Relevance, advantages and limitations of animal models used in the development of monoclonal antibodies for cancer treatment

Critical Reviews in Oncology/Hematology 62 (2007) 34–42

Relevance, advantages and limitations of animal models used in the development of monoclonal antibodies for cancer treatment Severine Loisel a , Marc Ohresser b , Marc Pallardy c , David Dayd´e b , Christian Berthou a , Guillaume Cartron b , Herv´e Watier b,d,∗ a

Universit´e de Bretagne Occidentale, Laboratoire de Th´erapie Cellulaire et d’Immunobiologie du Cancer, EA 2216, 29609 Brest Cedex, France b Universit´ e Francois-Rabelais de Tours, EA 3853 IPGA (Immuno-Pharmaco-Genetics of Therapeutic Antibodies), France c Universit´ e Paris-Sud 11, INSERM UMR 749, Facult´e de Pharmacie, Rue JB Cl´ement, 92290 Chˆatenay-Malabry, France d CHRU de Tours, France Accepted 24 November 2006

Contents 1. 2.

3. 4. 5.

6.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression of the target antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Antigen cross-reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Rodent models with surrogate antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Targeting human antigens in murine models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Antigen expression and use of primate models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunogenicity of therapeutic antibodies in animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacokinetics and biodistribution of therapeutic antibodies in animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacodynamics of therapeutic antibodies in animals: interaction with the host immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Interaction with host complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Interaction with Fc␥R-expressing host effector cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 35 35 36 36 37 37 38 38 38 38 40 40 40 40 42

Abstract Antibody humanisation through recombinant DNA technology was a key step in allowing monoclonal antibodies (mAbs) to reach the clinic, particularly for the treatment of cancer. As a consequence, they are less adapted to animal studies, although these studies continue to be important tools to study antibody distribution and action at the level of a whole organism. Moreover, preclinical studies in animals are mandatory before the approval of biologics license applications for mAbs by the U.S. Food and Drug Administration (FDA) or European Agency for the Evaluation of Medicinal Products (EMEA). Different parameters should be taken in consideration before starting animal experiments with recombinant mAbs, including antibody cross-reactivity, immunogenicity, pharmacokinetics, and possible interactions with the host immune system. The various interspecies differences are reviewed and discussed in light of the pharmacological properties expected in patients. In doing so, this article aims to provide a critical review of the animal models used in preclinical studies of mAbs for cancer treatment. In particular, their relevance, advantages and limitations will be discussed. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Monoclonal antibodies; Preclinical studies; Animal models; Cross-reactivity; Toxicity; Immunogenicity; Pharmacokinetics; Pharmacodynamics; Fc␥R ∗ Corresponding author at: Universit´ e Francois-Rabelais de Tours, EA 3853 IPGA (Immuno-Pharmaco-Genetics of Therapeutic Antibodies), Facult´e de M´edecine, 10 BoulevardTonnell´e, 37032 Tours Cedex, France. Tel.: +33 247 47 38 74. E-mail addresses: [email protected] (S. Loisel), [email protected] (H. Watier).

1040-8428/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2006.11.010

S. Loisel et al. / Critical Reviews in Oncology/Hematology 62 (2007) 34–42

1. Introduction Recombinant monoclonal antibodies (mAbs) have renewed biological research and clinical practice in oncology, illustrating nicely the concept of targeted therapies. Thirty years ago, the development of murine mAbs for cancer treatment was already based on the principles of a “targeted therapy” because mAbs are highly specific for epitopes on their target antigens. However, naked murine mAbs did not have clinical success because of their immunogenicity, their poor half-life in humans, and their low ability to recruit human immune effector mechanisms (i.e. complement and cells bearing receptors for the Fc portion of IgG, Fc␥R). These drawbacks were circumvented through recombinant DNA technology, by combining antibody variable domains (murine, humanised or human) involved in antigen recognition to human antibody constant regions, usually ␬ and ␥1, in order to obtain chimaeric, humanised or fully human IgG1, respectively. In contrast to mAbs with murine Fc regions, recombinant mAbs with human Fc regions are less immunogenic in humans and behave like human antibodies in terms of pharmacokinetics and pharmacodynamics. Monoclonal antibody treatment is therefore a unique type of targeted therapy because it can possibly combine the recruitment of immune effectors (Fc-based functions) to molecular/antigen targeting (Fab functions). Because antibody humanisation is the key for clinical success, these antibodies are less adapted for use in animals; however, animal studies always constitute important steps in preclinical development: determining the proof of concept, toxicity studies, evaluation of pharmacokinetics, and determination of antibody distribution at the level of a whole organism. For toxicity and pharmacokinetic studies, conventional animal models commonly found in the field of regulatory toxicology are used, but pharmacological activity of the product to be tested has to be first demonstrated in these animal species. This approach has to be applied regardless of the intended indication of the antibody (e.g. cancer, inflammatory diseases, etc.). Preclinical notes are available to provide guidance and a basic framework for the safety testing of mAbs. Within the U.S. Food and Drug Administration (FDA), the Center for Biologics Evaluation and Research (CBER) provides, and regularly updates, “points to consider (PTC) in the manufacture and testing of monoclonal antibody products for human use” (http://www.fda.gov/cber/gdlns/ptc mab.pdf). To promote international harmonisation of regulatory requirements, the European Agency for the Evaluation of Medicinal Products (EMEA), Japan, and the United States organised the International Conference on Harmonisation of Technical Requirement for Registration of Pharmaceuticals for Human Use (ICH), which led to the publication of a note concerning preclinical safety evaluation of biotechnology-derived pharmaceuticals, the ICH S6 guideline “Note for guidance on preclinical safety evaluation of Biotechnology-derived pharmaceuti-

35

cals” (http://www.emea.eu./pdfs/human/ich/030295en.pdf). These guidelines address some of the issues inherent to mAbs: antigenic specificity, pharmacokinetics, pharmacodynamics and toxicological studies in relevant animal species. Obviously, none of the available animal models are able to fully reproduce the situation in patients. Before conducting preclinical studies in animal models, four questions need to be answered: (1) Does the antibody directed to a human target recognise the animal antigen, and on which type of cells? Consequently, is it necessary to “implant” the human antigen/target cell in the chosen animal model? (2) Does the antibody raise an immunogenic response in the animal model, and if so, how will the induced antibodies affect the results? (3) Can the antibody half-life and the biodistribution be extrapolated to the human situation, and what are the identified causes of possible differences (e.g. soluble receptors, FcRn, etc.)? (4) How does the antibody interfere with the animal immune system (e.g. ADCC, complement activation, targeting of NK cells)? Obviously, these questions are not specific to mAbs used in oncology, and are relevant to all antibodies developed for any clinical use. Therefore, examples cited in this review will be predominantly drawn from cancer studies, but in some cases from other fields of mAb development. However, two particular features are specific to anti-cancer mAbs that target membrane antigens: (1) the possible use of tumour xenografts implanted into immunodeficient animals and (2) the importance of cytolytic mechanisms that are recruited by the mAbs once bound to their target.

2. Expression of the target antigen 2.1. Antigen cross-reactivity “Cross-reactivity” is the ability of an antibody to recognise an antigen in a species different from the one where the antigen was isolated. It is extremely difficult to establish rules concerning mAb cross-reactivity. If the antigen is a glycan, identical biochemical structures can be found even in very distant species, and the antibody can easily crossreact with the antigen in animals. However, the glycan can be coupled to different proteins or lipids, or can be expressed on different cell types or organs. If the antigen is a protein or a glycoprotein, which is the common situation, crossreactivity usually decreases with phylogenetic distance; the more distant the divergence between species, the higher the dissimilarity between orthologous genes. Linear or conformational epitopes on target antigens are easily lost as soon as one amino acid residue is different. Generally, based on the experience with leukocyte antigens, human epitopes are well conserved in apes (e.g. chimpanzees), usually conserved

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S. Loisel et al. / Critical Reviews in Oncology/Hematology 62 (2007) 34–42

Table 1 Cross-reactivity of approved mAbs against cancer in primates and rodents Antibody

Rituxan/MabThera Campath Herceptin Erbitux Avastin

Target antigen

CD20 CD52 erbB2 erbB1 VEGF

Cross-reactivity with

References

Apes

Old world monkeys

New world monkeys

Rodents

+ + ND ND ND

+ ± + + −

ND − ND ND ±

− − − − −

in old world monkeys (e.g. macaques, baboons, etc.), sometimes conserved in New World monkeys (e.g. marmosets) and not conserved in rodents (Table 1). Even if the epitope is preserved in the species under consideration, the expression of the protein could differ from the human situation. It is therefore advisable to consider each mAb as a particular case and to establish cross-reactivity before initiating any experiment. The most common approach to assess antibody cross-reactivity is immunohistochemistry or immunofluorescence using animal and human tissues to allow comparison of the results. However, as suggested by the FDA, appropriate newer technologies should be employed as they become available and validated. In the future, antibody profiling on protein microarrays could be one of these tools [1]. 2.2. Rodent models with surrogate antibodies Because therapeutic mAbs do not always cross-react with animal tissues, experiments in animal models such as rodents could be performed using a surrogate antibody, i.e. a mAb directed against the rodent antigen. Several “surrogate” mAbs recognising mouse antigens have been developed against murine CD20 [10] or murine EGF-R [11], which can be considered as surrogates of rituximab and cetuximab, respectively. In the case of an anti-CD11a developed for immunomodulation, the rat mAb recognising the murine antigen has even been chimaerised as a rat/mouse mAb to get closer the murine system [12]. Alternatively, murine mAbs directed to murine CD20 have been developed by immunising mice deficient in this antigen [10]. The main problem with surrogate antibody models is that the compound studied differs from the one being developed clinically with regard to production process and pharmacology. Consequently, extrapolation of the results to the human situation is not simple. In theory, conducting appropriate and extensive studies to define the pharmacology of the analogous protein in great detail and comparing it to the product in development could reduce the impact of this disadvantage. Indeed, in some cases, surrogate models have shown good correlation with the clinic and provided hypotheses concerning the mechanism of action in humans. In mice, CD20 mAbs rapidly deplete circulating and tissue B cells in an antibody subclass-restricted manner with a hierarchy of efficacy (IgG2a > IgG1 > IgG2b  IgG3) in relation to their ability to recruit distinct immune effector molecules [13]. However, developing a strictly comparable antibody

[2,3] [4–6] [7] [8] [9]

remains highly difficult to achieve. For example, a major part of the super-agonistic anti-CD28 mAb (TGN1412) preclinical development was based on a rat mAb recognising a similar epitope on murine CD28 and displaying similar super-agonistic properties in vitro [14]. However, neither this surrogate mAb, nor the drug itself used in macaques was able to predict the dramatic cytokine storm which occurred in March 2006 in six human volunteers [15]. Even with a surrogate antibody behaving similarly, inter-species differences are frequently found in the respective biological functions of the target antigen in animals and humans or in its tissue distribution. For toxicology studies, the use of surrogate models for predicting safety in humans has been performed for antibodies that are active only in humans or in chimpanzees. To date, analogous antibodies have been used for the safety assessment of two approved products not developed for cancer treatment: infliximab (anti-TNF-␣) and efalizumab (anti-LFA-1). 2.3. Targeting human antigens in murine models An alternative to surrogate antibodies that allows the study of the drug itself is to “transplant” the human antigen in the animal, either in the form of human cells in immunodeficient animals (xenogeneic models) or after gene manipulation (syngeneic and transgenic models) Immunodeficient mice are useful as hosts for propagation of xenogeneic malignant cells and tissues as they do not reject human tumours. The hairless nude mouse mutants (nu/nu homozygotes) and SCID (severe combined immunodeficiency) mice are commonly used, although subtle differences can be observed between these models (see below). Xenografts could be derived from either cell lines or patient biopsies. In respect to antigen expression, cell lines are more homogenous and could be better adapted to control experimental conditions. On the other hand, tissues derived from the patient tumour better reflect the clinical situation. Subcutaneous implantation is the most common method for xenografting, but this model has advantages and limitations. This volumetric model remains of great value, both for efficiency and pharmacodynamic studies of mAbs. In contrast, the drawbacks of the subcutaneous model for mAb studies are that tumours are characterised by a lack of invasion and metastasis, and that mAb efficiency depends upon recruitment of the host’s immune system to induce antibody-dependent cell-mediated cytotoxicity (ADCC). As

S. Loisel et al. / Critical Reviews in Oncology/Hematology 62 (2007) 34–42

an alternative to xenografts, syngeneic murine models can also be used once expression of the desired human antigen has been achieved through transfection or transduction. For example, the anti-CD20 mAb rituximab was shown to cure immunocompetent mice from murine lymphomas stably expressing human CD20 [16,17]. Transgenic animals expressing human antigens have been also developed to evaluate the efficacy of novel therapeutic strategies. Several mice transgenic for the human carcinoembryonic antigen (CEA) have been generated [18,19]; they express CEA accurately, in a cell- and tissue-specific manner in spontaneous or induced tumours [20–23]. These models proved to be valuable to the study of anti-CEA recombinant mAbs. 2.4. Antigen expression and use of primate models Even in the case of cross-reactivity in marmosets, macaques or baboons, there are many limitations for the design of relevant experiments in non-human primates. Tumours cannot be studied, and only normal tissues can be targeted. Primate models are usually used for pharmacokinetic and toxicity studies. The toxicity studies are particularly relevant when “tumour” antigens are also expressed in others tissues, which is actually a common situation, leading to the necessity of evaluating the impact of mAb treatment on normal tissues. However, toxicity studies in primates are not always predictive of what will occur in human trials. Toxicological studies conducted in cynomolgus monkeys with bevacizumab did not show any systemic toxicity, suggesting a lack of side-effects on the quiescent vasculature, but revealed abnormalities at the level of the growth plate and ovary, two tissues characterised by active angiogenesis [9]. Safety data from clinical trials of bevacizumab are now available and indicate that serious adverse reactions, including hypertension, bleeding episodes, and thrombotic events, are sometimes observed, although these were not predicted by the primate models [24]. Cardiotoxicity was another unanticipated toxic complication observed in patients treated with the anti-erbB2 trastuzumab, especially when it was combined with anthracyclines [25,26]. Besides toxicity studies, monkeys can sometimes be used to evaluate efficacy when a target cell is easily analysable. This is particularly true for anti-leukocyte antibodies, especially the anti-CD20 mAbs which recognise (and sometimes kill) normal B cells in blood and secondary lymphoid organs [27]. B lymphocyte depletion in this model was considered to be a good marker of rituximab efficacy, and the macaque model is now an obligatory step in the development of other anti-CD20 antibodies. As with the CD20 antigen, the antiCD52 mAb alemtuzumab recognises the CD52 orthologs in old world monkeys such as baboons and macaques [28]. However, erythrocytes in baboons and rhesus monkeys also express CD52 antigen. Due to the risk of vascular haemolysis, these species were excluded. In fact, the cynomolgus monkey was the only possible non-human primate model for

37

alemtuzumab, and only after exclusion of individuals whose red blood cells expressed CD52. Finally, the recent accident with the anti-CD28 superagonist inducing a cytokine release syndrome in human volunteers is a dramatic example of an inappropriate extrapolation of results obtained in monkeys to the human situation [15].

3. Immunogenicity of therapeutic antibodies in animals The high degree of humanisation of recombinant mAbs has rendered these drugs immunogenic in animals, including primates, because of the divergence of IgG protein sequences. Immunogenicity can also be viewed as being proportional to the phylogenetic distance. For example, humanised antibodies are more immunogenic in macaques than in chimpanzees [29]. This is also true in rodents, with rat IgGs being immunogenic in mice [30]. Consequently, human IgGs are particularly immunogenic in rodents as evidenced in the early 1980s when mice were immunised to produce mAbs directed against different portions and determinants of the human IgG [31]. The Fc regions of human IgG, always present in recombinant mAbs, are known to be particularly immunogenic [32–34]. It is also well known that human monomeric IgGs can be tolerogenic in mice unless they are complexed to their antigen [35], which is the natural goal of a targeted therapy. Immunogenicity of humanised mAbs in mice is only a problem in immunocompetent animals. SCID mice, which are devoid of B and T cells, are totally unable to develop an immune response to heterologous proteins. Nude mice (athymic) are only deficient in conventional T cells, but are also unable to develop antibodies to human IgG since it is a T cell-dependent antigen [36]. As such, immune responses against a therapeutic IgG in immunocompetent animals (rodents or primates) will occur with some delay, and should not interfere with very short-term experiments. The risk of developing antibodies to the drug increases with repeated administrations. The induced antibodies probably behave in vivo like human anti-murine (HAMA) and even human anti-chimaeric antibodies (HACA), i.e. they could neutralise the pharmacological activity of the therapeutic mAb and also accelerate its elimination, thereby shortening its in vivo half-life [37]. All of these phenomena result in a loss of efficacy, which needs to be recognised by performing adequate measurements. Animal models are not suitable for predicting the immunogenicity of therapeutic mAbs in humans, and transposition of the immunogenic potential of therapeutic antibodies in animals to the human situation has no scientific rationale, even in primates. In the field of antibodies for oncolytic indications, the percentage of patients with immune responses to humanised/human antibodies ranged from less than 1 to 12% (one human mAb derived from phage display), whereas most of these antibodies were clearly immunogenic in animals [38].

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4. Pharmacokinetics and biodistribution of therapeutic antibodies in animals Conducting pharmacokinetic studies in animals before administration in humans is required for dose calculation and dose escalation, and also to evaluate the animal exposure using area under the curve (AUC) and the maximum concentration (Cmax ). Again, extrapolation to the human situation requires a careful evaluation that takes into account the immunogenicity of the antibody in animals and the lack of soluble target that can be secreted by human tumours. Despite these difficulties, the measurements of Cmax and AUCs in animals and in humans are pivotal for a better extrapolation of the results from animal to man. How pharmacokinetic parameters could be used to define a safe starting dose in humans? For chemical products, but also for inhibitory therapeutic antibodies, the conventional approach use either the no observed adverse effect level (NOAEL) which is the dose where no toxic effects are observed or, the NOEL (no observed effect level) which refers to any effect based on clinical symptoms, clinical chemistry or histopathological examinations. Generally, a safe starting dose is defined on the NOAEL or the NOEL and, in order to provide a margin of safety for protection of human subjects receiving the initial clinical dose, a safety factor is then applied. However, in light of the recent dramatic TGN1412 phase I clinical trial, it appeared that the straightforward approach outlined above is not suitable for determining a safe starting dose in humans. A new concept has emerged that could be applied to agonistic antibodies such as TGN1412 based on the pharmacological activity of the antibody to be tested. This approach, termed minimal anticipated biological effect level (MABEL), uses the dose provoking a pharmacological effect in a responsive species, instead of the no observed adverse effect level (NOAEL) observed in toxicological studies for calculation of the initial dose in humans. Other parameters could also play a role. FcRn, a MHC class I-related Fc receptor, is responsible for the maintenance of serum IgG levels by binding them at acidic pH in endothelial endosomal compartments, protecting them from lysosomal degradation and recycling them to the vascular lumen [39]. Experimental data in mice [40] and macaques [41] indicate that FcRn indeed regulates IgG serum half-life and antibody pharmacokinetics. Human FcRn binds murine IgGs with lower affinity than murine FcRn does [42], explaining the poor pharmacokinetics of murine mAbs in patients. Such inadequacies result from phylogenetic divergences of both FcRn and IgGs, and nicely illustrate the pitfalls of heterologous situations. Fortunately, mouse FcRn has a good affinity for IgG regardless of the mammalian species, including humans [42]. Besides vascular endothelia, FcRn is also expressed broadly, notably in epithelial cells where it ensures a bidirectional transport of IgG and small immune complexes [43,44]. Therefore, FcRn is probably involved in mAb biodistribution in organs and the whole body, although this function is not well defined. Of note, it has been observed

that human FcRn is expressed in intestinal epithelial cells in adults, whereas only during suckling in young mice [44]. It is not known whether this major difference also exists in other epithelial tissues and/or has a detrimental effect on murine experiments, but this will have probably have to be taken into account in future experiments.

5. Pharmacodynamics of therapeutic antibodies in animals: interaction with the host immune system 5.1. Interaction with host complement Recent studies have shown, using human CD20transduced murine cell lines grafted into immunocompetent mice, that C1q is a fundamental element for rituximab therapeutic activity in vivo. It was shown that the effect of rituximab was abolished in C1q KO mice, and not in NK cellor macrophage-depleted mice, demonstrating the major role of complement in these models [16,17]. The C1q molecule is highly conserved throughout mammalian species [45,46] and displays the same haemolytic activity and specific Fc-binding property whatever the species of either C1q or IgG [45]. Initiation of the classical pathway of the complement cascade does not restrict the use of animals. This is in contrast with the fact that extensive studies of complement haemolytic activities have highlighted the phenomenon of “homologous restriction” of complement lysis. Indeed, antibody-sensitised erythrocytes are much less readily lysed by serum from the same species [47]. Homologous restriction appears dependent upon multiple parameters: the nature of the target cell, the pathway through which complement is being activated, the species source of the activating antibody and the source of complement [48]. The membrane complement regulatory proteins have been involved in the phenomenon of homologous restriction, but the considerable cross-species activity of each regulatory protein suggest that they are not primarily responsible for homologous restriction of lysis. The efficiency of complement activation is influenced by differences in charge and other surface features involved in the three pathways of complement lysis. Differences in convertase stability due to difference in microenvironment or to binding of the fluid regulator factor H to the membrane, and differences in the efficiency of MAC initiation and assembly could explain the phenomenon of homologous restriction [48]. In conclusion, complement activation by therapeutic antibodies in animal models is strongly influenced by a variety of parameters and does not necessarily reflect the human situation. 5.2. Interaction with FcγR-expressing host effector cells Receptors for the Fc portion of IgG (Fc␥R) play a key role in immune defense by linking humoral and cellular immunity. They are expressed on several hematopoietic cells but have different signalling capabilities depending on

S. Loisel et al. / Critical Reviews in Oncology/Hematology 62 (2007) 34–42

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Table 2 Human Fc receptor expression and IgG-Fc recognition specificity for effector ligands Fc␥RIa

Fc␥RIIa

Fc␥RIIb

ITIM

Human CD antigen

CD64

Signalling pathways

ITAM FcR-␥

ITAM

B Lymphocytes Dentritic cellsa Macrophages monocytes NK cells Neutrophils Mast cells

+ + − +

+ +

IgG isotype IgG1 IgG2 IgG3 IgG4 a b c

Fc␥RIIc

Fc␥RIIIa

ITAM

ITAM FcR-␥

+

+ + +

CD32

Fc␥RIIIb

CD16

+

+++ − ++++ ++

+

+ −/++b ++ −

+ + +

+

+

++ − ++ +

+ − + −/+c

+ − + −

Fc␥R expression depends on their activation status. Depending on the Fc␥RIIa allotype. Depending on the Fc␥RIIIa allotype.

whether they are associated with tyrosine-based activatory or inhibitory motifs (ITAM or ITIM, respectively) [49,50]. We have demonstrated the role of Fc␥R in the mechanism of action of rituximab in patients by showing for the first time a genotype/phenotype relationship between a functional polymorphism in the Fc␥RIIIa-coding gene and both the clinical response [51] and the lysis of rituximab-sensitised target cells by Fc␥RIIIa-expressing NK cells [52]. The relationship between the Fc␥RIIa-coding gene polymorphism and the response to therapeutic antibodies is more controversial [53,54] but it cannot be excluded that several of the eight Fc␥Rs present in humans are involved in this response (Table 2). Unless human cytotoxic cells (e.g. NK, macrophages, etc.) are transferred into animals such as SCID mice, the Fc␥R-expressing immune effector cells that will interact with recombinant-mAb-sensitised target cells will be of animal origin. It is therefore reasonable to raise questions about the similarity of animal Fc␥R with their human counterparts in terms of function and cell expression, and their affinity for IgGs having a human Fc (of ␥1 origin). Very few things are known about receptors in nonhuman primates such as macaques or baboons. Some cDNA sequences have been isolated (Fc␥RIIa, Fc␥RIIb and Fc␥RIIIa) that they are very similar to their human orthologs ([50], and M. Ohresser, unpublished results). Moreover, monkey peripheral blood mononuclear cells are reactive with human CD16, CD32 and CD64 mAbs, similarly to their human counterparts [55]. These data suggest that the monkey immune system is close enough to the human to allow relevant pharmacodynamic studies. The best example is given with the anti-CD20 antibodies: the human IgG4 chimaeric version of the 2B8 mAb was unable to eliminate peripheral blood B cells in rhesus monkeys, whereas the IgG1 chimaeric version (rituximab) did [56]. In vivo behaviour of IgG4 in monkeys parallels the properties of IgG4 in humans, i.e. a

very low affinity for human Fc␥RIa only, and an inability to activate complement. The situation is strikingly different in mice. Not only are the numbers of Fc␥Rs different, but so are their cell expression and functions (Tables 2 and 3). Nevertheless, each murine Fc␥R recognises human IgG1, and murine models can therefore be used to study the involvement of the different Fc␥Rs, although translation to the human situation has to be done with caution. For example, rituximab and trastuzumab are able to control CD20-expressing or erbB2-expressing tumour xenografts in normal mice, respectively, in contrast to mice deficient in FcR-␥ [57]. FcR-␥ is the ITAM-bearing accessory chain associated with the entire activatory mouse Fc␥R (Fc␥RI, Fc␥RIII, and Fc␥RIV) and is required for its components’ expression and function. These results suggest the involvement of effector cells expressing one or several of these activatory receptors in the control of xenograft tumours. In addition, Fc␥RII knock-out mice have an increased ability to kill xenogeneic tumour cells in the presence of rituximab or trastuzumab [57]. Fc␥RII, which is the murine ortholog of human Fc␥RIIb, is an ITIM-containing Fc␥R able to inhibit activatory Fc␥Rs when they are co-engaged. Macrophages, which express both the inhibitory Fc␥RII and activatory Fc␥Rs, are therefore supposed to be recruited and/or activated more effectively in Fc␥RII KO mice [57]. These mouse models have contributed greatly to showing that the response to recombinant mAbs results from a balance between activatory and inhibitory Fc␥ receptors. The use of immunodeficient mice in xenograft models should also be considered in interpreting experimental data. These mice (SCID and nude) have developed mechanisms of compensation due to their immune defect and they have an increase in their NK and macrophage functions, and therefore an increased in ability to kill cancer cells through Fc␥Rdependent mechanisms. Moreover, MHC class I molecules expressed on cancer cells may not be recognised by MHC

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Table 3 Murine Fc receptor expression and human IgG-Fc recognition specificity for effector ligands

Signalling pathways B lymphocytes Dentritic cells Macrophages Monocytes NK cells Neutrophils Mast cells Human IgG class IgG1 IgG2 IgG3 IgG4

Fc␥RI

Fc␥RII

Fc␥RIII

Fc␥RIV

ITAM FcR-␥

ITIM

ITAM FcR-␥

ITAM FcR-␥

+

+ +

+ + +

+

+ + −

++ ND

class I-specific inhibitory receptors on the murine effector cells. This could lead to an overestimation of the role of these mechanisms.

6. Conclusion In conclusion, it is clear that performing preclinical studies of therapeutic antibodies in animals is no more than a complex study of somewhat artificial interactions of a xenogeneic protein with the host immune system, and that an even higher level of complexity arises from the use of xenograft models. Recent tragic events show that it is very difficult to circumvent many of those drawbacks, and all these animals models must be considered as models only [58]. Reviewers Sergey M. Kipriyanov, Ph.D., Head of R&D, Affimed Therapeutics AG, Technologiepark, Im Neuenheimer Feld 582, Heidelberg 69120, Germany. Daniel Wierda, Ph.D., Research Fellow, Lilly Research Laboratories, Eli Lilly and Company, Toxicology Division, 2001 W Main Street, GL45, Greenfield, IN 46140, United States. Acknowledgements This work has been supported in part by the INCa (Institut National du Cancer; MAb IMPACT network), CANCEN and the foundation Langlois. David Dayd´e is granted by the R´egion Centre. References [1] Predki PF, Mattoon D, Bangham R, Schweitzer B, Michaud G. Protein microarrays: a new tool for profiling antibody cross-reactivity. Hum Antibodies 2005;14(1–2):7–15.

+

+++ +++ − ND

+++ ++ − ND

− ND − ND

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David Dayd´e is a Ph.D. student in Immuno-PharmacoGenetics in the Therapeutic Antibodies Laboratory, EA 3853, directed by Herv´e Watier.

Biographies

Christian Berthou, M.D., Ph.D. is currently the chief of the Haematology Unit at the Morvan Hospital, Brest (France). Since 2001, he has been the director of the Cellular Therapy and Cancer Immunology Laboratory at the University of Brest.

Severine Loisel, Ph.D. Since 2001, she has been a Research Engineer at the University of Brest and she is working on preclinical studies of targeted antitumour therapy in mouse models. Marc Ohresser, Ph.D. is a Research Engineer at the University of Francois-Rabelais in Tours, EA 3853 IPGA (Immuno-Pharmaco-Genetics of Therapeutic Antibodies), France. Marc Pallardy, Ph.D. is currently Professor of Toxicology at the University of Paris-Sud 11. He is heading a research group in the INSERM UMR 749 laboratory with special emphasis on signal transduction in immune cells. Since year 2000 he is also the Research Director of the School of Pharmacy of the University Paris-sud 11.

Guillaume Cartron, M.D., Ph.D. is a Hospital Practitioner at the University Hospital of Tours in France. Currently, he works at Centre Jean Bernard, Le Mans, France. Herv´e Watier, M.D., Ph.D. In 1986, he joined Prof. P. Bardos’ group in Tours to work on transplantation immunology and tolerance. In parallel, he discovered that the Fc␥RIIIa gene polymorphism is associated with the response to rituximab and other monoclonal antibodies in non-Hodgkin’s lymphoma patients. In 2001, he created a research group called “Immuno-Pharmaco-Genetics of Therapeutic Antibodies” consisting of clinicians and colleagues working in Pharmacology at the University of Tours.