Advances in the Assessment and Control of the Effector Functions Of

PersPecTIves

the class of receptors most relevant for the OPINION function of therapeutic antibodies. FcγRs are divided into three types — FcγRI Advances in the assessment and (CD64), FcγRII (CD32) and FcγRIII (CD16)5,6 — and have traditionally been control of the effector functions of categorized according to their affinity for specific IgG subclasses and the type of signalling pathway that they trigger; that is, therapeutic antibodies whether it is inhibitory or activating6. In general, FcγRI has been considered to be a ‘high Xu-Rong Jiang, An Song, Svetlana Bergelson, Thomas Arroll, Bhavin Parekh, affinity’ receptor that binds both monomeric Kimberly May, Shan Chung, Robert Strouse, Anthony Mire-Sluis and IgG and immune complexes, whereas the Mark Schenerman affinity of FcγRII and FcγRIII for IgGs is low and intermediate, respectively5,6. Abstract | The Fc (crystallizable fragment) region of therapeutic antibodies can However, assigning affinity to the FcγRs have an important role in their safety and efficacy. Although much is known is not simple. The classification of receptor about the structure–activity relationship of antibodies and the factors that affinity as ‘high’ or ‘low’ was first introduced in early studies using cell lines and human influence Fc effector functions, a process has not yet been defined to clearly myeloma proteins with an undefined glyco- delineate how Fc functionality should be assessed and controlled during form pattern associated with the receptor antibody development and manufacturing. In this article, we summarize the molecule. FcγRI was assigned as a ‘high current knowledge of antibody Fc functionality, provide a strategy for assessing affinity’ receptor because bound radio- the effector functions of different classes of therapeutic antibodies (including labelled IgG could be detected on the surface Fc fusion proteins) and propose a path for routine testing and controls for of cells following washing; this was not possible for FcγRII or FcγRIII. However, both manufacturers of antibody products. FcγRII and FcγRIII were shown to bind to immune complexes and become activated, as is the case for FcγRI. The development and manufacture of heavy chain domain combinations and Fluorescence-activated cell sorting analy- therapeutic antibodies, including Fc glycoengineering, in order to modulate sis now enables us to observe direct binding (crystallizable fragment) fusion proteins, effector functions and to optimize safety of FcRs to immune complexes. Therefore, represent a significant segment of the and efficacy3,4. the terms used to define receptor affinity are biopharmaceutical industry and have Therapeutic antibodies rely on two relative and not absolute. The use of these resulted in substantial benefits to public types of functionalities to achieve clinical terms needs to be reconsidered given that health. To date, more than 20 unconjugated efficacy: target-specific binding by the Fab it is now known that the ‘affinity’ of afuco- recombinant therapeutic antibodies (antigen-binding fragment) domain and sylated IgG1 for FcγRIIIa is greatly increased have received regulatory approval for the immune-mediated effector functions — compared with fucosylated IgG1. On the treatment of various diseases, and all of such as antibody-dependent cell-mediated other hand, if the FcγRIIIa molecule is not these antibodies are of the immunoglobulin cytotoxicity (ADCC) and complement- glycosylated at N162, there is no difference G (IgG) class1. In the 1980s, therapeutic dependent cytotoxicity (CDC)3,4 — via in the binding affinity for the different IgG antibodies were of murine origin, highly interaction of the Fc domain with glycoforms7. Both cases illustrate that over- immunogenic, suboptimal in eliciting receptors on various cell types5,6. The simplification of affinity designations can Fc effector functions and had short half- Fc portion of a therapeutic antibody may occur if the nature of the glycosylation pat- lives in humans2. These issues drove therefore have an important role in its tern of either the antibody or the receptor is the evolution of antibody technology mechanism of action through its influence not considered. and led to the development of chimeric, on either ADCC or CDC. The binding of IgG to FcγRs is a criti- humanized and human antibodies that cal step for the initiation and control of are less immunogenic, more efficient at Fc receptor and immunoglobulin categories. cell-mediated effector functions, and each promoting desired effector functions and Fc receptors (FcRs) are a group of glyco- subclass of IgG (IgG1, IgG2, IgG3 and have longer half-lives. More recently, a proteins belonging to the immunoglobulin IgG4) exhibits a distinct profile of effector new generation of therapeutic antibodies superfamily and are organized into classes, function, which is dictated by differential has been engineered, through techniques such as FcαR, FcγR and FcεR; FcγRs are binding to each of the FcγRs5,6. Whereas such as amino acid mutagenesis, altered expressed mostly on leukocytes and they are both IgG1 and IgG3 bind relatively strongly

Table 1 | Classification and effector function potential of FDA-approved therapeutic antibodies and Fc fusion proteins generic name (trade Format Targets Approved Proposed Therapeutic initial name; sponsoring indications mechanisms of antibody assessment companies) action* class of effector function potential|| Alemtuzumab Humanized IgG1κ cD52 cLL Induction of ADcc I High (campath; Genzyme/ Bayer) cetuximab (erbitux; chimeric (murine/ eGFr Metastatic Inhibition of eGFr I High Imclone/Bristol-Myers human) IgG1κ colorectal cancer, signalling; induction of squibb/Merck–serono) and head and neck apoptosis and ADcc; cancer sensitization of cells to chemotherapy and radiotherapy Ofatumumab Human IgG1κ cD20 cLL Induction of cDc and I High (Arzerra; Genmab/ ADcc GlaxosmithKline) rituximab (rituxan; chimeric (murine/ cD20 Non-Hodgkin’s Induction of apoptosis, I High Genentech/roche/ human) IgG1κ lymphoma, rA and ADcc and cDc; Biogen Idec) cLL sensitization of cells to chemotherapy Trastuzumab Humanized IgG1κ Her2 Her2- Inhibition of Her2 I High (Herceptin; overexpressing signalling; induction of Genentech/roche) breast cancer ADcc; sensitization of cells to chemotherapy Adalimumab (Humira; Human IgG1κ TNFα‡ rA, JIA, PA, cD, As Neutralization of TNFα II Moderate Abbott) and plaque psoriasis activity

Alefacept (Amevive; LFA3 ecD–Fc (IgG1) cD2 Plaque psoriasis Inhibition of II Moderate Astellas) fusion protein cD2-dependent lymphocyte activation Basiliximab (simulect; chimeric (murine/ cD25 Acute transplant Inhibition of IL-2- II Moderate Novartis) human) IgG1κ rejection mediated activation of lymphocytes Daclizumab (Zenapax; Humanized IgG1κ cD25 Acute transplant Inhibition of IL-2 II Moderate roche) rejection -mediated activation of lymphocytes efalizumab§ (raptiva; Humanized IgG1κ cD11a Plaque psoriasis Inhibition of II Moderate Genentech) cD11a-associated leukocyte adhesion Muromonab-cD3 Mouse IgG2aκ cD3 Acute transplant Blockade of II Moderate (Orthoclone OKT3; rejection cD3-associated T cell centocor Ortho functions Biotech) Tocilizumab (Actemra; Humanized IgG1κ IL-6r‡ rA Inhibition of IL 6r II Moderate Genentech/roche) signalling Golimumab (simponi; Human IgG1κ TNFα‡ rA, PA and As Neutralization of TNFα II Moderate centocor Ortho activity Biotech) Infliximab (remicade; chimeric (murine/ TNFα‡ cD, rA, PA, Neutralization of TNFα II Moderate centocor Ortho human) IgG1κ ulcerative colitis, As activity Biotech) and plaque psoriasis etanercept (enbrel; TNFr2 ecD–Fc (IgG1) TNFα‡ rA, JIA, PA, As and Neutralization of TNFα II Low63 Amgen/Pfizer) fusion protein plaque psoriasis activity Abatacept (Orencia; cTLA4 ecD–Fc, cD80 and rA and JIA Inhibition of II Low Bristol-Myers squibb) mutated IgG1 Fc cD86 cD80/86-dependent T cell activation Denosumab (Prolia; Human IgG2κ rANKL‡ Osteoporosis Neutralization of II Low Amgen) rANKL activity Natalizumab (Tysabri; Humanized IgG4κ α4 subunit of Multiple sclerosis Inhibition of leukocyte II Low Biogen Idec/elan) α4β1 and α4β7 and cD adhesion mediated by integrins α4β1 and α4β7 integrins

Table 1 | (contd) Classification and effector function potential of FDA-approved therapeutic antibodies and Fc fusion proteins Panitumumab (vectibix; Humanized IgG2κ eGFr Metastatic Inhibition of eGFr II Low Amgen) colorectal signalling; induction of carcinoma apoptosis romiplostim (Nplate; Thrombopoietin Thrombopoietin Thrombocytopaenia Activation of II Low Amgen) receptor-binding receptor in patients thrombopoietin peptide–Fc with idiopathic receptor (aglycosylated IgG1) thrombocytopaenic fusion protein purpura Bevacizumab (Avastin; Humanized IgG1κ veGFA Metastatic Neutralization of III Low Genentech/roche) colorectal cancer veGFA activity canakinumab (Ilaris; Human IgG1κ IL 1β cAPs Neutralization of IL-1β III Low Novartis) activity eculizumab (soliris; Humanized IgG(2/4)κ c5 complement Paroxysmal Inhibition of cleavage III Low Alexion) protein nocturnal of c5 to c5a and c5b haemoglobinuria Omalizumab (Xolair; Humanized IgG1κ Ige Allergic asthma Inhibition of binding of III Low Genentech) Ige to FcεrI Palivizumab (synagis; Humanized IgG1κ F protein of rsv Prevention of rsv Neutralization of rsv III Low MedImmune) infection in children rilonacept (Arcalyst; IL-1rI/IL-1rAcP IL-1 cAPs Neutralization of IL-1 III Low regeneron) ecD-Fc (IgG1) fusion activity protein Ustekinumab (stelara; Human IgG1κ p40 subunit of Plaque psoriasis Neutralization of IL-12 III Low centocor Ortho IL-12 and IL-23 and IL-23 activities Biotech) ADcc, antibody-dependent cell-mediated cytotoxicity; As, ankylosing spondylitis; cAPs, cryopyrin-associated periodic syndrome; cD, crohn’s disease; cDc, complement-dependent cytotoxicity; cLL, chronic lymphocytic leukaemia; ecD, extracellular domain; eGFr, epidermal growth factor receptor; Fc, crystallizable fragment; FcεrI, high affinity immunoglobulin ε receptor; Her2, human epidermal growth factor receptor 2 (also known as erBB2); IgG, immunoglobulin G; IL, interleukin; JIA, juvenile idiopathic arthritis; PA, psoriatic arthritis; rA, rheumatoid arthritis; rANKL, receptor activator of nuclear factor-κ B ligand; rsv, respiratory syncytial virus; TNFα, tumour necrosis factor-α; veGFA, vascular endothelial growth factor A. *Based on Us Food and Drug Administration (FDA)-approved label information from the Drugs@ FDA website. ‡exists in both soluble and transmembrane forms. §Withdrawn from the market. ||Fc effector function potential was determined based on the strategy outlined in this paper. The ranking and resulting control strategy can be revised based on additional non-clinical and clinical information obtained on that product. to all FcγRs, IgG1 binding to FcγRIIa/b In ADCC, an antibody first binds to its IgG1 antibody rituximab (Rituxan; isoforms is slightly weaker than IgG3 bind- target on tumour cells, and following this, Genentech/Roche/Biogen Idec) than those ing. By contrast, IgG2 only binds signifi- the Fc portion is recognized by FcγR on patients carrying the FcγRIIIa-F158 allo- cantly to one of the allelic forms of FcγRIIa effector cells. Interaction of the Fc with the type11,12. similar findings were also reported (FcγRIIa-H131, possessing a histidine at FcγR activates effector cells, resulting in the for patients with breast cancer who were position 131), whereas it binds weakly to the release of molecules contained in cytotoxic treated with the humanized HeR2-specific other FcγRs5. IgG4 demonstrates intermedi- granules, such as perforin, granulysin and IgG1 antibody trastuzumab (Herceptin; ate binding to FcγRI and weak binding to granzymes, which leads to lysis of tumour Genentech/Roche)13,14. Taken together, these FcγRIIb. Owing to the relatively lower affin- cells. The ability of therapeutic antibodies results indicate that ADCC is an important ity of IgG2 and IgG4 towards FcγRs, these to induce ADCC depends on their binding mechanism contributing to the clinical effi- two subclasses are sometimes chosen as the affinity to both the target and to the activat- cacy of these therapeutic antibodies. therapeutic antibody backbone when effec- ing FcγR. Another important effector activity tor functions are not desired. It is known that the genetic polymor- mediated by therapeutic antibodies is CDC. phism corresponding to the expression of The binding of the C1q component to the Fc Effector functions. Following binding of valine (v) or phenylalanine (F) at amino is the crucial initial step of the complement immunoglobulins to the various FcRs, acid 158 on the FcγRIIIa greatly influences cascade, leading to CDC. The classical acti- activating FcγRs induce ADCC, endocy- its affinity for IgG1. The FcγRIIIa-v158 vation pathway of CDC is triggered when tosis of immune complexes followed by allotype binds to IgG1 with a higher the C1 complex binds the antibody–antigen antigen presentation, phagocytosis, and affinity than the FcγRIIIa-F158 allotype, complex. Following a cascade of comple- release of cytokines or pro-inflammatory and immune effector cells bearing the ment protein activation, a membrane attack mediators. By contrast, inhibitory FcγRs FcγRIIIa-v158 allele have been shown to complex is formed, leading to lysis of target regulate immune responses by inhibiting mediate more potent ADCC activity than cells. Three approved anticancer antibodies the activation of B lymphocytes, mono- those bearing the F allele in an in vitro utilize this mechanism: rituximab, ofatumu- cytes, mast cells and basophils by activating ADCC assay10. Furthermore, genetic mab (Arzerra; Genmab/GlaxosmithKline) receptors5. studies in FcγR-deficient mice analyses have revealed that patients with and 131I-tositumomab (Bexxar; support the critical role of the different non-Hodgkin’s lymphoma carrying the GlaxosmithKline)3. several approaches have FcγRs in the functional effects of therapeutic FcγRIIIa-v158 allotype responded better to succeeded in enhancing CDC by facilitating antibodies8,9. treatment with the chimeric CD20-specific the binding of the antibody constant region

%NCUU+/1# %NCUU++/1# %NCUU+++/1# produced in CHO cells are fucosylated, and %GNNDQWPFCPVKIGP %GNNDQWPFCPVKIGP 5QNWDNGCPVKIGP a small change in Fc fucosylation may have YKVJFGRNGVKQP YKVJDNQEMKPI YKVJDNQEMKPI a significant effect on the FcγRIIIa binding activity. Recent studies also indicate that the increased ADCC by antibodies with an afucosylated Fc glycan was only observed with natural killer cells as effector cells, and not with the polymorphonuclear cells27. The effect of a terminal galactose on Fc function is less well understood and appears to be antibody-specific. The presence of terminal galactose has been shown to affect C1q binding and CDC without changing ADCC28. However, a recent study performed with different, modified antibodies containing varying levels of terminal galactose suggested that the presence of terminal galactose does affect FcγR binding29. Terminal sialylation also has multiple effects on IgGs. Increased sialylation was shown to reduce binding to FcγRIIIa and as a conse- #RQRVQUKU %&% quence, decrease ADCC30,31. On the other hand, the anti-inflammatory activity of IgG is dependent on sialylation of the N-linked glycan of the IgG Fc fragment30; higher levels 0-EGNN #&%% of antibodies lacking terminal sialic acid and galactose residues were found in patients Figure 1 | classification of therapeutic antibodies based on their putative mechanisms of with autoimmune disorders32. action. Therapeutic antibodies can generally be classified into three categories. class I antibodies recog- Depending on the desired mechanism nize and bind to cell-bound antigens and the complex remains stable. The Fc0CVWT (crystallizableG4GXKGYU fragment)^&TWI&KUEQ effecXG- T[ of action, effector functions of a given tor functions are generally part of the mechanism of action (MOA) for antibodies in this class. examples of therapeutic antibody can be modulated by 11 class I molecules include rituximab (rituxan; Genentech/roche/Biogen Idec) and alemtuzumab either altering the amino acid sequence and/ (campath; Genzyme/Bayer). class II antibodies also recognize and bind to cell-bound antigens but their or through glycoengineering3,10,15,16,33–35, proposed MOA does not involve Fc effector functions. examples of class II molecules include basiliximab although developers would also have to (simulect; Novartis) and efalizumab (withdrawn from the market). class III antibodies bind to and neutralize soluble antigens, and their MOA often involves blocking the soluble ligand from binding to its cognate consider the impact of such changes on the receptor. examples of class III antibodies include bevacizumab (Avastin; Genentech/roche)50 and palivizu- immunogenic potential of the molecule. mab (synagis; MedImmune). ADcc, antibody-dependent cell-mediated cytotoxicity; cDc, complement- Glycosylation alone has rarely been shown dependent cytotoxicity; NK, natural killer. to influence the immunogenic potential of a therapeutic antibody, except in cases in which antibodies are produced in murine to C1q. engineered amino acid mutations resulting from covalent and several non- cells and a specific non-human immu- inserted into either the Fc or the hinge covalent interactions of sugar residues with nogenic epitope is present (for example, region have resulted in antibody constant the protein surface. Thus, whereas glycosyla- Neu5Gc and gal-α1,3gal)36. Cetuximab regions with improved C1q binding15,16. tion of IgG–Fc has been shown to be essen- (erbitux; ImClone/Bristol-Myers squibb/ Binding of IgG to the neonatal FcR tial to the binding and activation of FcγRs, Merck–serono) — a chimeric IgG1κ anti- protects the antibody from degradation, an X-ray structure of the IgG–Fc–FcγRIII body — has a murine glycoform located in thereby prolonging the half-life of IgG in complex reveals minimal contact between the antigen-binding region and it has been the serum17. However, neonatal FcR bind- the oligosaccharide and the receptor20. The shown to induce reactive allergic responses ing does not involve ADCC or CDC, and is requirement for glycosylation is the main in patients who were previously environ- therefore outside the scope of this article. reason why most glycoprotein therapeu- mentally immunized against murine glyco- tics, including antibodies, are produced in form epitopes36,37. Fc glycosylation. Fc glycosylation is neces- eukaryotic systems (for example, Chinese sary for therapeutic antibodies to elicit effec- hamster ovary (CHO) cells, NS0 or Sp2/0 Assessing effector functions. Fc effector func- tor functions18,19. It has long been recognized cell lines) rather than in prokaryotes21. tions can be critical to the safety and efficacy that the glycans attached to asparagine at the The Fc glycan structure is characterized of some therapeutic antibodies. However, 297 position in the CH2 domain of IgG are by its heterogeneity, which has a direct effect there is little published guidance specifying critical for complement activation and FcγR on effector functions of therapeutic antibod- when and to what extent Fc functionality binding18. uniquely, X-ray crystallography ies22. Removal of the core fucose can signifi- should be assessed during the development shows that the IgG Fc glycan is ‘sequestered’ cantly enhance the FcγRIIIa binding affinity life cycle, and whether and when glycosyla- within the protein structure and it has a of IgG1s, resulting in increased ADCC23–26. tion monitoring and control should be defined secondary and tertiary structure, Over 90% of the recombinant antibodies implemented. In addition, our ability to

delineate an appropriate knowledge-based (ADCP) and Fc-dependent apoptosis (FIG. 1). be considered based on the target biology, control strategy has been continuously Class I antibodies can be of the IgG1 or IgG3 the introduction of mutations into the Fc challenged by new scientific information. subclass, and they can either be unmodified, region, or by altering the glycoform It is important to note that antibody glyco- or modified through the introduction of Fc pattern3,10 (TABLE 2). sylation may affect pharmacokinetics and amino acid mutations and/or glycoengineer- pharmacodynamics in addition to effector ing to enhance their effector functions. One Class III. Class III therapeutic antibodies functions, although there are ambiguities common characteristic of this class of anti- are designed to bind soluble antigens and among the published studies38–40. A compre- bodies is their reliance on Fc-mediated func- their MOA often involves blocking the solu- hensive evaluation of efficacy would include tionality to achieve cytotoxic or apoptotic ble ligand from binding to its receptor. For the assessment of effector functions as well responses as the main focus of their MOA this class of IgG1 or IgG3 antibodies, as effects on pharmacokinetics and pharma- (beyond simple antigen binding). These it may be helpful to investigate whether codynamics, but the scope of this article is therapeutic antibodies are designed to have a there are membrane-bound forms of the limited to the impact of glycosylation on Fc high effector function potential (TABLE 2). ligand on any cell types. If this is the case, effector functions. the antibody may need to be classified as With the aim of aiding future develop- Class II. Class II therapeutic antibodies also a Class II antibody with moderate effector ment of therapeutic antibodies, the remain- recognize cell-bound antigens; however, they function potential because there may be der of this article describes a strategy that are not designed to kill antigen-expressing some safety concerns associated with bind- may be used to assess and monitor the target cells. Thus, effector functions are not ing to a cell-bound ligand. IgG2 and IgG4 effector function of therapeutic antibodies, part of their MOA (FIG.1). The main MOA antibodies (or IgG1 and IgG3 antibodies including Fc fusion proteins. Therapeutic of this class of antibodies is to block ligand– with modifications to minimize effector antibodies are classified on the basis of their receptor interactions, and in other cases, to functions) are in the ‘low potential’ category putative mechanism of action (MOA), and act as agonists. (TABLE 2). ranked according to their effector function The Class II category may include anti- potential; the recommended strategy is bodies with moderate or low effector function ADCs. ADCs are constructed by chemically based on the ranking of Fc effector function potential. Because Fc-mediated function is conjugating one or more cytotoxic potential and a suitable control strategy that not part of the intended MOA of Class II small-molecule drugs or radioisotopes reflects this classification — a ‘graduated’ antibodies, IgG2 or IgG4 isotypes (which to an antibody that binds to the desired approach. such an approach is intended to have a lower propensity to elicit effector cell surface antigen target42. The primary help ensure product quality, manufactur- functions) are often selected. However, function of antibodies used to prepare ing consistency and maximum efficacy, some human polymorphisms can be an ADCs is to guide the highly toxic while minimizing the safety risk to patients. exception to this general observation18. conjugated drugs specifically to tumour Although these recommendations may be unmodified IgG1 or IgG3 isotypes within cells, in order to minimize potential applicable to the majority of therapeutic this class are considered to have a moder- systemic toxicity. In cases where there is antibodies and Fc fusion proteins, some ate effector function potential because evidence that the antibody alone (before molecules may require a modified strategy they contain an Fc region that is capable conjugation) has effector functions, because of unique factors that may affect of binding to FcγRs41. However, modula- the evaluation strategy appropriate for effector functions. tion of effector function potential should either Class I or II molecules should be

MOA-based classification of antibodies On the basis of their putative MOA, Table 2 | Ranking of Fc effector function potentials for therapeutic Abs most therapeutic antibodies (including Therapeutic Ab type class i: cell-bound class ii: cell- class iii: soluble Fc fusion proteins) can generally be clas- antigen stable upon bound antigen. antigen. MOA sified into three categories, from which Ab binding. MOA MOA not not involving Fc their potential for Fc functionality can be involving Fc effector involving Fc effector function function (ADCC, CDC, effector function (blocking) ranked (FIG. 1). excluding Fab fragments ADCP) and antibody–drug conjugates (ADCs), there are currently 27 antibodies and Fc IgG1 and IgG3 High Moderate Low fusion proteins approved by the us Food IgG1 and IgG3 with Fc High Not applicable Not applicable and Drug Administration (FDA) for thera- mutations to enhance Fc peutic purposes. Based on FDA-approved functionality label information (available at the Drugs@ Afucosylated IgG1 High Not applicable Not applicable FDA website) and the guidelines described IgG1 and IgG3 with Fc Not applicable Low Low below, the proposed classification and effec- mutations to reduce tor function potential of these molecules are Fc functionality, or aglycosylated IgG1 and IgG3 summarized in TABLE 1. IgG2 and IgG4; IgG2 and Not applicable Low Low Class I. Class I therapeutic antibodies recog- IgG4 with Fc mutations to reduce Fc functionality, or nize cell surface antigens and are designed aglycosylated IgG2 and IgG4 to kill target cells through Fc-mediated Ab, antibody; ADcc, antibody-dependent cell-mediated cytotoxicity; ADcP, antibody-dependent cellular effector functions, including ADCC, CDC, phagocytosis; cDc, complement-dependent cytotoxicity; Fc, crystallizable fragment; IgG, antibody-dependent cellular phagocytosis immunoglobulin G; MOA, mechanism of action.

(+*CRRNKECVKQP #RRTQXCN

4GUGCTEJ 2TGENKPKECN 2JCUG+ 2JCUG++ 2JCUG+++ 2QUVOCTMGV

r'ȭGEVQTHWPEVKQP r'ȭGEVQTHWPEVKQPOQPKVQTKPI /1# OQPKVQTKPIYKVJ +PKVKCNGXCNWCVKQP TQWVKPGOQPKVQTKPICPFQT EQPȮTOGF CPFFGOQPUVTCVKQP EJCTCEVGTK\CVKQPCUUC[U XCNKFCVGFQT r+FGPVKȮECVKQPCPFOQPKVQTKPI EJCTCEVGTK\CVKQP *KIJ QHGȭGEVQT HWPEVKQPUVQDG QH%3#UKPENWFKPIECTDQJ[FTCVGU CUUC[U KPENWFGFKP CȭGEVKPIGȭGEVQTHWPEVKQP r/QPKVQTKPI%3#U (+*RCEMCIG RQVGPVKCN TQWVKPGOQPKVQTKPI KPENWFKPIECTDQJ[FTCVGU CPFQTEJCTCEVGTK\CVKQPCUUC[U CȭGEVKPIGȭGEVQT HWPEVKQPRQVGPVKCN YKVJXCNKFCVGFQT (EGȭGEVQTHWPEVKQP EJCTCEVGTK\CVKQPCUUC[U PQVENKPKECNN[FGOQPUVTCVGF

r'ȭGEVQTHWPEVKQPEJCTCEVGTK\CVKQP 0Q 6JGTCRGWVKE HQTEQORCTCDKNKV[CPF EJCPIGKP(E CPVKDQFKGUQT +PKVKCNGXCNWCVKQP OCPWHCEVWTKPIEQPUKUVGPE[ RQVGPVKCN (EHWUKQP 'ȭGEVQTHWPEVKQP QHGȭGEVQT r+FGPVKȮECVKQPCPFEJCTCEVGTK\CVKQP EJCTCEVGTK\CVKQPHQT RTQVGKPU /QFGTCVG HWPEVKQPUVQDG QH%3#UKPENWFKPIECTDQJ[FTCVGU (EGȭGEVQT EQORCTCDKNKV[CPF KPENWFGFKP CȭGEVKPIGȭGEVQTHWPEVKQP OCPWHCEVWTKPIEQPUKUVGPE[ HWPEVKQP (+*RCEMCIG RQVGPVKCN EJCTCEVGTK\CVKQPCUUC[U RQVGPVKCN HQTEQORCTCDKNKV[CPF OCPWHCEVWTKPIEQPUKUVGPE[

(EGȭGEVQTHWPEVKQP FGOQPUVTCVGFENKPKECNN[

+PKVKCNFGOQPUVTCVKQP QHTGFWEGFQT 0QPGGFVQOQPKVQT(EGȭGEVQTHWPEVKQPWPNGUU .QY CDNCVGFGȭGEVQT PGYFCVCEJCPIKPIVJG(ERQVGPVKCN HWPEVKQPUVQDG KPENWFGFKP (+*RCEMCIG /GODTCPGDQWPFHQTOKPCFFKVKQP VQUQNWDNGVCTIGVKFGPVKȮGFDWV PQGȭGEVQTHWPEVKQPUJQYP

Figure 2 | Assessment and control strategies for effector functions. affects effector functions. With the progress of drug development, data This decision tree summarizes the assessment process and recommended from clinical studies or other investigations0CVWT mayG4G provideXKGYU additional^&TWI&KUEQ inforXGT[- strategies for therapeutic antibodies. Based on the mechanism of action mation on the MOA of the therapeutic antibodies, and this may prompt (MOA), immunoglobulin G isotype and engineering status, therapeutic reclassification of the molecule and re-evaluation of the Fc effector func- antibodies can be ranked into three groups with high, moderate or low tion potential. In this case, assessment and control strategies appropriate effector function potential (TABLE 1). It is recommended that an initial to the reclassified group should be applied. It is optimal to have appropri- evaluation, mostly using in vitro methods to demonstrate the presence ate assays in place before testing material intended for the pivotal clini- or absence of effector functions, should be carried out at the preclinical cal studies. When a product reaches the marketing application stage, a stage of development, and the data supporting MOA — including control strategy should be in place and appropriate assays should be vali- Fc (crystallizable fragment) effector function potential — should be dated to support commercial quality control testing. The knowledge included in the package for first-in-human (FIH) applications. During gained during development should be explained in the marketing appli- clinical development and manufacturing, different strategies appropriate cation and it should form the basis of the ‘Justification of specifications’ to each potential group can be devised to assess effector functions and section as well as other sections (for example, in ‘Process controls’), critical quality attributes (cQAs), particularly Fc glycosylation, which where relevant. considered. Although the intended scope discovery, development, and, in some cases, has less of a role. Conversely, a moderate of this paper covers only unconjugated post-marketing. The classification described potential antibody or an IgG2 antibody antibodies, the principle for assessment above is a general one and relies on both the may be able to mediate effector functions of Fc effector functions and the resulting intended use of the antibody (for example, under certain circumstances, as described control strategy can also be applied when effector function is desired as part previously18. In this case, the antibody to ADCs. of the MOA) and the known properties of should be reclassified into the higher the molecule. For example, a therapeutic potential group. Reclassification of therapeutic antibodies. antibody may be expected to be in Class I in IgG1 and IgG3 antibodies targeting solu- understanding the MOA of a therapeutic preclinical studies, but subsequent clinical ble ligands have low Fc effector function antibody is a continuous process throughout studies may show that effector function potential. However, if emerging evidence

Box 1 | Methods for assessing Fc (crystallizable fragment) effector functions could be either intentionally or uninten- tionally involved in their MOA. Antibody Antibody-dependent cell-mediated cytotoxicity engineering often involves an amino acid The conventional antibody-dependent cell-mediated cytotoxicity (ADCC) assay is often highly mutation or a glycomodification to enhance variable, owing to the use of effector cells isolated from normal human peripheral blood mononuclear effector functions. For molecules in this cells (PBMCs). Assay variation can be reduced by using selective donor pools with defined Fcγ group, a thorough evaluation of effector receptor IIa (FcγRIIa) or FcγIIIa genotypes, or by replacing PBMCs or PBMC-derived natural killer (NK) functions using the assays described in cells with a CD16a (Fc RIIIa)-expressing cell line (for example, engineered NK92). Killing of the target γ BOX 1 is recommended before and during cells can be assessed by measuring the release of specific probes from prelabelled target cells, using 51Cr or fluorescent dyes such as calcein-AM44,45, carboxyfluorescein succinimidyl ester (CFSE)46,47, the early phases of clinical development. 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)48 or Europium49, or by measuring the Ideally, this evaluation should be completed release of cytosolic enzymes such as lactate dehydrogenase (LDH)50 or ATP. before the first-in-human studies and if at all Antibody-dependent cellular phagocytosis possible, at the stage where clonal selection Antibody-dependent cellular phagocytosis (ADCP) measures the destruction of target cells via is occurring, in order to support a desired monocyte or macrophage-mediated phagocytosis. ADCP assays use PBMC-derived cells or U937 MOA. efforts to confirm the MOA and to cells differentiated to the mononuclear type. The phagocytosis readout requires tracking understand the properties of the molecule fluorescently tagged target cells by either flow cytometry47 or confocal microscopy51. that impact effector functions are war- complement-dependent cytotoxicity ranted throughout the product development The complement-dependent cytotoxicity (CDC) assay can be performed with non heat-treated serum life cycle43. or commercially available complement fractions10. Target cell killing can be assessed by several cell During clinical development, relevant viability reagents such as Alamar Blue10,15, CellTiter-Glo52, LDH release53 or calcein-AM release54. effector function assays, both cell-based Fc-dependent apoptosis and binding, are used to assess the activity This is one of the effector activities in which target cells are destroyed55. Apoptosis can be assessed of manufactured lots in order to help pro- by an Annexin V assay or a caspase 3 or caspase 7 assay55. vide an overall understanding of the criti- Fcγrs and c1q binding cal quality attributes of the molecule. If the Surrogate binding methods may be used for the assessment of Fc effector functions. They measure effector function is proven to be involved complementarity determining region (CDR) binding to the cell surface target antigen, and Fc in the MOA, it may be necessary to imple- binding to FcγRs, either separately or simultaneously. Various flow cytometry and plate-based ment one or more effector function assays formats can be used for CDR and FcγR binding assays10. Recombinant protein receptor binding as part of routine monitoring for manufac- and C1q binding assays have been developed with a number of platforms, including surface turing, depending on the role that effector 56 15 plasmon resonance and enzyme-linked immunosorbent assays . functions have in antibody efficacy. The Fcγr polymorphism selected assay (or assays) for routine testing Several clinical studies with therapeutic antibodies have demonstrated increased clinical activity also need to be suitable for implementation 57–60 in patients expressing the high-affinity 158 (V/V) phenotype in FcγRIIIa . In light of the potential in a quality control environment. These impact of position 158 polymorphisms in Fc RIIIa on the efficacy of therapeutic antibodies, γ tests should be qualified and subsequently characterization of the binding affinity of therapeutic antibody candidates to the high and low validated at the pivotal study phase and affinity versions of FcγRIIIa may be warranted. A functional polymorphism has also been identified on FcγRIIa. Thus, if primary PBMCs or NK cells are used for ADCC or other effector function assays, before filing the marketing application. the donor’s FcγRIIa and FcγRIIIa genotype should be determined so that the resulting phenotype However, not all Fc functional assays are can be considered when interpreting the assay data. easily implemented in routine GMP (good manufacturing practice) testing. Therefore, if effector function is part of the proposed points to the existence of a membrane- necessarily have a high effector function MOA, the developer should develop a suit- bound form of the ligand, or if the ligand– potential because of its target biology, or an able control strategy to test for any specific antibody complexes bind to the cell surface, IgG2 antibody may only have some effector attribute (or attributes) of the molecule the effector function potential of the IgG1 function in a subset of patients with certain that play a part in Fc function, in addi- and IgG3 therapeutic antibodies should be polymorphisms. tion to any validated Fc functional assay. re-evaluated, and, if necessary, they should strategies to monitor and control struc- However, Fc functional assays that are not be reclassified into the moderate potential tural attributes and/or effector functions suited to quality control lot release along group. Any relevant knowledge of target during antibody manufacturing should be with other relevant tests can be utilized biology and MOA data from non-clinical customized according to the corresponding for product characterization during non- and clinical studies can prompt reclassifica- effector function potentials (high, moder- clinical and clinical development and for tion of therapeutic antibodies at any stage ate or low). Below, we propose a strategy for comparability studies. of development, and should lead to a re- assessing Fc effector functions of therapeutic Methods of assessing Fc effector func- evaluation of effector function potential and antibodies and Fc fusion proteins, based on tions of therapeutic antibodies are sum- control strategies. their Fc effector function potential ranking marized in BOX 1. Cell-based assays (such (FIG. 2). as ADCC, ADCP and CDC) or surrogate Strategies for assessing effector functions binding assays (when cell-based assays are Within each of the three classifications, Strategies for therapeutic antibodies with not feasible) can be used for either routine molecules can exhibit different levels of high effector function potential. The high monitoring or characterization. similarly, effector functions depending on their nature effector function potential group refers to product quality attributes that may have and design. For example, an IgG1 antibody unmodified or engineered IgG1 or IgG3 a role in effector function, such as glyco- that binds to a cellular antigen may not antibodies in which Fc effector functions form content and structure, should also be

Box 2 | Methodology for assessing Fc (crystallizable fragment) glycosylation Fc functionality on either safety or efficacy. In this case, there is no need to implement Effector function testing should be tightly coordinated with other analytical assessments of Fc functional assays in routine monitoring. product quality attributes. The characterization of immunoglobulin G1 (IgG1) Fc glycosylation is of However, an understanding of the attributes utmost importance, owing to the established correlation between fucosylation and effector of the molecule that may modify (for exam- function. IgG1 fucose content can be measured at the monosaccharide or oligosaccharide level. Monosaccharide analysis involves the complete hydrolysis of N-glycans into individual ple, enhance) the effector function potential monosaccharides61. One method for separating and detecting monosaccharides utilizes high is still warranted. The focus here is to select performance anion exchange chromatography followed by pulsed amperometric detection 61. An the appropriate methods (BOX 1) to support alternative method involves labelling of monosaccharides with the fluorescent dye product characterization and comparability, 2-aminobenzoic acid amide followed by reversed phase high performance liquid chromatography and to ensure manufacturing consistency. (HPLC) separation and fluorescence detection61. However, this method is complicated by the difficulty in achieving consistent labelling between neutral and amino sugars. Strategies for therapeutic antibodies with In general, monosaccharide analysis does not offer glycosidic linkage information and it is low effector function potential. The low limited by relatively low sensitivity. Oligosaccharide analysis allows for better detection of minor effector function potential group of thera- glycovariant species with some glycosidic linkage information that may be important in some peutic antibodies includes unmodified IgG1 cases for characterizing product quality attributes. These methods involve release of oligosaccharides from the antibody using PNGase F (also known as peptide: N-glycosidase F). and IgG3 that target only soluble ligands, Several methods can be used to quantify fucose content in free oligosaccharides. One method IgG2 and IgG4 subclasses that have much involves labelling of oligosaccharides with the fluorescent dye 2-aminobenzamide, followed by less potential for effector functions, and normal phase or reversed phase HPLC separation and mass spectrometry detection61. A second mutated or aglycosylated IgG1 or IgG3 sub- method can be performed by labelling glycans with the charged fluorescent dye classes that are specifically designed to not 1-aminopyrene-3,6,8-trisulphonate (APTS), followed by separation by capillary zone have any effector function. It is important to electrophoresis and detection by laser-induced fluorescence62. note that although mutated or aglycosylated IgG1 or IgG3 antibodies are intentionally designed to ablate effector function, this appropriately controlled. The methodology the antibody itself — for example, whether it should be confirmed using appropriate for assessing Fc glycosylation is summarized contains a functional Fc region — other fac- methods (BOX 1). For most of these low in BOX 2. tors, many of them target-related, will prob- potential molecules, an initial evalua- It is also worthwhile to comment on non- ably contribute to the final outcome; that is, tion using an appropriate in vitro system fucosylated therapeutic antibodies. These whether effector function will actually occur. is recommended, in order to confirm the molecules have FcγR binding properties For example, IgG1 targeting cell surface decreased or absent effector functions, and that are significantly different from their antigens may not induce any effector func- this should preferably be carried out at the fucosylated counterparts, and their efficacy tions, and the absence of this activity could earlier stages of product development. The and safety profiles may be distinct. However, be attributable to insufficient expression of subsequent strategies may differ slightly for levels of fucose, which is one of the glycan the cell-bound target, rapid internalization different types of molecules. variables known to affect effector functions, and/or downregulation or shedding of the In the case of IgG1 and IgG3 antibod- may be well controlled, owing to production target upon antibody binding. ies that target soluble ligands, monitor- cell line engineering. Other glycan variations Transfected cell lines expressing high, ing and controlling effector function may may also have less of an impact because of the non-physiological levels of target are some- not be required if it is confirmed that the overall strong Fc functionality resulting from times used when performing the initial ligand only exists in the soluble form and afucosylation. Therefore, a refinement of the evaluation. In some cases, these transfected is not found on any cell surface. If emerg- overall strategies should be considered in the lines are the only source of cells available ing evidence points to the existence of the case of non-fucosylated antibodies, and rou- for such an assessment. In particular, when membrane-bound form of the ligand, or if tine glycan testing may not always be needed. low levels of effector function activity are the ligand–antibody complexes bind to the observed, these results should be cautiously cell surface, the effector function potential Strategies for therapeutic antibodies with interpreted because the in vitro activity may of the IgG1 and IgG3 therapeutic antibod- moderate effector function potential. or may not be relevant in vivo. ies should be re-evaluated, and, if necessary, Moderate effector function potential mol- Data obtained from the initial evaluation they should be reclassified into the moderate ecules can include unmodified IgG1 and will influence the subsequent development group with adaptation of the strategies rec- IgG3 antibodies. Although possessing a fully strategies, including in vivo study and safety ommended for that group. functional Fc, the effector functions of these assessment, as well as process development For aglycosylated IgGs produced from antibodies, if present, are not part of their and manufacturing. Throughout product Escherichia coli, or Fc aglycosylated IgGs desired MOA. Therefore, the concerns asso- development, any new data will continue to from CHO cells (created by introducing ciated with Fc functionality should focus influence the strategy. If a therapeutic anti- a mutation at the Fc N-linked glycosyla- on its potential impact on safety. similarly body initially designated as having moderate tion site), the initial assessment could be to the high potential molecules, it is recom- potential is later reclassified into the done with glycan assays to demonstrate the mended that an initial evaluation should higher potential group, strategies appropri- absence of Fc glycans, and with binding be conducted to analyse effector functions ate for high potential molecules should then assays to show the absence of binding activ- before the first-in-human studies. be applied. ity to FcγRs. subsequent assessment of effec- It is often challenging to prove the Alternatively, the molecule will remain in tor function is therefore not necessary. For absence of effector functions for this class of the moderate potential group if there are no IgG2 and IgG4 antibodies, and for mutated antibodies. In addition to characteristics of additional data demonstrating the impact of IgG1 and IgG3 antibodies with intact

glycan structures and decreased (but not impact of antibody effector function in first- The glycosylation profile of a therapeutic absent) effector functions, limited follow-up in-human studies, as well as for interpreta- antibody should be assessed for characteri- strategies may be needed. In each case, the tion of the data from preclinical studies. zation purposes during development, as strategy will depend on risk assessments for As development moves through the later well as during comparability studies. The patient safety and efficacy, as well as manu- stages, an understanding of whether effec- combination of understanding the effect of facturing experience. tor function (if present) plays a part in safety glycosylation and effector function and the The FcγR binding activity is not com- or efficacy should be established through impact of this on preclinical and clinical pletely absent in IgG2 and IgG4 antibodies, preclinical and/or clinical studies, as this will safety and efficacy profiles, in addition to a and these molecules may be able to mediate help dictate the most appropriate control thorough understanding of the criticality of Fc-related functions under certain condi- strategy, as illustrated in FIG. 2. It is always quality attributes and a robust manufactur- tions. As additional knowledge is gained, optimal to have appropriate assays in place ing process, can serve to ensure product especially relating to polymorphisms in before testing material intended for the piv- safety and efficacy. the general population, the effector func- otal clinical studies, because these studies are Xu-Rong Jiang, Robert Strouse and Mark Schenerman tion potential for IgG2 and IgG4 antibod- intended to confirm the safety and efficacy are at Analytical Biochemistry, MedImmune, ies should be considered, and appropriate profile of the product and thus strengthen the Gaithersburg, Maryland 20878, USA. strategies, including those described in this link between laboratory testing and the clinic. An Song and Shan Chung are at BioAnalytical Sciences, paper, need to be utilized. When a product reaches the marketing Genentech, South San Francisco, California 94080, USA. application stage, a control strategy based Svetlana Bergelson is at Analytical Development, A life cycle approach to effector functions on the accumulated knowledge regarding Biogen Idec, Cambridge, Massachusetts 02142, USA. It is clear from the discussion above that a effector functionality should be in place. Thomas Arroll is at Cellular Resources, Amgen, thorough understanding of whether a thera- Appropriate validated assays should also Seattle, Washington 98119, USA. peutic antibody does or does not have effector be in place to support commercial quality Bhavin Parekh is at Bioassay Development, function (and whether the effector function control testing. The knowledge gained dur- Eli Lilly and Company, Indianapolis 46221, USA. has a role in its MOA) is essential to define ing development should be explained in the Kimberly May is at Biologics Manufacturing Sciences an appropriate control strategy. However, it is marketing application and it should form the and Commercialization, Merck & Co., Union, recommended that developers also consider basis of the ‘Justification of specifications’ New Jersey 07083, USA. the stage of the development life cycle at section and other sections (for example, in Anthony Mire-Sluis is at Global Product Quality and which they should evaluate the presence or ‘Process Controls’), where relevant. Quality Sciences, Amgen, Thousand Oaks, California absence of Fc functionality and its role in the 91320, USA. Concluding remarks safety and efficacy of the molecule. Correspondence to X-.R.J. It is hardly surprising that it is in our The Fc region of therapeutic antibodies e-mail: [email protected] interest to thoroughly understand how can have an important role in safety and doi:10.1038/nrd3365 these molecules work, so that undesirable efficacy. In this article, we have presented a 1. Lee, J. I., Zhang, L., Men, A. Y., Kenna, L. S. & Huang, consequences do not occur during late strategy for assessing Fc effector functions of S. M. CYP-mediated therapeutic protein–drug development (or even more critically, post- therapeutic antibodies (including Fc fusion interactions — clinical findings, proposed mechanisms and regulatory implications. Clin. Pharmacokinet. 49, approval), as clinical studies are expensive proteins) based on a three-tiered ranking 295–310 (2010). and it is harder to redevelop molecules with- of Fc effector function potential. We rec- 2. Reichert, J. M. Antibodies to watch in 2010. 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