Immunotherapy of Malignant Disease with Tumor Antigen–Specific Monoclonal Antibodies

Home , Antigen presentation, Tumor antigen

Published OnlineFirst December 22, 2009; DOI: 10.1158/1078-0432.CCR-09-2345

Review Clinical Cancer Research Immunotherapy of Malignant Disease with Tumor Antigen–Specific Monoclonal Antibodies

Michael Campoli1, Robert Ferris2,3,6, Soldano Ferrone2,4,5,6, and Xinhui Wang2,6

Abstract A few tumor antigen (TA)–specific monoclonal antibodies (mAb) have been approved by the Food and Drug Administration for the treatment of several major malignant diseases and are commercially available. Once in the clinic, mAbs have an average success rate of ∼30% and are well tolerated. These results have changed the face of cancer therapy, bringing us closer to more specific and more effective biological therapy of cancer. The challenge facing tumor immunologists at present is represented by the identifica- tion of the mechanism(s) underlying the patients' differential clinical response to mAb-based immunotherapy. This information is expected to lead to the development of criteria to select patients to be treated with mAb-based immunotherapy. In the past, in vitro and in vivo evidence has shown that TA- specific mAbs can mediate their therapeutic effect by inducing tumor cell apoptosis, inhibiting the targeted antigen function, blocking tumor cell signaling, and/or mediating complement- or cell-dependent lysis of tumor cells. More recent evidence suggests that TA-specific mAb can induce TA-specific cytotoxic T- cell responses by enhancing TA uptake by dendritic cells and cross-priming of T cells. In this review, we briefly summarize the TA-specific mAbs that have received Food and Drug Administration approval. Next, we review the potential mechanisms underlying the therapeutic efficacy of TA-specific mAbs with emphasis on the induction of TA-specific cellular immune responses and their potential to contribute to the clinical efficacy of TA-specific mAb-based immunotherapy. Lastly, we discuss the potential negative effect of immune escape mechanisms on the clinical efficacy of TA-specific mAb-based immunotherapy. Clin Cancer Res; 16(1); 11–20. ©2010 AACR.

The concept that antibodies could be used for the treat- quate response rates (RR) observed with first generation ment of malignant disease originated more than a century TA-specific mAb most likely reflected their murine origins, ago (1). The first generation of antibody-based therapies resulting in high immunogenicity and poor ability to re- were based on the use of tumor antigen (TA)–specific al- cruit immune effector mechanisms (4–6). These hurdles logeneic, autologous, or xenogeneic polyclonal antibodies, have been recently overcome by the generation of chime- which were ill suited as cancer-specific therapies because ric, humanized, and human mAb, resulting in reduced or of their limited or lack of specificity and reproducibility. lack of immunogenicity and improved ability to recruit ef- It was not until the development of the hybridoma tech- fector cells (7). nology (2) that antibody-based immunotherapy of malig- Today, TA-specific mAb have been established as highly nant diseases became a practical reality. The hybridoma sensitive and reproducible probes in the diagnostic arena technology enabled the production of a large number of (8–13) as well as in clinically and commercially successful human TA–specific murine monoclonal antibodies therapies for a variety of malignant diseases (3). The average (mAb). The clinical application of some of them yielded clinical success rate of TA-specific mAb-based immunother- a handful of promising results that were however oversha- apy, which manifests itself as statistically significant disease- dowed by disappointing outcomes in early clinical trials free interval and survival prolongation as well as reduction implemented with TA-specific mouse mAb in patients of tumor mass in some of the treated patients, is ∼30% with with various types of cancer (3). In hindsight, the inade- ranges from 0% to 60% (3, 14). Only little is known about why merely a limited percentage of the treated patients re- spond clinically to TA-specific mAb-based immunotherapy. Authors' Affiliations: 1Department of Dermatology, University of Colorado The mechanisms underlying the patients' differential clini- Health Science Center, Denver, Colorado; Departments of 2Immunology, cal response to TA-specific mAb-based immunotherapy 3Otolaryngology, 4Surgery, and 5Pathology, University of Pittsburgh Cancer Institute, and 6Cancer Immunology Program, School of Medicine, represent a topic of intense research at present, because this Pittsburgh, University of Pittsburgh, Pennsylvania information has both basic research and clinical relevance. Corresponding Author: Soldano Ferrone, University of Pittsburgh Cancer It will contribute to our understanding of the molecular ba- Institute, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623- sis of the clinical efficacy of TA-specific mAb-based immu- 5040; Fax: 412-623-4840; E-mail: [email protected]. notherapy and it will lead to the development of criteria to doi: 10.1158/1078-0432.CCR-09-2345 select patients to be treated with TA-specific mAb-based ©2010 American Association for Cancer Research. immunotherapy. In this review, we first summarize the

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14). Clinical responses have been found to include com- Translational Relevance plete and partial responses as well as statistically significant increases in progression-free survival and in – Tumor antigen specific monoclonal antibodies disease-free interval and survival in patients with several (mAb) have been successfully implemented into stan- malignant diseases (Table 1). In general, compared with dard treatment regimens for patients with a variety of solid tumors, hematologic neoplasms have been proven malignant diseases. Despite the clinical successes, scant easier to target with TA-specific mAb-based therapies be- information is available about the variables underlying cause therapeutic efficacy can be achieved at lower doses patients' differential clinical response to mAb-based and tumor penetration is more readily achieved (3, 14). immunotherapy. The scant information in this area Moreover, radioimmunotherapy has been more successful has a negative effect on the optimization of the use for hematologic malignancies such as non–Hodgkin's – of tumor antigen specific mAb in therapeutic strate- lymphoma. However, fewer of these agents are entering gies and represents a major obstacle to the selection clinical trials due to complexities in manufacture and safe- of patients to be treated with mAb-based immunother- ty concerns compared with unconjugated mAb (3, 14). An apy. This review discusses the potential mechanisms additional limitation of radioimmunotherapy relates to underlying the therapeutic efficacy of mAb-based im- the delivery of the dose of radioactivity to the tumor com- munotherapy in patients with malignant disease. This pared with normal tissues (3, 14). The poor specificity in- information is expected to contribute to our under- dex is due to slow pharmacokinetics and slow tumor standing of the molecular basis of the clinical efficacy perfusion. of mAb-based immunotherapy and to lead to the de- Compared with traditional chemothearpy- and radio- velopment of criteria to select patients with malignant therapy-based regimens, in general, the side effects of disease to be treated with mAb-based immunotherapy. immunotherapy with nonconjugated TA-specific mAb are fairly mild (3, 14). Nonetheless, toxicities do occur and may be classified as mechanism-independent and TA-specific mAbs that have received Food and Drug Admin- mechanism-dependent (Table 2). Most of the toxicities re- istration approval. Second, we describe the potential me- lated to TA-specific mAb are mechanism-independent and chanisms underlying the therapeutic efficacy of TA-specific are related to allergic or hypersensitivity reactions caused by mAb with emphasis on the induction of TA-specific cellular a protein containing xenogeneic sequences (3, 14). These immune responses. Finally, we discuss the potential nega- reactions occur during or just after the first injection and tive effect of tumor immune escape mechanisms on the are summarized in Table 2. Moreover, human anti-mouse clinical efficacy of TA-specific mAb-based immunotherapy. immunoglobulin antibodies, including anti-idiotypic antibodies, can complex with circulating therapeutic mAb, TA-Specific mAb and inhibit their targeting of tumor cells. It should be noted that conjugated antibodies often have a lower therapeu- In recent years, the regulatory approval and sales of new tic index than nonconjugated TA-specific mAb, and for this human medicines indicates an increasing number of bio- reason, they often result in more side effects (3, 14). Rare, logical therapies, i.e., small molecular inhibitors and mAb but more serious side effects of TA-specific mAb-based im- specifically designed to target malignant cells (15).7 In munotherapy are often related to mechanism-dependent fact, the global sale of mAb-derived biological therapies toxicities and result from the binding of a therapeutic in 2006 was $20.6 billion dollars, indicating a major par- antibody to its target antigen (Table 2; refs. 3, 14). adigm shift in industrial research and development from pharmaceutical to biological therapies (15).7 At the end of 2007, >30 years of clinical studies have resulted in the Molecular Mechanisms Underlying the approval of six unconjugated, humanized, or chimeric TA- Therapeutic Efficacy of TA-Specific mAb-Based specific mAbs for cancer therapy along with one drug im- Immunotherapy munoconjugate and two radioisotope immunoconjugates (Table 1; refs. 3, 14). The results of clinical trials in pa- The majority of nonconjugated TA-specific mAb ap- tients treated with TA-specific mAb have shown that the proved for clinical use display intrinsic antitumor effects goal of cancer-targeted therapy is realistic and may be mediated by one or more of the mechanisms outlined superior to older nonspecific conventional chemotherapy- in Table 3. They can be broadly divided into those that and radiotherapy-based regimens, at a minimum enhanc- require immune effector cells and those that do not. It ing their activity. Once in the clinic, TA-specific mAb are should be noted that these mechanisms do not function well tolerated with an average success rate of 30% (3, independently, but extensively interact with each other. The relative importance of each mechanism varies with the type of tumor and the treatment administered. More- 7 Datamonitor. Big Pharma' Turns To Biologics For Growth To 2010: Fi- over, it should be stressed that TA-specific mAb have been nancial And Strategic Segmentation of The 'Big Pharma' Sector By Drug Technology. Datamonitor, May 4, 2006). http://www.marketresearch. clearly shown to be able to inhibit their specific receptor com/map/prod/1285675.html. and induce apoptosis in the targeted tumor cell without

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the influence of immune cells in vitro. Nevertheless, it is factor-α, chemokines, and opsonins that recruit immune still debated whether immune effector cells or receptor effector cells. Consequently, tumor cell proliferation and blockade is the dominant mode of action in vivo or if both angiogenesis are inhibited; antigen presentation is pathways need to cooperate to achieve therapeutic effect. increased; and tumor cells are lysed (31, 32, 34). TA-specific mAb can block activation signals that are Several of the clinically approved TA-specific mAbs, needed for continued malignant cell growth and/or viabil- such as rituximab, cetuximab, trastuzumab, alemtuzumab, ity by blocking the interactions between the ligand and its panitumumab, and 131I-tositumomab, have been shown receptor, inducing modulation of the receptor or interfer- to activate ADCC and CDC in vitro and when administered ing with ligand binding and/or dimerization of the recep- to mice transplanted with TA-expressing tumor cells. In tor (reviewed in refs. 3, 14). The latter mechanisms seem many cases, it is difficult to unravel whether the therapeu- to be particularly important for the epidermal growth tic efficacy of TA-specific mAb depends more on ADCC or factor receptor (EGFR)– (16–21), CD20- (22–26), and CDC; however, there has been some work in this area. It is vascular endothelial growth factor–specific (27–30) noteworthy that the most convincing evidence for ADCC mAb. Alternatively, some TA-specific mAb may exert their and CDC in the antitumor activity of TA-specific mAb effects through Fc-based mechanisms such as antibody- comes from hematologic malignancies, i.e., the CD52- dependent cell-mediated cytotoxicity (ADCC) and com- and CD20-specific mAbs alemtuzumab and rituximab, plement-dependent cytotoxicity (CDC; refs. 31, 32). respectively (3, 14). Whether this finding reflects the acces- ADCC and CDC are dependent on interactions of anti- sibility of tumor cells to both TA-specific mAb and plasma body Fc domains with cellular Fcγ receptors (FcγR) ex- proteins of the complement cascade as well as immune ef- pressed on immune accessory cells and with the classic fector cells remains to be determined. Nevertheless the complement-activating protein C1q, respectively. The role of CDC in the antitumor activity of mAb, which rec- potential to mediate ADCC and CDC is a function of an ognize antigens expressed by malignant lymphoid cells, is antibody's subclass and is also influenced by the nature of suggested by experimental and clinical findings. First, its glycosylation (33). Triggering of both ADCC and CDC alemtuzumab mediates significant CDC of chronic lym- not only activates natural killer (NK) cells, neutrophils, phocytic leukemia (CLL) cells in vitro (35). Second, the mononuclear phagocytes, and/or dendritic cells (DC), ability of rituximab to cure immunocompetent mice chal- but also induces secretion of IFN-γ, tumor necrosis lenged with murine lymphoma EL4 cells stably transfected

Table 1. FDA-approved TA-specific mAb for human cancers

mAb Target Isotype FDA-approved disease

Rituximab CD20 Chimeric IgG1 CD20 (+) low-grade lymphoma,* diffuse large B-cell lymphoma,* follicular lymphoma* 90Y Ibritumomab + tiuxetan CD20 Radiolabeled murine IgG1 CD20(+) low-grade lyphoma† 131I Tositumomab CD20 Radiolabeled murine IgG1 CD20(+) low-grade lyphoma† Alemtuzumab CD53 Humanized IgG1 Chronic lymphocytic leukemia‡ Gemtuzumab + ozogamicin CD33 Recombinant humanized Acute myelogenous leukemia§ IgG4-conjugated to calicheamicin Trastuzumab HER2/neu Humanized IgG1 Her2/neu (+) breast cancer∥ Cetuximab EGFR Chimeric IgG1 EGFR(+) Colon cancer¶ Panitumumab EGFR Fully human IgG2 EGFR(+) Colon cancer† Bevacizumab VEGF Humanized IgG1 Colon cancer,** recurrent or advance non–small cell lung cancer, metastatic breast cancer

Abbreviations: FDA, Food and Drug Administration; VEGF, vascular endothelial growth factor. *Low-grade lymphoma: second-line monotherapy; diffuse large B-cell lymphoma and follicular lymphoma: first-line chemoimmune therapy as well as maintenance for follicular lymphoma. †Second-line monotherapy. ‡First- and second-line monotherapy. §>60 y of age, second-line monotherapy. ∥Second-line monotherapy, adjuvant, and first-line chemoimmunetherapy. ¶Second-line monotherapy or chemoimmune therapy. **First-line chemoimmune therapy.

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Table 2. Toxicities related to TA-specific mAb for human cancers

Mechanism dependent Mechanism independent

Rituximab Transitory lymphocyte B depletion Hypersensitivity reactions* Reactivation of hepatitis B/C, cytomegalovirus, Urticarial or morbilliform and parvovirus B19 skin eruptions Reversible ventricular tachycardia Fever, chills, headache 90Y Ibritumomab + tiuxetan Transitory lymphocyte B depletion Gastrointestinal upset Myelosuppression† Anaphylactoid reactions 131I Tositumomab Hypothyroidism Hypotension Hematologic toxicities Angioedema Thrombocytopenia, neutropenia, and anemia Development of human Transitory lymphocyte B depletion anti-mouse Ig antibodies Alemtuzumab Pancytopenia Autoimmune idiopathic thrombocytopenia and hemolytic anemia Infections‡ Gemtuzumab + ozogamicin Myelosuppression Infection Trastuzumab Cardiac dysfunction§ Cetuximab Severe infusion reactions∥ Flushing, seborrheic dermatitis, and acneiform eruptions Panitumumab Severe infusion reactions∥ Pulmonary fibrosis Hypomagnesemia Bevacizumab Hypertension, proteinuria, minor bleeding, or thrombosis Bowel perforation¶ Wound healing complications and bleeding**

*Caused by mAb-containing xenogeneic sequences. These reactions occur during or just after the first injection. †Patients must have >25% bone marrow involvement to be considered eligible for treatment. ‡Pneumocystis jiroveci pneumonia and herpes virus prophylaxis is recommended for patients being treated with alemtuzumab. §When administered in combination with paclitaxel or an anthracycline and cyclophosphamide. ∥Rare and usually occur within minutes of the initial infusion. ¶Alone or in combination with chemotherapy. **In patients treated within 60 d from surgery.

with human CD20 is completely abolished in syngeneic tumor challenge using several antigen/antibody systems. knockout mice lacking C1q (36). Lastly, complement con- Furthermore, the removal of the Fc portion from TA- sumption has been observed after administration of the specific mAb reduces TA-specific mAb-related side effects CD20-specific mAb rituximab to patients with lymphoma as well as their antitumor activity. In contrast, deletion (32, 37). It is noteworthy that target antigen density seems of the inhibitory type II FcγR(FcγRIIb) results in an in- to be a critical factor for CDC, since Golay et al. (39) have creased protective effect, suggesting that FcγRIIb modu- shown that the success of rituximab in mediating CDC lates TA-specific ADCC activity in vivo (31). The role of against malignant B cells is highly dependent on CD20 interactions with cellular FcγR in the clinical efficacy of density. Whether the lack of convincing evidence for the TA-specific mAb-based immunotherapy is further sup- role of CDC in the antitumor activity of TA-specific mAb ported by the statistically significant correlation between used for solid tumors reflects inadequate TA density re- improved clinical RR to mAb-based immunotherapy and mains to be determined. particular “high responder” FcγR polymorphisms in pa- The role of ADCC in the antitumor activity of TA- tients with (a) CD20(+) follicular cell lymphoma treated specific mAb is also suggested by experimental and clinical with rituximab, (b) metastatic colon cancer treated with findings. First, transgenic mice that lack type I and type III cetuximab, or (c) metastatic breast cancer treated with FcγR have provided the conclusive evidence that mAbs are trastuzumab (3). Nevertheless, it should be stressed that capable of targeting immune effector cells to cancer cells the type of FcγR polymorphism does not seem to be asso- in vivo (31, 39). In this regard, FcγR(−)mice,unlike ciated with improved clinical RR in every patient with a wild-type mice, fail to show protective immunity against certain disease and in every disease. For example, Fcγ

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RIIIa-158F polymorphism is an indicator of improved immunity through the production of cytokines, chemo- clinical RR in patients with colon cancer and breast can- kines, and opsonins, which ultimately lead to (a) amplifi- cer treated with cetuximab and trastuzumab, respectively, cation of ADCC and CDC, (b) recruitment and activation of but is linked to poor RR in patients with hematologic immune effector cells, (c) maturation of APC, (d) enhance- malignancies treated with rituximab (3). Moreover, ment of antigen presentation, and (d)generationofTA- FcγRII and RIII polymorphisms are not associated with specific CD4(+) and CD8(+) T-cell immunity (Fig. 1). improved clinical RR in chronic lymphocytic leukemia Moreover, generation of cleavage products from compo- patients treated with alemtuzumab or rituximab, in dif- nents of the complement pathway, e.g., C3b, may acti- fuse large B-cell lymphoma patients treated with rituxi- vate complement receptor 3 on the surface of effector cells mab, and in follicular cell lymphoma non–Hodgkin's and induce complement receptor 3–dependent cellular lymphoma patients treated with sequential cyclophos- cytotoxicity (42). phamide-Adriamycin-vincristine-prednisone (Oncovin) The possibility that TA-specific mAb can enhance the and rituximab (3). Whether these conflicting findings re- immunogenicity of TA and induce TA-specific cellular flect the effect of other variables, such as characteristics immunity is supported by the following lines of preclin- of the TA-specific mAb used, induction of TA-specific ical and clinical evidence. First, incubation of ovarian CTLs, tumor sensitivity to apoptosis, and host immune cancer cells with the CA125-specific mouse IgG1 mAb cell dysfunction, on the role of FcγR polymorphisms in oregovomab in vitro can induce CA125-specific and the patients' clinical response to antibody-based immu- autologous ovarian TA-specific CTL (43). Second, cross- notherapy is not known. presentation mediated by FcγRs on human DC can en- At present, the variables underlying the differential clini- hance the presentation of multiple myeloma antigens to pa- cal RR of the patients treated with antibody-based immu- tient-derived T cells, thus suggesting that uptake of notherapy have not been identified. The data we have antibody-opsonized tumor cells and cellular fragments by summarized suggest that ADCC and CDC play a part in APCs could lead to antigen/epitope spreading and induction the clinical efficacy of at least some TA-specific mAb. How- of immunity to several TA (40). Third, in vitro incubation of ever, other mechanism(s) are likely to underlie the durable cells with trastuzumab results in augmentation of HER2- clinical responses observed in some patients treated with specific CTL killing of HER2(+) tumors, through the ability TA-specific mAbs, because every tumor cell is not complete- of trastuzumab to mediate HER2 internalization and to en- ly eradicated during therapy with TA-specific mAbs. Given hance HLA class I antigen–restricted presentation of endoge- the number of interactions that are predicted to occur nous HER2 through proteasomal processing (44). Fourth, among different components of the immune system, sever- immunization of mice with DC pulsed with antibody- al investigators have begun to examine the ability of TA-spe- opsonized TA acquired through either FcγR-mediated cific mAb to induce TA-specific cytotoxic T-cell responses in endocytosis (40, 42, 45) or phagocytosis (43) induces TA-specific mAb-treated patients. In this regard, TA-specific CD4(+) and CD8(+) T-cell–mediated tumor immunity. mAbs are likely to enhance antigen-presenting cell (APC) Lastly, induction of a TA-specific CTL response has been internalization and antigen presentation of TA, as well as recently documented in patients treated with TA-specific cross-priming of T cells through endocytosis and phagocy- mAb-based immunotherapy (46). Six of 10 evaluable tosis of TA-containing immune complexes and antibody- breast cancer patients showed augmented HER2-specific opsonized tumor target cells, respectively (40, 41). Further- CD4 T-cell responses during therapy with trastuzumab. Fur- more, the activation of both ADCC as well as CDC can furthermore, the number of patients with detectable HER2- ther augment and focus the generation of TA-specific T-cell specific antibodies among the 27 treated with trastuzumab

Table 3. Molecular mechanisms underlying therapeutic efficacy of TA-specific mAb-based therapy

Immune effector cell independent* Immune effector cell dependent

Induce apoptosis Activation of complement mediated: Induce alteration in intracellular signaling Phagocytosis Inhibit growth factor binding to its cognate receptor CDC Inhibit growth factor receptor activation Trigger antibody-dependent cellular cytotoxicity Induction of tumor cell necrosis or apopotsis leading to Presentation of TA by APC Activation of CD(+) T-cell–mediated kill Activation of B cells and eosinophils Activation of TA-specific CTL

*Ultimately results in inhibition of cancer cell proliferation, tumor-induced angiogenesis, cancer cell invasion and metastasis, as well as potentating antitumor activity of cytotoxic drugs and radiotherapy.

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Fig. 1. Triggering of immune effector mechanisms by TA-specific mAb-based immunotherapy. TA-specific mAb may participate in host-dependent immune effector mechanisms including (A) activation of (i) complement-mediated phagocytosis and/or (ii) CDC, (B) induction of ADCC, and/or (C) induction of tumor cell necrosis or apoptosis. The latter mechanisms result in the release of TA as well as the production of cytokines and opsonins that lead to (I) TA uptake, APC maturation and presentation of TA by APC, (II) generation of CD8(+) TA-specific CTL through CD4(+) T-cell help, and (III) activation of CD4(+) T helper cells which leads to activation of NK cells, granulocytes, and macrophage through release of Th1 cytokines, CD4(+) T-cell–mediated killing, and activation of B cells and eosinophils through release of Th2 cytokines.

increased from 8 (29%) before treatment to 15 (56%) dur- FcγR, have been reported to present HLA class I anti- ing treatment (P < 0.001). Similar results have been gen–restricted exogenous antigens (47, 48). An alterna- obtained by one of us (RLF) in studies in progress. Specifi- tive, although not exclusive, possibility is represented cally, HLA class I antigen–restricted, TA-specific CTL were by the induction of anti-idiotypic antibodies, i.e., anti- detected in four of seven HLA-A*0201+ patients with head bodies specific for the variable region of the administered and neck squamous cell cancer treated with the EGFR- TA-specific mAb (49, 50). In some cases, these anti-idio- specific mAb cetuximab. typic antibodies may mimic the TA recognized by the TA-specific mAb are likely to prime antigen-specific original TA-specific mAb and function as a surrogate an- CD4(+) and/or CD8(+) T cells by cross-presentation of tigen (49, 50). The anti-idiotypic antibody as such or TA released by dying cells. In this regard, once TA-specific complexed with the TA-specific mAb may be taken up mAb mediate tumor cell lysis by any of the mechanisms by professional APC and induce a TA-specific T-cell im- outlined above, they can also participate in the cross- mune response (49, 50). presentation of antigen to professional APC (Fig. 2). The term cross-presentation has been used to describe Tumor Cell Resistance to TA-Specific CTL the uptake and representation of cell-associated antigens primarily in the HLA class I antigen pathway of profes- Despite appropriate TA expression, patients may not sional APC (47, 48). Among them, B cells and most have a clinical response to TA-specific mAb-based immu- prominently DC and macrophages, all of which express notherapy and/or may develop resistance to therapy during

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the course of treatment. The multiple mechanisms by cells may change in the expression of molecules such as which TA-specific mAb may exert their antitumor effects TA, HLA class I antigens, and/or antigen-processing ma- make it difficult to determine which “escape” mechanism chinery components; all of them play a crucial role in (s) is/are most important in patients. Furthermore, the the generation of the HLA class I antigen–TA peptide com- intrinsic genetic instability of tumor cells (51) limits our plex, which mediates the recognition of tumor cells by the ability to predict how successful TA-specific mAb-based im- host's CTL. Approximately 10% to 30% of tumor cells may munotherapy will be. Nevertheless, several mechanisms of fail to express TA and a variable degree of interlesional tumor cell resistance to TA-specific mAb-based immu- and intralesional heterogeneity may also be present in notherapy have been suggested (reviewed in refs. 3, 14). a patient (52). Furthermore, tumor cells may present For the purpose of this review, we have focused on the abil- TA-derived peptide analogues with antagonist activity re- ity of tumor cells to evade immune recognition and de- sulting in suboptimal T-cell activation (53). These defects struction by TA-specific CTL. have been found to render malignant cells ineffective tar- If T cells do play a role in the clinical efficacy of TA- gets for TA-specific T cells. Moreover, HLA class I antigen specific mAb-based immunotherapy, then the escape me- downregulation or loss by tumor cells occurs with a fre- chanisms, which have been identified in patients treated quency of about 10% to 80% depending on the type of ma- with T-cell–based immunotherapies, will have relevance. lignancy (53). These abnormalities are caused by distinct The latter include changes in TA, HLA antigen, and anti- molecular mechanisms (reviewed in ref. 54). In some of gen-processing machinery component expression as well these cases, HLA class I antigen expression can be restored as CD4(+)CD25(+) T regulatory cells, each of which mod- by cytokines, providing the potential for benefit by combin- ulates the interaction between tumor cells and T cells. Spe- ing TA-specific mAb-based immunotherapy with adminis- cifically, because of their genetic instability (51), tumor tration of cytokines.

Fig. 2. Induction of TA-specific CTL responses by TA-specific mAb-based immunotherapy. TA-specific mAb may enhance the generation and promote the survival of TA-specific CTL through several mechanisms. TA-specific mAb may (i) induce tumor cell death or activate (ii) ADCC and (iii) CDC. The latter results in (a) the formation of the lytic membrane-attack complex (MAC); (b) the generation of opsonins (C3b), and (d) the release of the anaphylatoxins C3a and C5a. The culmination of the above events (i-iii) leads to the release of Th1 cytokines, (iv) the formation of TA-specific mAb-TA complexes, and (v) the uptake of TA and TA-specific mAb-TA complexes by APC. Ultimately, mature DC present processed TA to CD4(+) and CD8(+) T cells, and promote the generation of (vi) TA-specific CTL.

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It is noteworthy that the role of HLA class I antigen ab- mAb to induce more clinically effective TA-specific T-cell normalities in the clinical course of the disease is high- immune responses. First, the type of TA targeted by the lighted by their increased frequency in recurrent cancers TA-specific T-cell responses generated by TA-specific in patients treated with T-cell–based immunotherapy mAb-based immunotherapy may be very important. In (53). These findings have important implications if the this regard, most of the currently used TA in T-cell–based generation of TA-specific CTL underlies, at least in part, immunotherapy trials represent differentiation and/or the clinical efficacy of TA-specific mAb-based immu- shared TA that have been identified from tumor metasta- notherapy. Specifically, the loss of HLA class I antigens ses and CTL from patients who, in the majority of cases, will facilitate the outgrowth of tumor cells that have ac- have failed to reject their cancer. Moreover, many of these quired the ability to escape T-cell recognition because of TA are selected on the basis of their immunogenicity and these defects. If this interpretation is correct, the outgrowth tissue distribution without paying much attention to their of tumor cells with HLA class I antigen abnormalities is function in tumor cell biology. Whether any of the identi- likely to reflect escape of tumor cells from immune recog- fied TA are tumor rejection antigens and whether stage IV nition more than dormancy or ignorance of the patient's cancer patients represent the most appropriate source to immune system. This possibility is supported by the dis- identify clinically relevant TA is not known at present. Sec- ease progression frequently observed in patients with ma- ond, TA-specific mAbs have the potential to generate adap- lignancy in spite of the development and/or persistence of tive immune responses against the entire TA repertoire of a a TA-specific CTL response (54). Moreover, given the high particular patient's tumor because they are able to enhance rate of mutations in tumor cells, the generation of TA- cross-presentation of multiple TA through their direct anti- specific CTL through TA-specific mAb therapy, even when tumor effects. Third, the adaptive immunity potentially successful, is not likely to be able to completely eradicate a generated by TA-specific mAb provides a means to match patient's malignancy, because the eventual outcome is therapeutic regimens with changing tumor antigenic pro- likely to be outgrowth of tumor cells with HLA class I an- files that are likely to occur during the course of the dis- tigen abnormalities. ease. Fourth, the potential ability of TA-specific mAb to HLA class I antigen–TA peptide complex loss by tumor generate adaptive immunity against multiple TA increases cells may not solely be responsible for their immune resis- the chances to also target unique TAs that have been sug- tance because in several cases, TA-specific CTL coexist with gested to be more effective targets of T-cell–based immu- cancer cells expressing the target components required for notherapy than shared TA (60). Lastly, the polyvalent recognition (55). Factors such as the type of T-cell re- vaccine–like potential of TA-specific mAb not only elimi- sponse induced (i.e., tolerance), lack of CD4+ helper-T-cell nates the requirement for patient selection based on HLA activation, host immune cell dysfunction, activating type, but may also be able to counteract escape mechan- antigen-specific CD4(+)CD25(+) T regulatory cells, pro- isms caused by selective loss of HLA class I allospecificities duction of inflammatory and lytic molecules by tumor and TA. However, it should be noted that one disadvan- cells, and/or high tumor burden may also be responsible tage of the use of TA-specific mAb is the lack of knowledge for the lack of association between self–TA-specific T-cell about the identity of the TA-mediating tumor recognition responses and clinical outcome (55–59). that are targeted in adaptive immune responses; the lack of this information hinders the standardization of the assays Toward Improving Immunotherapy designed to monitor immune responses in patients treated with TA-specific mAb-based immunotherapy. Clearly we are at an early stage in our understanding of The ability of TA-specific mAb to induce more clinical- the molecular mechanisms by which TA-specific mAb- ly effective TA-specific T-cell immune responses may also based immunotherapy mediates effective clinical re- stem from their ability to activate multiple arms of the sponses. Although not conclusive, the information we host's immune system including ADCC, CDC, innate im- have reviewed has focused attention on the potential role mune effector cells, DC, NK, B cells, and T cells. This is in of TA-specific adaptive immunity in patients treated with contrast to the narrow view of strictly activating TA-spe- TA-specific mAb-based immunotherapy. Such responses cific CTL that has been attempted in the majority of the would be desirable, because they provide a mechanism T-cell–based vaccine strategies to date. In this regard, re- for long-term protection and immunologic memory. cent progress into our understanding of the mechanisms Moreover, the generation of TA-specific adaptive immuni- underlying activation and proliferation of the adaptive ty provides a mechanism to explain the improved clinical arm of the immune response provides support for the responses in at least some patients treated with repeated concept that tumor cells may directly and/or indirectly administrations of some TA-specific mAb. lead to dysfunction and/or death of immune cells in If T cells do play a role in the clinical efficacy of TA- the tumor microenvironment (55). Therefore, it is unlike- specific mAb-based immunotherapy, one might ask why ly that T-cell–based vaccination strategies will be success- T cells activated in this setting are effective at controlling ful in the setting of tumor cell–induced immune tumor cell growth but not when patients are vaccinated suppression. Through their ability to activate ADCC, with T-cell–based activation strategies. Several possibilities CDC, and APC, TA-specific mAbs may promote a Th1 cy- can be envisioned to underlie the ability of TA-specific tokine–rich environment, thereby enhancing both the

18 Clin Cancer Res; 16(1) January 1, 2010 Clinical Cancer Research

mAb-Based Immunotherapy, T Cells, Escape Mechanisms

number and function of DC, B cells, as well as NK cells Consequently, even when successful, it is likely that im- and ultimately preventing premature death of T-cell effec- munotherapy will facilitate the emergence and expansion tors. It is noteworthy that the potential ability of TA-spe- of tumor cell populations with TA and/or HLA antigen cific mAb to activate NK cells may prove significant, defects and eventually the recurrence of malignant le- because there is growing evidence that NK cells are re- sions. The latter suggests that immunotherapy for the quired for the activation of DC as well as the generation treatment of cancer may only be successful in a limited of antigen-specific T- and B-cell response both in vitro number of patients. Even when successful, it is likely that and in vivo (61–63). the selective pressure imposed by immunotherapy will If it is determined that the induction of such TA-specific facilitate the emergence and expansion of tumor cell po- T-cell immune responses does contribute to the clinical ef- pulations with TA and/or HLA antigen defects and, even- ficacy of TA-specific mAb-based immunotherapy, in vivo tually, the recurrence of malignant lesions. Therefore, it induction of ADCC or CDC leading to TA-specific CTL will be important to combine immunotherapy with other could be viewed as potential biomarkers of clinical re- types of immunologic and nonimmunologic strategies, sponse. In this regard, it should be noted that the ability which use distinct mechanisms to control tumor growth. of TA-specific mAb to induce ADCC, CDC, and TA-specific In this regard, the concomitant targeting of other cells in T-cell immune responses might have important implica- the tumor microenvironment, which are crucial for ma- tions for the development of future TA-specific mAb. As lignant cell survival and proliferation, may counteract noted above, the potential of a TA-specific mAb to medi- the negative effect of tumor cell genetic instability on im- ate ADCC and CDC is a function of not only its subclass munologic and nonimmunologic therapies. but also its degree and type of glycosylation (33). Notably, γ deglycosylated antibodies are unable to bind Fc R and the Disclosure of Potential Conflicts of Interest presence of sialic acid residues confers anti-inflammatory properties to mAb. Therefore, the type and amount of R. Ferris, commercial research grant, Amgen; consultant, glycosylation of TA-specific mAb is likely to play an im- Bristol Myers Squibb. portant role in balancing the proinflammatory versus anti-inflammatory properties of administered antibodies. It is possible that this vaccine-like property of TA-specific Grant Support mAb-based immunotherapy could be exploited to selec- tively amplify or bias the resulting adaptive immune re- ASDS Cutting Edge Research Grant (M. Campoli) and by sponse by promoting antigen presentation, host antibody PHS grants IPSO CA097190 (R. Ferris, S. Ferrone), production, and expansion of TA-specific CTL. Nonethe- RO1CA110249 (S. Ferrone), RO1CA113861 (S. Ferrone), less, it must be kept in mind that if indeed T cells are RO1CA10494 (S. Ferrone), R01CA105500 (S. Ferrone), major players in the clinical efficacy of TA-specific RO1 DE19727 (R. Ferris) and RO3 RFA PA-08-209 (X. mAb-based immunotherapy, we will continue to face Wang) awarded by the National Cancer Institute. the negative effect on the outcome of immunotherapy Received 8/27/09; revised 11/2/09; accepted 11/3/09; of escape mechanisms selected for by immune pressure. published OnlineFirst 12/22/09.

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20 Clin Cancer Res; 16(1) January 1, 2010 Clinical Cancer Research

Immunotherapy of Malignant Disease with Tumor Antigen− Specific Monoclonal Antibodies

Michael Campoli, Robert Ferris, Soldano Ferrone, et al.

Clin Cancer Res 2010;16:11-20. Published OnlineFirst December 22, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-09-2345

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