement (C5) TNF-a TNF-a TNF-a CD4
5G1.1 D2E7 CDP870 Infliximab IDEC-151
Humanized Human Humanized (Fab) Chimeric (IgG1) Primatizedb (IgG1)
II III II FDA approved (1999) II
CD4
MDX-CD4
Human (IgG)
Alexion Pharm CAT/BASF Celltech Centocor IDEC Pharm/SmithKline Beecham Medarex/Eisai/Genmab
Crohn’s disease
TNF-a TNF-a
Infliximab CDP571
Chimeric (IgG1) Humanized (IgG4)
Centocor Celltech
FDA approved (1998) II
Ulcerative colitis
a4b7
LDP-02
Humanized
LeukoSite/Genentech
II
Autoimmune disease
CD4
Humanized (IgG)
Ortho Biotech
II
CD3
OrthoClone OKT4A Smart anti-CD3
Humanized
Protein Design Lab
I/II
SLE
C5 CD40L CD40L Complement (C5)
5G1.1 Antova IDEC-131 5G1.1
Humanized Humanized (IgG) Humanized Humanized (IgG)
Alexion Pharm Biogen IDEC Pharm/Eisai Alexion Pharm
I II (on hold) II I/II
Multiple Sclerosis
VLA-4 CD40L
Antegren IDEC-131
Humanized (IgG) Humanized
Elan IDEC Pharm/Eisai
II II
Autoimmune haematological disorders
CD64 (FcgR)
MDX-33
Human
Medarex/Centeon
II
Asthma/Allergy
IL-5 IL-5 IL-4 IgE
SCH55700 SB-240563 SB-240683 rhuMab-E25
Humanized (IgG4) Humanized Humanized Humanized (IgG1)
II II II III
CD23
IDEC-152
Prim’ed
Celltech/Schering SmithKline Beecham SmithKline Beecham Genentech/Norvartis/ Tanox Biosystems IDEC Pharm
I
GVHD
CD147 CD2 CD2
ABX-CBL BTI-322 MEDI-507
Murine (IgM) Rat (IgG) Humanized
Abgenix Medimmune/BioTransplant Medimmune
III II I/II
Allograft rejection
CD3 CD3 CD25
Orthoclone/OKT3 SMART anti-CD3 Zenapax
Murine (IgG2a) Humanized Humanized (IgG1)
FDA approved (1986) I/II FDA approved (1997)
CD25 b2-integrin CD4 CD147 CD40L CD18
Simulect LDP-01 OKT4A ABX-CBL Antova Anti-LFA-1
Chimeric (IgG1) Humanized (IgG) Humanized (IgG) Murine (IgM) Humanized (IgG) Murine [F(ab´)2]
Ortho Biotech Protein Design Lab Protein Design Lab/ Hoffman-La Roche Novartis Pharm LeukoSite Ortho Biotech Abgenix Biogen Pasteur-Merieux/ Immunotech
Stroke
b2-integrin
LDP-01
Humanized (IgG)
LeukoSite
I/IIa
Glaucoma surgery
TGF-b2
CAT-152
Human
Cambridge Ab Tech
II
Anti-coagulant
Fact VII
Corsevin M
Chimeric
Centocor
I
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Table 1. A selection of naked mAb currently in clinical developmenta (cont.) Indication
Target Ag
Antibody name
Product type
Sponsors
Trial status
Complications of coronary angioplasty
Glycoprotein IIbIIIa receptor PDGFbR
ReoPro Abciximab CDP860
Chimeric (Fab)
Centocor/Lilly
FDA approved (1994)
Humanized F(ab´)2
Celltech
II
Myocardial infarction
CD18
anti-CD18
Humanized F(ab´)2
Genentech
II
Virus RSV
F protein
Synagis
Humanized (IgG1)
MedImmune
FDA approved (1998)
HIV
gp120
PRO542
CD4 fusion
II
Hep B
Hep B
Ostavir
Human
CMV
CMV
Protovir
Humanized (IgG1)
Progenics/ Genzyme transgenics Protein Design Lab/ Novartis Protein Design Lab/ Novartis
Toxic shock
TNF-a CD14
MAK-195 (SEGARD) Murine F(ab’)2 IC14 ?
Knoll Pharma/BASF ICOS Pharm
III I
VEGF
anti-VEGF
Humanized (IgG1)
Genentech
III
Ovarian
CA 125
OvaRex
Murine
Altarex
II/III
Colorectal
17-1A cell surface antigen
Panorex
Murine (IgG2a)
Glaxo Wellcome/ Centocor
Approved (1995) in Germany
Lung
anti-idiotypic GD3 epitope
BEC2
Murine (IgG) Merck KGaA
ImClone Sys
III
Head and Neck
EGFR
IMC-C225
Chimeric (IgG)
Imclone Sys
III
Breast
HER2/neu
Herceptin
Humanized (IgG1)
Genentech
FDA approved (1998) for metastizing tumours; Phase III for early tumours
Sarcoma
aVb3 integrin
Vitaxin
Humanized
Applied Molecular Evolution (formerly Ixsys)/ Medimmune
II
CLL
CD52
Campath1H/ (LDP-03)
Humanized (IgG1)
Leukosite
BLA
AML
CD33
Smart M195
Humanized (IgG)
Protein Design Lab/ Kanebo
III
NHL
CD20
Rituxan
Chimeric (IgG1)
FDA approved (1997)
CD22 HLA HLA DR
LymphoCide Smart 1D10 Oncolym (Lym-1)
Humanized (IgG) Humanized Radiolabelled murine
IDEC Pharm/Genentech Roche/Zenyaku Immunomedics Protein design Lab Techniclone
Cancer (general)
II (on hold) III (studies completed)
I/II I II/III
Adapted from Refs 10 and 50, and from individual company websites. Every effort has been made to check the current status of the antibodies listed in this table; however, we cannot guarantee the accuracy of these data. aAbbreviations: AML, Acute myeloid leukaemia; CD, cluster of differentiation; BLA, Biologics License Application; FDA, Food and Drug Administration; NHL, Non-Hodgkin’s lymphoma; RA, rheumatoid arthritis; IL-8, interleukin 8; ICAM, intercellular adhesion molecule; IgE, immunoglobulin E, TCR, T-cell receptor; FcgR, Fc receptors for IgG, LFA-1, leukocyte function-associated molecule 1; CMV, cytomegalovirus; Hep B, hepatitis B; EGFR, epidermal growth factor receptor; HLA, human leukocyte antigen; GVHD, graft-versus-host disease. bThese primatized mAbs contain variable regions from cynomolgus macaque (.90 % homologous with human consensus sequence) and human IgG1 constant domains10.
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cytokine, thereby providing clinical improvements in Crohn’s disease that were Multiple Abs show able to justify FDA approval. The placeboclinical efficacy Eli Lilly purchase controlled trials demonstrated improvements Hybritech ($350 m) in the severity as measured by a clinical Launch of multiple Molecular index17, endoscopic findings18 and the healAb-based companies, engineering ing of enterocutaneous fistulae19. In RA, studCancer trials First mAb (e.g., Hybritech) provides chimeric, show lack of ies of the antibody alone20 or in combination produced humanized and efficacy with methotrexate7 have demonstrated sighuman Ab Success of antinificant improvements in joint pain, inflamIdiotype in lymphoma mation and mobility when compared with placebo. 1975 1982 1986 1994 A similar blocking action underlies the use of anti-CD25 mAb for the suppression of FDA approve OKT3 Panorex (Germany) and FDA approve for acute allograft rejection ReoPro (FDA) approved for rejection following allotransplantation. As a six mAbs for (Ortho Biotech) colorectal cancer and acute treatment for rejection, the mAb is successclinical use coronary syndromes, ful, although its use in acute graft-versusrespectively host disease (GVHD) has had mixed results21. Immunology Today In almost all these applications the mAb Fig. 1. Key events and ‘landmarks’ in the therapeutic monoclonal antibody (mAb) industry. Abbre- appears to act by blocking either a key cytokine or a receptor–ligand interaction in the viation: FDA, US Food and Drug Administration. immune system (Fig. 2). A similar blocking half-life. Most rodent IgG have half lives of less that 20 h, whereas action probably occurs in most applications of mAbs to virus infecIgG with human constant regions will persist with a half-life of tions (Table 1). This is entirely in keeping with the primary function many days and sometimes closer to the 21 days measured for en- of Ab: defending the body by blocking and neutralizing the entry dogenous human IgG. Obviously, attainment of these long half lives and spread of pathogens. A key aspect of this process is that the can only occur once the antigen load in the patient has been exposure to antibody might only need to be brief in order to moderquenched by mAb. For example, in studies of anti-cancer mAbs, the ate the response, break the inflammatory cycle and allow the repair presence of bulky tumour deposits resulted in a shortening of the process to begin. In effect, mAbs are achieving the goal of ‘a longhalf-life for humanized antibodies to a figure much closer to that of term effect following a short-term treatment’ suggested by Waldmann the rodent types. However, once saturation of antigen has occurred, and colleagues25. the consequence of prolonged survival is a greatly increased biological dose of human mAb delivered to the target. We now understand how this biological survival is controlled16: the Fc regions of human Targeting IgG allow binding to specialized Fc receptors on endothelial cells, In contrast to their use in autoimmunity and immunosuppression, called FcRn or the Brambell receptors, which ensure that endocy- mAbs are required to perform a very different task when used in the tosed IgG is continually recycled back into the blood pool instead of treatment of cancer: cellular ablation. The original intent of tumour being broken down intracellularly. By contrast, rodent mAbs fail to targeting by mAbs was that when tumour-specific reagents could be bind to FcRn and are rapidly removed from the circulation into the made, they would opsonize the malignant cells and allow effectors intracellular degradation pathway. within the immune system to destroy them (Fig. 2), a prejudice that was supported by the early success of anti-Id mAbs in lymphoma. It is now less clear to what extent such Ab-effector mechanisms operMechanisms of action: blocking, targeting and ate in vivo and whether they ever have the capacity to deal with subsignalling stantial numbers of cancer cells. In the case of complement-mediated Blocking mAbs cytotoxicity for example, it is clear that regulatory proteins Most of the early clinical investigations with mAbs were conducted expressed by host cells are highly capable of protecting normal and in cancer patients, where they were used to target tumour cells and tumour tissues from Ab attack26. Likewise natural killer (NK) cells often to deliver an insult such as radiation or toxin. However, the possess a wide range of regulatory receptors that can switch off cymajority of unconjugated mAbs now in clinical development are for totoxicity if engaged by their ligand, major histocompatibility comautoimmunity and immunosuppression, where it appears that they plex (MHC) class I (Ref. 27). This is not to say that targeted cellular act by blocking or modulating responses. A good example is in effectors do not have a key role in the activity of some anti-cancer the use of anti-tumour necrosis factor a (TNF-a) mAb (Infliximab; mAbs, and the evidence from Fc receptor knockout mice suggests Centocor, Malvern, PA, USA) for treating RA and Crohn’s disease7,8. that in certain systems the binding of Fc regions is paramount for The mAb binds and blocks the action of this pro-inflammatory therapeutic activity28. Furthermore, it has recently been demonstrated Severe side effects halt multiple trials and Wellcome drops Campath 1
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C1q
Targeting in elegant experiments using human xenografts that loss of the activation Fc receptor results in reduced therapeutic effects from rituximab and trastuzumab against lymphoma and breast cancer models, respectively29. Conversely, the effects of these antibodies can be increased strikingly if the inhibitory Fc receptor, FcgRIIB, is disrupted by genetic knockout. These results are significant for two reasons: first, because they underline the central role of Fc receptorexpressing effectors in mAb-mediated tumour therapy; and second, because they indicate an important potential role for macrophages. Inhibitory FcgRIIB is expressed on macrophages but not NK cells, so that the enhanced effect in FcgRIIB2/2 mice must be attributed to cells other than NK cells. In the clinical setting, much less information is available regarding the importance of mAb targeting. Campath1H, which is currently involved in a Biological Licence Application for the treatment of chronic lymphatic leukaemia in the USA, is a potent recruiter of effector cells in vitro and is generally felt to operate via this mechanism in vivo30. Careful studies in patients have shown that the isotype of Campath 1H is crucial to its therapeutic success, and that its interaction with host effectors might well relate to the strong surface expression and the stability of the target antigen, CD52 (Ref. 31).
Signalling
Effector cell
Target cell
mAb crosslinking
Reactive cell (recognizing target cell) Blocking Soluble factor receptor Crosslinked receptor Target-molecule for Ab-attack Target-molecule for reactive cell
Signalling
mAb
Soluble factor
Signal
Fc receptor
Target-molecule ligand
Block Immunology Today
Fig. 2. Mechanisms operating with therapeutic monoclonal antibodies (mAbs). A number of potential mechanisms have been identified that allow mAbs to operate in vivo. Antibodies have traditionally been seen as glycoproteins that protect the body by blocking invasion by microbes. The current success of mAbs in the treatment of a range of diseases (Table 1) demonstrates that this blocking function is highly efficient and can operate at a number of levels. Thus, the ‘blocking’ section shows therapeutic mAbs preventing access of a growth factor, cytokine, or other soluble mediator by binding directly to the soluble factor itself or to the factor’s receptor. Similarly, mAbs can prevent cell–cell crosstalk by blocking receptor–ligand interaction. Once microbes have invaded, then Ab can target effector systems, such as complement and Fc receptor-bearing cells. The ‘targeting’ section shows activation of the classical pathway of complement (C1q) and recruitment of a cellular effector against a target cell. It is these mechanisms that have become accepted as the most likely processes that operate when tumour cells become coated with IgG mAbs. In vitro data suggest that such Abs work best when the targets are highly expressed and tend to remain at the cell surface when bound by mAbs (i.e., they do not internalize). The B-cell marker CD20 is an example of such a molecule and has proved an ideal target to treatment of B-cell lymphoma with mAbs (Refs 22,23). Finally, the ‘signalling’ section shows an alternative mechanism that might operate in vivo with certain mAbs, such as anti-Id and anti-CD20. In this situation, the mAb appears to crosslink the target molecule and deliver transmembrane signals that control cell division. It appears that crosslinking of neighbouring receptors is a prerequisite of such activity and that mAbs might become more active in this regard when hypercrosslinked by Fc receptor-bearing effector cells24.
With more successful anti-cancer mAbs, such as anti-Id, rituximab (anti-CD20) and trastuzumab (anti-HER2/neu), there is a growing feeling that while they are able to recruit effectors, they might also employ direct signalling mechanisms to achieve their cytotoxic effect4. The first clinical evidence for such activity came from anti-Id studies, which showed a strong positive correlation between the therapeutic efficacy of a patient’s anti-Id mAb and the ability of that mAb to trigger tyrosine phosphorylation of intracellular proteins32. Such results were consistent with in vitro studies showing that crosslinking of the B-cell receptor (BCR) with Ab results in growth arrest and apoptosis in both normal and neoplastic B cells. A number of mouse lymphoma models also support a role for BCR signalling33, for example BCL1 tumour cells emerging after long periods of anti-Id-induced dormancy still express surface Id, but often show
alterations in levels of vital signalling proteins such as Syk, HS-1 and Lyn (Ref. 34). The role of transmembrane signalling produced by other anticancer mAbs is less clear4. Both rituximab and trastuzumab carry human Fc regions and, in vitro at least, recruit complement and cellular effectors. However, this activity does not distinguish them from a range of mAb reagents that performed well during in vitro
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development, but then failed to perform in vivo. In the case of trastuzumab (anti-Her-2/neu), mAb binding has been shown to induce a number of signalling events that might play a role in controlling tumour growth35. Given that the antigen is a member of a family of growth factor receptors that provide important mitogenic signals, it is likely that the mAb could block an important ligand–receptor interaction normally responsible for promoting tumour growth. In the case of the CD20 target, crosslinking with various mAbs, including rituximab, induces a number of signalling events including increased protein tyrosine phosphorylation, activation of protein kinase C and upregulation of Myc (Ref. 4). In vitro, these changes can regulate B-cell growth and appear to depend upon the association of CD20 with an ill-defined factor that couples it to the Src-kinases Lyn, Fyn and Lck (Ref. 36). Although the function of CD20 is still unknown and CD20-knockout mice do not show a prominent phenotype37, it does appear to play a role in Ca21 transport as cells transfected with CD20 increase their resting levels of intracellular Ca21. A number of studies have shown that certain mAbs, including rituximab, can induce apoptosis in some B-cell lines. Interestingly, the level of such activity is increased when the mAb is crosslinked by a second layer of Ab or by Fc receptor-bearing effectors, such as macrophages24. Some of the most interesting data on CD20 signalling comes from work showing that when crosslinked by mAb, CD20 is rapidly reorganized in the lipid membrane38. CD20 might be one of many signalling molecules, including the kinases Lyn, Fyn and Lck, able to partition into the sphingolipid and cholesterolrich microdomains so important in antigen receptor signalling in lymphocytes.
Function of the Fc region of therapeutic mAbs Interestingly, most anti-cancer mAbs, at least in animal studies, do not function therapeutically when used as Fab9 or F(ab9)2 fragments. This lack of activity has been cited as evidence to support the role of whole mAbs in recruiting effectors, although an alternative explanation might be that Fc receptor interactions with normal cells increase the density of Ab crosslinking on the targets. In studies with anti-lymphoma mAbs it has been found that F(ab9)2 fragments from anti-Id and anti-CD40 mAb are not therapeutic, even when all efforts are made to compensate for their shorter half-life (M.J. Glennie and P.W.M. Johnson, unpublished). The importance of Fc receptors was most clearly seen in the ‘CD40-culture’ system developed by Banchereau and colleagues39. Here, normal B cells could be maintained in culture for extended periods with anti-CD40 and IL-4, but only if a feeder layer of CD32-expressing L cells was provided to present and crosslink the mAb. The ability of trimeric soluble CD40 ligand to produce the same effects is an indication that crosslinking rather than the presence of other cellular interactions is crucial in this process40. Thus, it is likely that the presence of Fc receptor-expressing effector cells, not as cytotoxic effectors but as providers of IgG crosslinking, could prove to be an important factor in immunotherapy and explain the lack of activity normally seen with F(ab9)2 fragments.
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How will antibody-based therapies be used in cancer treatment? Although current research in illnesses ranging from autoimmunity to infections suggests that the future of therapeutic, unconjugated, mAbs is on an upward trajectory, their application in cancer is more problematic. Despite the successes seen in clinical trials, with tumour regressions produced by rituximab and trastuzumab, it does not appear that these approaches will prove curative for advanced malignancies. Patients with low-grade B-cell lymphomas that respond to rituximab have a median time to recurrence of approximately one year22,23. The use of mAbs in the setting of low-volume residual disease, for example, as adjuvant therapy after surgery to remove a primary tumour, might be more likely to succeed in curing some patients. The trials currently underway to examine the effects of trastuzumab and the chimeric 17-1A antibody in breast and colorectal cancer, respectively, will be important in addressing this question14.
Conjugated mAbs in tumour therapy Although outside the scope of this review, an expanding field in antibody-based cancer therapy is the use of mAbs to direct selective cytotoxic agents. Radionuclides, toxins and prodrug-converting enzymes have all been conjugated to mAbs and are at various stages of development in clinical trials. The most promising results have been seen with radioimmunotherapy for lymphoma, where there appears to be synergy between the signalling induced by anti-CD20 and the delivery of beta-radiation, an effect also seen in mouse lymphoma models41. High response rates have been seen in both newlydiagnosed patients42 and those previously treated extensively with chemotherapy43,44. There is early evidence from a randomized trial to confirm the impression that this approach is superior to the use of ‘naked’ rituximab45; whether it will prove superior to naked antibody plus external-beam radiation is not known. The approaches of immunotoxin therapy and ADEPT (antibody-directed enzyme prodrug therapy) are at an earlier stage of clinical development, and the next few years will show whether these can achieve a useful therapeutic effect5,11.
Target selection for mAb therapy For any mAb-based cancer therapy, target selection appears crucial and to date most mAbs have not made useful therapeutics. If, as we believe, a direct cytotoxic action by mAb is important, we need to know more about the normal function of cellular targets and whether they are likely candidates for growth control or induction of apoptosis. Perhaps one of the most exciting prospects for the future is the development of a new class of anti-cancer mAb that operate not via a direct effect on the tumour, but as a result of agonistic interaction with key receptors on cells of the immune system (Fig. 3). In this way, they might be able to potentiate weak, ineffective, antitumour responses to a level that provides effective therapy. Such mAbs have so far only been evaluated in animal models, a situation that will change very rapidly. The most exciting reagents described include anti-CTLA4 (Ref. 46), anti-CD40 (Ref. 47), anti-CD137
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loads are greater and have evolved to escape immune detection, and where tumours might be less immunogenic.
T-cell-based response Tumour
Stimulatory mAb (e.g., a-CD137 a-CTLA4 a-BAT)
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CTL
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Co-stimulation (e.g., CD28–B7)
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Fig. 3. Monoclonal Ab (mAb) in the potentiation of immune responses. Recent data show that mAbs, by crosslinking certain trigger molecules on cells of the immune system, can stimulate rapid, powerful, T-cell responses to unknown tumour antigens. Such reagents can perform as either agonists, in which case they appear to stimulate antigen presenting cells (APCs) or lymphocytes [e.g., anti-CD40 (Ref. 46) and anti-CD137 (Ref. 47)], or as antagonists that block the inhibitory signals delivered by certain receptors, such as CTLA-4 (Ref. 48). Interestingly, a number of these stimulatory targets happen to belong to the tumour necrosis factor (TNF) receptor superfamily and function at the level of the APC, for example, anti-CD40 mAb, or the responding lymphocytes, such as anti-CD137. In most cases the stimulatory mAbs act like a surrogate ligand and, at least in a functional sense, mimic the crosslinking activity of the natural ligand. The great potential of such reagents lies not only in their ability to augment existing ineffectual responses, but in their ability to leave the treated host immune to tumour rechallenge. Only time will tell if similar reagents will function in humans. Abbreviations: CTL, cytotoxic T lymphocyte; Ag, antigen. (Ref. 48) and anti-BAT (Ref. 49). With these, the mAb provides blocking or crosslinking function on cytotoxic lymphocytes or APC in such a way that anti-tumour T-cells and inflammatory cells residing in the immune system are stimulated and expand to a level capable of eradicating metastatic disease. In most cases, the mAb binds to its target on T cells or APC, and mimics the natural ligand of the target molecule. Thus, in the case of anti-CD40 mAb, it probably mimics the action of the CD40L on helper T cells and, by activating CD401 APC, promotes the efficiency with which tumour antigens are processed and presented to CD8 cytotoxic T cells47. Anti-CTLA4 works in a different way, by blocking the inhibitory effects of CTLA4–B7 interaction, it appears to alter the balance of T-cell costimulation so that the expansion-inducing effects of CD28 predominate. Regardless of the initiating mechanism, the result of treatment is the rapid and, in some cases, massive expansion of CD8 cytotoxic T cells that recognize and destroy the tumours, and leave the animals partially or completely immune against tumour rechallenge. The next step will come in seeing whether similar immune stimulation can be achieved in patients, where tumour
Concluding remarks Technologies now in place allow the production of chimeric or human reagents, with long half-lives, reduced chance of stimulating anti-antibody responses and efficient interaction with natural effectors. In the future it is likely that we will see an expansion of human mAbs, made either by phage technology or in human-Ab transgenic mice, to act as blocking reagents for a variety of immune and infectious diseases. This technology is already being licensed for a plethora of new targets. These reagents will be directed at a variety of cytokines, chemokines or their receptors involved in controlling the immune system, and at extracellular receptors and ligands that control cell–cell interactions. In the case of cancer, progress might be somewhat slower with ‘naked’ mAbs. Specificity appears to be the key issue, and selecting reagents that can modulate weak, existing, immune responses is an exciting way forward.
We thank Tenovus, Cardiff, the Cancer Research Campaign and the Leukaemia Research Fund for their support, and are indebted to colleagues for help and discussion of the manuscript.
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