Regimen
Disease status
n
ORR
CR
Survival
Reference
R-GEM
High-grade B-NHL (elderly: 64–78 years)
7 (1 previously untreated)
71 %
29 %
PFS:10 m
[344]
OS:11 m
R-GEMOX
Aggressive NHL
46
83 % at the end of fourth cycle
50 %
OS: 66 %
[345]
74 % at the end of treatment
72 % at the end of treatment
EFS:43 %
FU:28 m
R-GIFOX
Aggressive NHL
13
77 %
54 %
EFS:80 %
[346]
FU: 6 m
GaRD
Aggressive NHL
19
79 %
42 %
NG
[103]
GaRD
Aggressive B-NHL
22
55 %
27 %
NG
[347]
R + E
DLBCL
6
67 %
50 %
NG
[348]
R + E
DLBCL
15
47 %
33 %
PFS:6 m
[139]
FU:11 m
R + CMD
DLBCL (elderly: 65–79 years)
30
74 %
57 %
OS:45 %
[349]
PFS:37 %
FU:2 years
R + TTP
Aggressive NHL
71
70 %
25 % in primary refractory
DR:21 m
[350]
56% in relapsed
FU:26 m
R + TTP
DLBCL
10
60 %
30 %
NG
[351]
R + ADOX
DLBCL
20
70 %
25 %
Median survival: 11 m
[352]
R-CHOP
DLBCL
108
NG
NG
Significant improvement vs. CHOP
R-CHOP-like
DLBCL
194 (97 GCB vs. 97 bob-GCB)
NG
NG
OS (GCB: 70 % vs. non-GCB: 47 %)
[104]
FFS, 59 % vs. 30 %,
FU: 52 m
R-TCOP
DLBCL
38
NG
NG
OS and FS both significantly greater compared to TCOP alone
[354]
R-CHOP-like
DLBCL
113
NG
NG
OS: 77 %
[355]
EFS:59 %
R-DHAOX
DLBCL
42
42.85 %
26.19 %
OS: 71 %
[102]
PFS: 44 %
FU:2 years
8.3.3 Targeting CD20 with New Anti-CD20 mAbs
Despite the promising results with rituximab, resistance is expected to be observed in about 60 % of the previously responding FL patients, shedding light on the importance of developing other anti-CD20 mAbs [13]. To stem the tide of resistance to CD20 mAb, a new mind-set has been adopted, leading to the development of second- and third-generation CD20 mAbs. The presence of humanized or completely human CDR in second-generation anti-CD20 mAbs leads to significant reduction in the formation of human anti-chimeric Abs, preventing therapy resistance. Third-generation anti-CD20 mAbs are not only reinforced by a humanized CDR region, but also a modified Fc region is present, leading to stronger activation of the complement system or effector cells [13].
The development of novel anti-CD20 Abs aims at constructing new Abs which exhibit significant activity in patients with rituximab-resistant lymphoma or possess increased efficacy in comparison with rituximab in head-to-head comparative trials. Nonetheless, available data in rituximab-refractory patients is scarce, and further studies are needed to establish their clinical application. According to FDA guidelines, rituximab resistance is defined as disease on progression during rituximab monotherapy or rituximab chemotherapy. Progressive disease or relapse less than 6 months after the last rituximab infusion or after the last course of rituximab chemotherapy is also considered as rituximab resistance.
Anti-CD20 mAbs are divided into two subgroups, types I and II. Type I anti-CD20 mAbs trigger the complement system through aggregating CD20 molecules into lipid microdomains upon binding [74]. Conversely, type II antibodies do not activate CDC. Instead, they lead to the induction of direct cell death, through homotypic adhesion and actin-dependent lysosome-mediated cell death [105]. In the following, brief description is provided on the new generations of CD20 mAbs applied in lymphoma. Comparison between CD20 mAb variants is provided in Tables 8.2 and 8.3.
Table 8.2
Comparison between the different properties of different generations of anti-CD20
Anti-CD20 | Generation | Localize CD20 to lipid rafts | ADCC activity | CDC activity | Direct induction of cell death | Homotypic aggregation | Number of binding sites on CD20 | ORR | Reference |
|---|---|---|---|---|---|---|---|---|---|
Type I | |||||||||
Rituximab | I | + | ++ | +++ | ± | – | 100 % | 38 % | [108] |
Reengineered rituximab | I | + | ++ | +++ | ± | – | 100 % | NG | |
Ocrelizumab | II | + | ++ | +++ | ± | – | 100 % | 38 % | [109] |
Ofatumumab | II | + | ++ | +++ | ± | – | 100 % | 42 % | [111] |
Veltuzumab | II | + | ++ | +++ | ± | – | 100 % | 44 % | [110] |
AME-133 | III | + | ++ | +++ | ± | – | 100 % | NG | |
PRO131921 | III | + | ++ | +++ | ± | – | 100 % | NG | |
Type II | |||||||||
B1 (tositumomab) | I | – | ++ | – | +++ | + | 50 % | NG | |
GA101 | III | – | ++ | – | +++ | + | 50 % | (Low dose) 1/13 (8 %) | [119] |
(High dose) 6/11 (55 %) | |||||||||
Small modular immunopharmaceutical anti-CD20 protein | |||||||||
TRU-015 | ++ | + | + | – | NG | ||||
Table 8.3
Clinical trials on novel anti-CD20
Monoclonal Ab | Generation | IgG type | Patients | Disease status | Regimen | ORR | Adverse events | Reference |
|---|---|---|---|---|---|---|---|---|
Ofatumumab (OFA) | 2nd | IgG1 Kappa | 116 | Refractory FL | Ofatumumab | 13 % 10 % | Infections, rash, urticaria, pruritus, neutropenia, anemia, thrombocytopenia | [356] |
500 mg 1,000 mg | ||||||||
59 | (Untreated FL) | O-CHOP | 90 % 100 % | Leucopenia, neutropenia | [357] | |||
500 mg 1,000 mg | ||||||||
Veltuzumab | 2nd | IgG1 | 82 | Relapsed/refractory B-cell NHL | Veltuzumab | 44 % (FL) 83 % (MZL) 43 % (DLBL) | Fatigue, pruritus, asthenia, fever, dyspnea, headache, infection | [110] |
80–750 mg/m2 | ||||||||
Ocrelizumab | 2nd | IgG1 | 47 | Relapsed/refractory FL | (Ocrelizumab) | 38 % | Infusion-related reaction, nasopharyngitis, asthenia, lymphopenia, infection | [109] |
750 mg/m2 | ||||||||
Obinutuzumab (GA-101) | 3rd | IgG1 | 21 | Refractory B-cell NHL | Obinutuzumab | 60 % 35 % | Infusion-related reaction, neutropenia, anemia, thrombocytopenia, tumor lysis syndrome | [358] |
1,600/800 mg 400/400 mg | ||||||||
28 | Relapsed or refractory FL | G-CHOP | 94 % | Infusion-related reaction, neutropenia, neuropathy, infection | [358] | |||
1,600/800 mg 400/400 mg | ||||||||
28 | Relapsed or refractory FL | G-FC | 93 % | Infusion-related reaction, neutropenia, rash, infection | [358] | |||
1,600/800 mg 400/400 mg | ||||||||
PRO131921 | 3rd | IgG1 | 24 | Relapsed/refractory B-cell NHL | PRO131921 | 27 % | Infusion-related reaction, upper respiratory tract infection, neutropenia | [359] |
25–800 mg/m2 | ||||||||
Ocaratuzumab (AME-133v) | 3rd | IgG1 | 56 | Relapsed/refractory FL | Ocaratuzumab | 36 % | Infusion-related reaction, nasopharyngitis, asthenia, lymphopenia, infection | [360] |
100 mg/m2 375 mg/m2 |
8.3.4 First-Generation Anti-CD20 mAb
8.3.4.1 Reengineered Rituximab
A variety of reengineered rituximab variants have been developed recently. The triple variant, constructed by three-point mutations in the CDR region, targets the same epitope as rituximab. Nonetheless, it is more efficient in the induction of apoptosis. In a rituximab-resistant lymphoma mouse model, the triple variant led to prolonged survival [106]. Furthermore, a genetically engineered tetravalent version of rituximab has been constructed which possesses a stronger anti-lymphoma and antiproliferative effect in vitro and in a lymphoma mouse model [107].
8.3.4.2 Tositumomab (B1)
Tositumomab, a clinically used type II murine IgG2a mAb with a covalently bound iodine-131 radioisotope, has yielded promising results in the treatment of low-grade lymphoma. It is an efficient caspase-independent mAb with antiproliferative effect. However, it acts through a non-apoptotic manner. The direct effect of antibody binding, as well as the cytotoxic effect of irradiation, has led to its potentiality [117]. Tositumomab proved more efficacious in a mouse model compared to rituximab [118]. Even though tositumomab lacked rituximab’s efficiency in CDC induction, it displayed the same ability in the activation of Fc-bearing effector cells, possessing the same binding affinity and half-life in vivo. Furthermore, it significantly surpassed rituximab in B-cell depletion from the periphery and from secondary lymphoid organs [118]. Its ability to directly induce nonclassical apoptosis can explain its superiority. In a clinical study, administration of 900 mg of unlabeled mAb prior to therapeutic anti-CD20 radioimmunoconjugate exerted an efficient tumor-reduction effect [117]. It has provided the opportunity to treat lymphoma through CDC-independent pathways [118].
Low-dose type II antibody, GA101 (400 mg/infusion on days 1, 8, and 22 and subsequently every 3 weeks for a total of nine infusions), yielded an ORR of 8 %. High-dose GA101 treatment (1,600 mg on days 1 and 8, followed by 800 mg thereafter) resulted in significant activity (55 % ORR) in rituximab-refractory patients. Therefore, a dose–effect relationship is proposed [119].
8.3.4.3 Veltuzumab (hA20, IMMU-106)
Veltuzumab, a mAb constructed on the framework regions of the humanized anti-CD22 mAb epratuzumab, consists of CDRs taken from the murine A20 anti-CD20 mAb. During preliminary studies, no difference was observed between the effectiveness of rituximab and veltuzumab. Further studies demonstrated a slower off-rate with superior CDC and significantly improved therapeutic outcomes with veltuzumab in different lymphoma models, as well as potent anti-B-cell activity in cynomolgus monkeys [120].
Low doses of veltuzumab at short infusions were well tolerated with no serious adverse events in 55 pretreated patients with FL in a multicenter phase I/II trial [110], which were comparable with the ranges in rituximab re-treatment in patients with relapsing disease [108]. Subcutaneous injection of veltuzumab resulting in a slow release pattern over several days with a rapid depletion of B cells was studied in a phase I/II study, yielding an OR of 55 % and a CR rate of 20 % [121]. A modified hexavalent antibody named hex-hA20 has been developed with interesting properties of both type I and II antibodies, capable of inducing lipid raft formation (type I), in addition to its antiproliferative and apoptotic properties and homotypical adhesion induction (type II); nonetheless, it has a shorter half-life in comparison with its origin [122].
8.3.4.4 Ocrelizumab (PRO70769, rhuH27)
Ocrelizumab, a type I mAb with an IgG1 isotype generated from the murine 2H7 anti-CD20 Ab, predominantly acts through ADCC mechanism, while it has a reduced capacity of CDC compared to rituximab [123]. Its efficient B-cell removal, from the periphery and to a lesser extent in secondary lymphoid organs, has been observed in monkeys achieving results comparable to rituximab [79]. Even though it is postulated to be more effective than rituximab due to its enhanced ADCC capabilities, mild infusion-related toxicity with doses up to 750 mg/m2 was experienced in a phase I/II dose-escalating trial administered to previously rituximab-treated FL patients [109]. However, decreased intravascular complement activation was proposed to be attributable to the observed toxicity; moreover, the safety profile was similar to rituximab [109].
8.3.5 Second-Generation CD20 mAb
Second-generation type I anti-CD20 mAbs, including ofatumumab, veltuzumab, and ocrelizumab, have been constructed which are effective in patients with FL previously treated with rituximab. Nonetheless, in a comparison between the clinical responses to these mAbs with the responses of patients who received rituximab re-treatment or maintenance therapy, no significantly improved survival was demonstrated. In comparable patient populations from different studies, the ORR for rituximab re-treatment was 38 % [108], 38 % for ocrelizumab [109], 44 % for veltuzumab [110], and 42 % in those receiving ofatumumab [111].
8.3.5.1 Ofatumumab (Arzerra, HuMax-2F2)
Ofatumumab, a completely human anti-CD20 mAb, is constructed in a human transgenic mouse. Remarkable activation of the complement system is its outstanding property. In addition, it is favored by the ability to kill target cells with much lower numbers of CD20 molecules on the cell surface, requiring less human serum, as it binds two to three times more than rituximab to C1q [112]. Binding of ofatumumab to the small 7-mer extracellular loop of the CD20 molecule close to the cell membrane accounts for its exceptional efficiency in complement fixation [112]. Nonetheless, rituximab is significantly more efficient in ADCC activation pathway in patients with NK cells expressing the FcγRIIIa-158V allotype than the low-affinity receptor FcγRIIIa-158F allotype [113]. Promising results were obtained with in vitro and in vivo administration of ofatumumab against rituximab-resistant, as well as rituximab-sensitive models [114]. Heavily pretreated patients with relapsed/refractory FL in the first phase I/II clinical trial yielded an ORR of 42 %. In addition, it is considered as a safe agent [111]. These results were in the same range with rituximab re-treatment patients [108]. In addition, an 11 % OR with a mean duration of 6 months was achieved with ofatumumab monotherapy in 116 heavily pretreated FL patients refractory to rituximab; patients refractory to rituximab monotherapy achieved a 22 % ORR [115]. Furthermore, promising results have been obtained with ofatumumab in combination with CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) chemotherapy. An ORR up to 100 % was achieved, as well a 69 % complete response with favorable toxicity profiles [116].
Overall, some studies have demonstrated an ORR of 11–17 % in rituximab-refractory patients with the application of novel type I antibodies, ofatumumab and ocrelizumab, which is quite modest.
8.3.6 Third-Generation CD20 mAb
8.3.6.1 PRO131921 (RhumAb v114)
To overcome the less favorable disease outcome in patients with low-affinity receptor (FcγRIIIa) expressed on NK cells after rituximab monotherapy, a modified version of ocrelizumab (PRO131921)was developed; it achieved a 30-fold better binding to FcγRIIIa (FF or FV) than rituximab. An up to tenfold stronger ADCC was demonstrated in in vitro studies [124, 125]. Efficient B-cell depletion in cynomolgus monkeys was observed in a dose-escalating study. However, adverse events including dose-dependent reversible neutropenia and thrombocytopenia were also manifested [125]. In a dose-escalating phase 1 trial on patients with relapsed or refractory indolent lymphoma who were previously treated with rituximab, single-agent administrations of PRO131921 were well tolerated [126].
8.3.6.2 AME-133v (LY2469298)
AME-133v, a third-generation IgG1 anti-CD20 mAb, was developed in an attempt to enhance ADCC of anti-CD20 mAb. It possesses increased affinity to the FcgRIIIa on NK cells, as well as a ten times stronger B-cell killing. Targeted insertion of a synthetic oligonucleotide pool into a human germ line sequence was used for the construction of the CDRs. AME-133v proved as an effective activator of NK cells which induced the same degree of ADCC with lower doses as compared to rituximab in in vitro studies [46].
8.3.6.3 GA-101 (RO5072759, Obinutuzumab)
GA-101, derived from the murine IgG1-k antibody B-lyl, is the first Fc-engineered type II anti-CD20 mAb which mediates homotypic adhesion and does not relocate CD20 into lipid rafts after binding to CD20 [42]. In comparison with rituximab, a distinct but overlapping epitope on CD20 is recognized by GA-101; in addition, it binds in a different orientation and on a larger surface area; additionally, it possesses increased induction of direct cell death after binding. GA-101 exhibited more efficient direct cell death induction and nonclassical apoptosis in comparison with tositumomab. Significantly more efficient killing of lymphoma was observed in whole blood sample assays. In addition, GA-101 was more efficient in killing normal and malignant B cells in several murine models and in monkeys, and contrary to rituximab, it proved capable in B-cell elimination from the spleen and lymph nodes [42]. During a dose-escalating phase 1 clinical trial in 25 patients with NHL, receiving 9 infusions of GA-101 starting at 50–2,000 mg per dose, promising results were obtained, with a toxicity profile comparable to rituximab and no dose-limiting toxicities. An ORR of 36 % was achieved [127]. Likewise, doses up to 2,000 mg of GA-101were well tolerated, and an ORR of 22 % was achieved in pretreated patients with NHL in a phase 1 clinical trial [128]. Furthermore, single-agent GA-101 had a high response rate in heavily pretreated patients with indolent NHL, which demonstrated a possible dose–effect relation; an ORR up to 55 % was observed in patients with rituximab-refractory disease [129]. Obinutuzumab in combination with chlorambucil was compared with chlorambucil alone or rituximab and chlorambucil in a phase III randomized study in patients with previously untreated CLL. The median PFS was 23 months in patients treated with obinutuzumab plus chlorambucil and 11.1 months for patients treated with chlorambucil alone. This led to the FDA approval of obinutuzumab in CLL in November 2013. A survival advantage was also demonstrated in the obinutuzumab and chlorambucil arm compared with rituximab and chlorambucil (Goed V et al., Abstrace #6, Blood, ASH conference 2013).
8.3.7 Small Modular Immunopharmaceutical Anti-CD20 Protein
8.3.7.1 TRU-015
Small modular immunopharmaceuticals are encoded by a single-chain protein expressed as homodimers. TRU-015, a very small protein, is generated from the heavy and light chain variable regions from murine anti-CD20 mAb 2H7, which are linked to HuIgG1 CH2 and CH3 domains [130]. TRU-015 manifests comparable ADCC and a reduced CDC activity compared to rituximab during in vitro studies. Noteworthy, it was found superior to rituximab in terms of reduction of tumor mass and prolonged survival of mice with human lymphoma. Moreover, a dose-dependent and durable B-cell depletion was experienced with escalating single-dose injections of TRU-015 in cynomolgus monkeys [130]. Remarkably, TRU-015 proved as a safe B-cell-depleting agent in patients with rheumatoid arthritis in a dose-dependent manner during a dose-escalating phase I/II trial [130]. However, its safety in lymphoma patients remains to be defined.
8.4 CD22
CD22, a 135 kDa molecule, is exclusively expressed on the transmembrane of mature (IgM and IgG) B cells [131]. It plays a crucial role in the regulation of B-cell activation, survival, and BCR and CD19 signaling, after being phosphorylated [132]. Prolonged contact hypersensitivity reactions have been observed in CD22-deficient B cells, implying its inhibitory role within the immune system [133]. Apoptotic pathways are activated upon binding of an antibody or the natural ligands (sialylated glycans) to CD22 and its internalization [134]. CD22 is expressed on more than 90 % of the FLs.
8.4.1 Epratuzumab
Epratuzumab is derived from the murine IgG2a LL2 anti-CD22 mAb, which was previously used for the radioimmunodetection of NHL. LL2 has been humanized by CDR grafting techniques, and the murine IgG2a has been replaced with human IgG1; hence, its immunogenicity is reduced, and an efficient immunotherapeutic mAb is resulted. Epratuzumab and CD22 are rapidly internalized upon binding, possibly leading to its phosphorylation and downstream signaling [135]. In vitro studies have demonstrated no complement system activating capability, no clear direct cytotoxic effect, and only a modest ADCC activity. Nonetheless, significant antiproliferative effect has been observed in vitro. Noteworthy, a stronger antiproliferative effect has been observed for the combination of epratuzumab and rituximab compared with rituximab alone [135, 136]. Single doses of epratuzumab were well tolerated and resulted in transient B-cell depletion in a dose-escalating phase I trial in patients with different subtypes of NHL. Patients with FL revealed the best clinical response (43 %) among other subtypes at a dose of 360 mg/m2/week [137]. A multicenter study in patients with various subtypes of refractory/relapsed NHL assessed the effect of the combination of epratuzumab with rituximab; the injection of weekly doses of 360 mg/m2 epratuzumab and 375 mg/m2 rituximab yielded promising results in the FL group (ORR, 54 %; CR/CRU, 24 %; median response duration, 13.4 months) [138]. In addition, a phase II clinical trial revealed similar results in patients with indolent lymphoma. Epratuzumab (360 mg/m2) combined with rituximab (375 mg/m2) administered weekly for four consecutive weeks yielded comparable toxicities to rituximab alone with an ORR of 64 % (24 % CR/CRu) with a response duration of 14 months in patients with refractory/relapsed FL [139]. The combination of epratuzumab and rituximab with CHOP chemotherapy has been studied in a phase II study in patients with untreated DLBCL; six cycles of treatment obtained an OR of 87 % and CR/Cru of 67 % [140]. In a Raji lymphoma mouse model, the combination of anti-CD20 mAb veltuzumab and epratuzumab obtained no significantly different results compared to veltuzumab alone [141]. On the other hand, an anti-CD20/anti-CD22 bispecific mAb was developed, which obtained remarkable antiproliferative effects in in vitro, in contrast to the parental Abs (veltuzumab and epratuzumab or combined) [142]. Even though the cross-linking of CD20 and CD22 does not lead to significant activation of the complement system, marked ADCC activity is obtained. Moreover, the bispecific Ab was demonstrated to be superior to anti-CD20 in a Daudi lymphoma model [143]. Hexavalent bispecific anti-CD20/anti-CD22 antibody manifested similar results [142]. Due to its rapid internalization after binding to CD22, epratuzumab is considered an ideal immunoconjugate for drug delivery, resulting in increased potency; nonetheless, increased toxicity may be incurred [144].
8.4.2 Inotuzumab Ozogamicin (CMC-544)
Inotuzumab, an IgG4 humanized anti-CD22 mAb which is derived from the murine mG5/44 Ab, resides on to human acceptor frameworks. The antibody–antigen complex is internalized after binding to CD22 [144]. No toxic effect has been reported from CMC-544 [145]. By conjugation of inotuzumab to ozogamicin (calicheamicin), a potent antitumor antibiotic, growth of CD22 B cells has been inhibited in vitro. Moreover, dose-dependent significant anti-lymphoma effect has been attributed to this immunoconjugation in vivo [145]. In addition, it possessed greater cytotoxicity in comparison with rituximab conjugated to ozogamicin [146]. On the other hand, an even stronger anti-lymphoma effect in similar B-cell lymphoma mouse models was attributed to the combination of rituximab and inotuzumab ozogamicin [147]. The safety and efficacy of inotuzumab ozogamicin were studied in patients with pretreated NHL in a multicenter, dose-escalating (0.2–2.4 mg/m2) phase I study; a maximum dose of 1.8 mg/m2 was well tolerated. Moreover, adverse events consisting of thrombocytopenia (90 %), asthenia (67 %), nausea (51 %), and neutropenia (51 %) were all reversible. Subgroup analyses on FL patients demonstrated an objective response rate of 68 % with a 32 % CR/CRu rate and a PFS of 10.4 months [148]. The combination of inotuzumab, ozogamicin, and rituximab in patients with FL resulted in similar reversible adverse events in another clinical study [149]. The immunoconjugate inotuzumab ozogamicin (CMC544) with rituximab obtained an ORR of 87 % with a 23.6 months’ response duration in 38 patients with recurrent/refractory lymphoma [149].
8.5 CD19
CD19, a member of the immunoglobulin superfamily, consists of two extracellular immunoglobulin-like domains with an extensive cytoplasmic tail [158]. It is exclusively expressed on B-cell lineage from the very early B cell and is lost upon differentiation to plasma cells [158]. Due to its high, homogeneous expression in nearly all different subtypes of lymphoma, it is considered a potential target for immunotherapy [159]. Even though CD19 was one of the first targets for immunotherapy [160] and its safety and efficacy were approved, its development has been stagnated by the more promising results obtained by anti-CD20 mAbs [160].
8.5.1 XmAb5574
XmAb5574, a novel humanized anti-CD19 mAb, mediates significantly higher ADCC compared with rituximab, owing to its engineered Fc domain [161]. It acts through modest induction of apoptosis and activation of the phagocytic system [161, 162]. This mAb has excellent preclinical features, and further studies should be awaited.
8.5.2 Blinatumomab (MT102/MEDI-538)
Blinatumomab (MT102/MEDI-538), a new anti-CD19-CD3 bispecific Ab, which gathers lymphoma (CD19) and effector T cell (CD3) together, leads to efficient elimination of lymphoma cells. Despite the promising clinical results of previous anti-CD19/CD3 bispecific Abs [163], they proved ineffective in clinical trials [164]. However, blinatumomab yielded promising results in a phase I clinical study [165]. In vitro, it demonstrated efficient anti-lymphoma cytotoxicity at extremely low doses (10 pg/ml, 100,000-folds lower than rituximab) and low effector/target cell (2:1) ratio [166]. Clinically, in 38 patients with relapsed NHL (FL, CLL, and MCL), doses ranging from 0.0005 to 0.06 mg/m2/day were found safe and effective. Complete remission was achieved in four and seven patients with doses starting at 0.015 mg/m2/day with 13 months’ duration of response in one patient [165].
8.5.3 hu-DM4/SAR3419
Various phase I studies have evaluated the application of anti-CD19 mAbs coupled to immunotoxins. Binding of the tubulin inhibitor maytansinoid derivate DM4 to the humanized IgG1 anti-CD19 mAb, huB4 (huB4-DM4/SAR3419), has yielded promising results. After binding to CD19, SAR3419 undergoes internalization, resulting in intracellular release of DM4, eventually leading to cell death. SAR3419 was found superior to rituximab in preclinical xenograft models [167]. In addition, its safety was proved in a dose-escalating phase I study in patients with different types of lymphoma. However, dose-limiting toxicities including severe transient blurred vision, associated with microcystic epithelial corneal changes, were observed. Finally, 53 % of the patients refractory to rituximab experienced remission [168].
8.6 CD30
CD30, a member of TNF receptor superfamily, is expressed on the cell surface of 10 % of NHLs [23]. It is considered a diagnostic immunomarker and a potential target for immunotherapeutic approaches for anaplastic large cell lymphoma (ALCL) [24]. The prognosis of ALCL is significantly correlated to serum CD30 level [25]. Other NHL subtypes including DLBCL, primary mediastinal large B-cell lymphoma, FL, and Epstein–Barr virus (EBV)-positive lymphomas express lower levels of CD30 expression [26]. A variety of CD30 mAbs are applied in the NHL including M67, SGN-30, Ki-1, M67, and Ber-H2 [27, 28], most of which are effective on ALCL cells. A summary of trials on the application of CD30 mAbs in NHL is summarized in Table 8.4.
Table 8.4
Anti-MHC-II monoclonal antibodies
Anti-MHC-II | Origin | Notable comments | Adverse events |
|---|---|---|---|
Anti-CD74 | |||
Milatuzumab | Murine LL1 | Acts through direct growth inhibition, apoptosis | No serious adverse event |
No CDC or ADCC | Reversible T-cell reduction | ||
HLA-DR | |||
Apolizumab | Murine 1D10 | Acts through APC, ADCC, apoptosis | No serious adverse event |
Type I hypersensitivity | |||
IMMU-114 | Humanized IgG4 | Leads to disease-free survival | No serious adverse event |
LYM-1 | Murine IgG2a | Acts through CDC | No serious adverse event |
Dose-limiting thrombocytopenia | |||
SHAL | Very rapid blood clearance | Non reported | |
Suitable carrier for radio-isotypes | |||
8.6.1 M67
M67, a monoclonal anti-CD30 developed in 1994 by Gruss et al., was established as an efficient growth inhibitor of ALCL cell lines in vitro [29, 30]. In addition, a significant correlation was observed between its antitumor effects and the differences in the constitutive NF-kB signaling in ALCL and HD cell lines [31]. As evident in in vitro studies, ALCL cells undergo apoptosis in the presence of M67 due to their inability to activate the transcription factor NF-kB, whereas HD cell lines (L428, KM-H2, L591) were resistant to M67, attributed to constitutive expression of NF-kB [8].
8.6.2 SGN-30
SGN-30, a chimeric IgG1 mAb derived from the murine AC10 anti-CD30 mAb, was demonstrated an antiproliferative effect in vitro and a potent anti-HL effect in xenografts [32]. Macrophages play a critical role in the activity of SGN-30, proven by the abolished effect of SGN-30 in the absence of macrophages in experimental studies [32]. It has proved as a safe and well-tolerated mAb, yielding optimal results in patients with (cutaneous) ALCL [33–35].
8.7 CD37
CD37, a heavily glycosylated 40–52 kDA glycoprotein, is a member of the tetraspan transmembrane family of proteins [169, 170], which internalizes and displays modest shedding in transformed B cells expressing the Ag. It is expressed in cells progressing from pre-B to peripheral mature B cell. Nonetheless, it is lost during B-cell development in terminal differentiation to plasma cells. It is considered an optimal target for immunotherapy in B-cell NHL and other B-cell malignancies, owing to its high selectivity [55].
8.7.1 Tetulomab (HH1)
Tetulomab (HH1), a murine IgG1 Ab, was the first anti-CD37 developed in the 1980s [171]. The binding properties to various NHL subtypes of tetulomab has been compared with the chimeric IgG1 antibody rituximab, and significant therapeutic effect of 177Lu-tetulomab was established with tolerable toxicity [55].
8.8 CD40
CD40, a member of the TNF receptor family, is constitutively expressed on antigen-presenting cells (B cells, dendritic cells, and macrophages), acting as a co-stimulatory molecule, which interacts with CD40L (CD154) expressed by activated T cells. In addition, endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells express CD40 on their membrane. In addition, malignant cells such as NHL, multiple myeloma, and various solid tumors express CD40 in considerable amounts. Stimulation of CD40 leads to immunoglobulin isotype switching and activation of B cells. Expression of CD40L can also be found on activated B cells, NK cells, monocytes, dendritic cells, endothelial cells, and smooth muscle cells. CD40–CD40L interaction plays a general role in the immune regulation (apoptosis and enhancing cell survival) [150]. In addition, soluble CD40L (sCD40L) has been obtained from serum of patients with lymphoma, CLL, MM, and autoimmune diseases. It is recognized as an independent risk factor for some hematological malignancies [151]. Since CD40 and CD40L can be co-expressed on B-cell lymphoma, it is postulated that this system may act as an autocrine–paracrine survival loop of malignant hematopoietic cells [152]. CD40 has the structure of a typical type I transmembrane molecule with a large extracellular part, acting as a binding side for anti-CD40 mAbs which acts as a target for Abs [150].
8.8.1 Dacetuzumab (SGN-40)
Dacetuzumab, a humanized mAb with CDRs of murine S2C6 in the human IgG1 framework sequences, is found to have potent anti-lymphoma effects, including growth arrest upon cross-linking, induction of apoptosis, and ADCC that is Fc dependent in vitro assays [153, 154]. Dacetuzumab significantly increased the survival of mice compared to controls in a Daudi mouse model [154]. It has been found to result in transient decrease of T cells and NK cells and a persistent decrease in CD20-positive cells, after injection into cynomolgus monkeys. Dacetuzumab was well tolerated in patients with CLL, MM, and relapsed/refractory NHL, as demonstrated in different phase I studies conducted on different CD40-positive malignancies [155]. Nonetheless, no response was seen in patients with FL in a dose-escalating trial for NHL, whereas an ORR of 12 % was maintained in patients with pretreated DLBCL [156].
8.8.2 Lucatumumab (HCD122, CHIR-12.12)
Lucatumumab, a fully human anti-CD40 mAb, is generated in a human IgG1 transgenic mouse by immunizing mice with the extracellular domain of recombinant human CD40. Effector cells are more potently activated by Lucatumumab compared to rituximab [157].
8.9 CD52
CD52, a low molecular weight glycoprotein (21–28 kDa) of unknown function [172], is exclusively expressed on mature B and T lymphocytes, NK cells, monocytes, and dendritic cells and is absent on hematopoietic precursors.
8.9.1 Alemtuzumab (CAMPATH-1H)
Alemtuzumab, a humanized rat IgG CD52 mAb, is created by transferring the antigen-specific CDRs of the rat mAb onto a human framework. It is known to act through CDC, ADCC, and apoptosis induction in in vitro, yet its exact mechanism for the in vivo killing remains to be unveiled [172]. Studies have revealed its efficacy in cutaneous T-cell lymphoma and peripheral T-cell lymphoma [173]. Although CD52 is also expressed on FL cells, no clinical trials have been conducted in this regard [13]. It has proved as a competent mAb in combination with chemotherapy. In a recent study on patients with relapsed or refractory advanced T-cell NHL (age range: 11–65), who had previously received remission induction by cladribine, cytosine, arabinosine, and etoposide combined with granulocyte colony-stimulating factor support (CLAEG), patients received medium doses of alemtuzumab in combination with carmustine, etoposide, cytosine, arabinoside, and melphalan (BEAM) treatment; BEAM and alemtuzumab appeared beneficial in 20 patients from the overall 21 patients receiving CLAEG induction therapy. Nine patients experienced CR, and 50 % did not achieve CR by the time of hematopoietic stem cell transplantation (HSCT). After HSCT, 20 patients reached CR during a median follow-up of 11 months. Overall, this study revealed that reduced-intensity BEAM-alemtuzumab conditioning and allogeneic HSCT proceeding intense reinduction therapy provide curative potential in patients with advanced T-cell lymphomas, even for those not in remission [174].
8.10 CD80
CD80 (B7-1), a protein expressed on NHL cells [175], is normally limited to the cell surface of activated antigen-presenting cells including B cells, dendritic cells, and monocytes [176]. It is considered as a co-stimulatory molecule for CD28 and is expressed on T cells. Since the extracellular part of CD80 contains two Ig-like domains, it is recognized as a suitable target for mAb therapy. CD80 together with CD86 stimulates Cd28 and T-cell receptor, leading to the activation and clonal expansion of T cells. Moreover, the interaction between CD80 (and CD86) and CTLA-4 (CD152) expressed on activated T cells results in decreased T-cell response. Nonetheless, the intrinsic function of CD80 is unclear [176].
8.10.1 Galiximab (IDEC-114)
Galiximab, a chimeric IgG1 anti-CD80 mAb derived from the cynomolgus monkey and man, includes both human constant regions and monkey variable regions. Due to its structure similarity to human Abs, an immune response in patients is less likely. Galiximab has demonstrated antiproliferative and anti-apoptotic properties, as well as a dose-dependent ADCC induction in in vitro studies [177]; galiximab prolonged survival in mice compared to controls, to the same degree as rituximab in in vivo human lymphoma mouse models. Furthermore, the combination of rituximab and galiximab increased survival of mice compared to rituximab alone [177]. After binding of galiximab to CD80, CTLA-4 is blocked, which may induce an anti-lymphoma environment [178]. A dose up to 1,200 mg/m2 galiximab in monkeys for 5 months exhibited no adverse events [177]. In addition, its safety and efficacy were evaluated in a multicenter, dose-escalating phase I clinical trial of 37 patients with relapsed or refractory FL who received four weekly infusions of galiximab. Even though it was well tolerated, it resulted in an ORR as low as 11 % with CR in only two patients. Remarkably, delayed response was observed in some cases. Despite its 2–4 weeks half-life, its efficacy may last for years, conferring to its further immune response induction [179]. Safety and efficacy of the combination of galiximab and rituximab were evaluated in 75 patients with relapsed or refractory FL, in which 500 mg/m2 of galiximab was recommended in combination with standard doses of rituximab. An ORR of 66 % (33 % CR/CRu; 33 % partial response) with a PFS of 12.1 months was achieved, and no adverse event was observed [85].
8.11 CD74 and HLA-DR
CD74 and HLA-DR are both members of MHC class II. CD74, the cell surface form of the invariant chain, acts as a chaperone molecule for MHC class II. CD74 binds to HLA-DR within the endoplasmic reticulum; the complex is then transferred to the late endosomal compartment, where CD74 is cleaved into peptide fragments and is dissociated from DR. Peptides form complexes with DR and are transported to the cell surface for antigen presentation to T cells [180]. Besides aiding peptide presentation, CD74 functions as a signaling molecule. Anti-CD74 mAbs have led to maturation of B cells through a direct signaling pathway involving NF-kB [181]. It also acts as a high-affinity receptor for the proinflammatory cytokine macrophage migration inhibitory factor [182]. In addition to expression on APCs, CD74 is a marker on various tumor cells, including B-cell lymphomas, gastric cancer, renal cancer, and non-small cell lung cancer. Its expression has been found to contribute to poor prognosis, possibly explained by the suppressive effects on the immune system [180]. It is regarded as an efficient target for mAb therapy, resulting in the development of different anti-CD74 Abs. Nonetheless, clinical experience is lacking. Various CD74 monoclonal antibodies are discussed below and summarized in Table 8.4.
8.11.1 Milatuzumab (IMMU-115, hLL1), Naked and Conjugated
Milatuzumab, an IgG1k anti-CD74 mAb, is derived from the murine LL1 and is humanized by CDR grafting. Rapid internalization is resulted in both CD74 and milatuzumab upon binding and is replaced by newly synthesized CD74. It has exhibited growth inhibition and apoptosis induction in in vitro, whereas no CDC or ADCC is exerted by milatuzumab. Significant prolonged survival was observed in human Burkitt lymphoma xenograft mouse model compared to control mice by administrating milatuzumab [141]. In single-dose and multidose experiments, no serious adverse events were experienced with only a reversible decrease in T cells, B cells, NK cells, and monocytes in cynomolgus monkeys [180]. Due to its rapid internalization (106–107 molecules/cell/day), it is a perfect target for conjugation with radioisotopes, drugs, or toxins. In vitro studies on murine versions of milatuzumab conjugated to different radioisotopes revealed high efficiency in eliminating B-cell lymphomas. Auger emitters (111In and 67Ga) demonstrated a potent anti-lymphoma effect and prolonged survival compared to unlabeled LL1. No significant toxicity has been observed; however, further clinical trials are warranted to establish its efficacy [180]. Milatuzumab (BR96-Dox, hLL1-Dox, IMMU-110) combined to doxorubicin (dox) manifested as a lethal combination upon binding to CD74-positive cells. A 100 % survival rate was exhibited by the administration of HLL1-Dox to mice bearing Raji lymphoma; in addition, no toxicity was observed in mice models [180, 183]. Doses up to 30 mg/kg were well tolerated in cynomolgus monkeys, and the first signs of bone marrow toxicity were observed with doses of 30 mg/kg [183]. The combination of milatuzumab and the toxin ranpirnase, a frog RNase that results in the degradation of tRNA, yielded similar results, with respect to protein synthesis, inhibition, and apoptosis [184]. To reduce the effect of Fc-expressing cells, milatuzumab was altered into IgG4 (2L-Rap-hLL1-g4P), which manifested more potentiality in vitro. High remission rates were observed in Daudi and Raji mouse models. High doses of the conjugated antibody resulted in hepatotoxicity in mice. While highly efficient anti-lymphoma effects have been observed in mice, clinical trials are needed to study its safety and efficacy in clinic [184].
8.11.2 Apolizumab (Hu1D10, Remitogen)
HLA-DR, a heterodimer comprising of DRa and DRb subunits, presents antigen to the TCR on CD4-positive T cells, hence initiating a humoral immune response. In addition to APCs, most neoplastic cells, including FL, express HLA-DR. Apolizumab is derived from the murine 1D10 mAb and is humanized by CDR grafting. The polymorphic determinant on HLA-DRb is the target of apolizumab; nonetheless, the HLA-DRb is not shed or internalized from the cell surface after binding [185]. Its capable mediation of CDC, ADCC, and apoptosis has been demonstrated in in vitro studies [186]. Bolus infusions in rhesus macaques resulted in type I hypersensitivity reactions; nonetheless, no serious adverse event was experienced with slow infusions, except transient decrease in B cells [187]. Doses ranging from 0.15 to 15 mg/kg were found to be safe and were well tolerated in patients with relapsed NHL in a phase I dose-escalating study. Nevertheless, it showed no clinical efficacy in relapsed/refractory FL [188]. In addition, it appeared more effective in combination with rituximab in a phase I study in 35 patients with relapsed/refractory NHL. An ORR of 28 % with 17 % CR/Cru was achieved. Toxicities were mostly minor and reversible, with atypical hemolytic uremic syndrome in some patients [189]. Overall, apolizumab monotherapy seems clinically ineffective, while its efficacy may be enhanced in combination with other mAbs [13].
8.11.3 IMMU-114 (hL243g4P)
IMMU-114 is a novel humanized mAb developed with an IgG4 isotype, which targets the HLA-DRa chain, leads to direct binding, and eventually leads to antiproliferative effect and apoptosis induction [190]. An increased antiproliferative effect was observed with the combination of IMMU-114 and rituximab [191]. It revealed a disease-free survival in mice bearing CD20-resistant lymphoma cells in human lymphoma mouse models [192].
8.11.4 LYM-1
LYM-1, a murine IgG2a mAb generated by immunizing mice with Raji Burkitt lymphoma cells, targets the polymorphic variants on the HLA-DR10 b chain, activates complement, effector cells and induces apoptosis in vitro [193]. In a study of ten patients with refractory NHL, limited results were obtained, with only small reduction in lymph node size in some patients but with good safety profiles [194]. LYM-1 conjugated to 131-iodide has been extensively tested in two phase I/II trials in patients with therapy refractory NHL in which unconjugated LYM-1 was injected prior to the administration of escalating doses of 131-I-LYM-1. Thrombocytopenia was the only dose-limiting toxicity. In the low-dose trial, 85 % of patients obtained tumor regression with 10 % CRs. In the maximum tolerated dose trial, 52 % of the patients experienced remission, and 33 % achieved CR. A significant correlation between the levels of human anti-mouse antibodies which developed after the administration of LYM-1 and clinical response was observed [195]. LYM-1 combined with other radioisotopes including yttrium-90 [196] or copper-67 [197] yielded similar results. A synergistic anti-lymphoma effect was induced by adding LY-M1, yttrium-conjugated Lym-1, and a chimeric form of LYM-1 to rituximab in vitro [193, 198].
8.11.5 Selective High-Affinity Ligands (SHALs)
Small molecules, called selective high-affinity ligands (SHALs) or antibodies, which mimic the binding of LYM-1 on HLA-DR, have been generated by linking two ligands (molecules) that recognize the LYM-1 epitope based on computational and experimental methods and are 50 times smaller than mAbs. Despite long residence time in the circulation and toxicities caused by their combination with radioisotopes, SHALs are favored by their very rapid blood clearance, making them suitable carriers for radioisotopes [199]. Studies on mice have demonstrated that radioisotopes coupled to SHAL located and targeted the tumor cells with a rapid blood clearance and no toxicity [200]; yet, no direct anti-lymphoma effect has been attributed to SAH [201]. The production and selection of SHALs need to be optimized, and the anti-lymphoma activity of radioisotopes coupled to SHALs needs to be tested in in vivo models before drawing a comprehensive conclusion [13].
8.12 CD1d and NK Cells
8.12.1 CD1d
CD1d, normally expressed on hematopoietic cells of myelomonocytic and B-cell lineages, is a marker for malignancies originating from the corresponding tissues.
B-cell malignancies have also been found to display CD1d. Studies on murine models have demonstrated the expression of CD1d on many leukemia and lymphoma cell lines. Moreover, NKTs have exhibited a protective role in the A20 murine B-cell lymphoma model [202], which is correlated to the level of CD1d expression on lymphoma cells and was lost in NKT-deficient mice. Studies on human lymphomas have revealed that CD1d is expressed on the surface of Reed–Sternberg (RS) cells in half of the cHL cases and in 30 % NHLs [203]. Notably, NKTs were present at high frequencies in primary cHL tumors and reactive lymph nodes irrespective of CD1d expression on tumor cells. However, the functional role of tumor-infiltrating NKTs in cHL biology and disease outcome is yet to be determined. It is postulated that NKTs may co-localize with CD1d-positive tumor-associated monocytes/macrophages (TAMs) in the microenvironment of CD1d-negative tumors [204]. In addition, the increased number of TAMs is significantly correlated to decreased survival rates in patients with cHL [205]. Targeting both RS cells and TAMs for immunotherapy with NKTs and/or their ligands seems a promising approach [206].
8.12.2 Function of NK Cells in NHL
The strongest known risk factor for the development of lymphoma is immunosuppression, predominantly NK cell dysfunction. NK cells are critical effectors in tumor immunology and were usually regarded as effector cells of innate immunity. However, more recently it has been shown that they attribute to both innate and adaptive immunity, playing a regulatory role in shaping antigen-specific T- and B-cell responses [207]. A study evaluating NK cell activity in patients with NHL and HL prior to therapy applied lactate acid dehydrogenase (LDH) release cytotoxicity assay and revealed that decreased NK cell activity in NHL patients is significantly correlated to unfavorable histology, with the lowest activity in very aggressive forms. The clinical stage of the disease also contributed to the degree of NK cell dysfunction. NK cell activity is significantly impaired in HL compared to controls, irrespective of histological type and clinical stage. Notably, the most profound NK cell dysfunction, present and persistent in HL and present in very aggressive NHL, is associated with increased LDH release activity from peripheral blood mononuclear cells. NK cell function is greatly impaired in HL and in very aggressive NHL; in addition, impaired NK cell activity is associated with increased spontaneous release activity of LDH from patients’ PBL, which is indicative of cell membrane damage, followed by the release of cytotoxic proteins, and eventually impaired NK cell activity [7].
8.12.3 Adoptive Transfer of Highly Cytotoxic NK Cells
ADCC is considered one of the major effector functions of mAbs, which is triggered following the binding of the antibody Fc region to the Fcγ receptor (FcγR) on effector cells. Most NK cells express CD16 (FcγRIII), a receptor that binds to the Fc region of IgG1, and are the major effector cells related to ADCC [208]. Given the capability of NK cells in controlling tumor growth and metastatic dissemination [209–213], novel NK cell-based cancer immunotherapies are promising [214]. The adoptive transfer of highly cytotoxic NK cells has emerged as a promising strategy in immunotherapy, which have been expanded from peripheral blood mononuclear cells (PBMC) by a feeder-cell-free expansion method [215]. Given the absence of cancer feeder cells or genetically modified cells, it is considered a safe method [216, 217]; in addition, greater NK cell enrichment and higher expansion fold than other reported methods [218–220] are achieved. T lymphocytes expansion is also accomplished. Lines of evidence suggest that the efficacy of cancer treatment is enhanced by combining mAb drugs with adoptively transferred ex vivo expanded NK cells [221]. Cytotoxicity and ADCC functions of expanded NK cells in combination with rituximab against CD20+ lymphoma cell lines were compared with that of freshly isolated NK cells, which revealed that expanded NK cells ex vivo are significantly more efficient in the induction of activating receptor expression, production of IFN-γ and TNF-α, as well as cytotoxicity against various cancer cell lines including CD133+ primary cancer cells, as compared to freshly isolated NK cells [215]. The emergence of other therapeutic mAbs including trastuzumab [222–224], cetuximab [225], and alemtuzumab [226] and their potential combinations with expanded NK cell therapy are hypothesized to be broadly applicable to a wide range of malignancies [215].
8.13 Therapeutic Efficacy of Antibody-Targeted Cytokines
Various limitations have led to trivial application of antibody-targeted cytokines in NHL. Some cytokines possess high systemic toxicity, hence limiting their application for specifically targeted tumor tissues. On the other hand, decreased function is observed when fused to other protein domains. Therefore, the potentiality of the antibody-targeted cytokine depends on the binding avidity and the immunomodulatory capacity of the fusion protein [10]. Various cytokines have been applied in the treatment of NHL, as discussed in the following.
8.13.1 Interferon-α (IFN-α)
IFN, a natural glycoprotein, is subdivided into three subgroups of IFN-α, IFN-β, and IFN-γ; IFN-α is produced by leukocytes, whereas fibroblasts secrete IFN-β, and IFN-γ is a product of activated T and NK cells. They are described to have immunoregulatoy activity combined with antiproliferative effects [227]. Monoclonal immunotherapy with IFN-α has yielded promising results in considerable number of patients with low-grade lymphoma [228]. Pilot studies were designed to assess the efficacy of IFN-α in low-grade lymphomas [229]. It yielded partial remission in one patient among four in a clinical trial. Another clinical trial resulted in 4 remissions among 13 patients [230]. A phase II trial studied the safety and efficacy of rituximab and IFN-α-2a in 38 patients with relapsed or refractory low-grade or follicular B-cell NHL. A dose of 2.5 MIU of IFN-α-2a, three times weekly for 12 weeks, combined with 375 mg/m2 rituximab starting at week 5, yielded an OR of 45 %, with CR in 11 %. No toxicities were observed. Adverse events included asthenia, chills, fever, headache, nausea, and myalgia [231]. The GELA-GOLELAMS FL2000 study investigated the efficacy of rituximab combined with CHVP (cyclophosphamide, adriamycin, etoposide, and prednisolone) chemotherapy and IFN in patients with FL as the first-line treatment. An EFS of 53 % was achieved which was significantly greater than those receiving the same regimen without rituximab [232]. Moreover, a meta-analysis investigated the effect of adding IFN-α2 to chemotherapy in patients with newly diagnosed FL. The regimen was found to be most efficacious when relatively intensive initial chemotherapy was applied, when doses ≥5 MIU with a cumulative dose ≥36 MIU per month were applied, and when it was applied in combination with chemotherapy rather than as maintenance therapy. In addition, remission duration was significantly greater when IFN-α2 was added to the regimen [233].
8.13.2 Interleukin-2 (IL-2)
The pleiotropic cytokine, interleukin-2, a 15–17 kd glycoprotein, is secreted by T lymphocytes and plays a crucial role in their proliferation. Three types of membrane components, the α, β, and γ chains, comprise the receptor. A variety of receptor types with different binding affinities are formed by different combinations of the α chain (CD25, 55 kd glycoprotein), the β chain (75 kd), and the γ chain (50–64 kd). Remarkably, hematopoietic malignancies have been described to express a high level of IL-2 receptor [234]. Therefore, it was among the initial immunotherapeutic agents. It plays a crucial role in the augmentation NK cell cytotoxicity, induction of lymphokine-activated killer (LAK) cells, and activation of T and B lymphocytes, as well as monocytes. Serum-soluble interleukin-2 (sIL-2R) level has been found to possess a prognostic value in patients with DLBCL [235] and T-cell lymphoma [236]. In addition, its correlation with the tumor burden at diagnosis and during the clinical course of therapies in patients has been recently established [237]. Notably, it has been mostly studied in HL patients as discussed in the previous chapter.
8.13.3 Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)
TRAIL (Apo-2), a member of the TNF superfamily, consists of 28 receptors and 18 ligands. Similar to all other members of the TNF superfamily, it regulates cell survival and cell death upon infection or malignant transformation. A death-inducing signaling complex is formed, and eventually apoptosis is triggered after binding of TRAIL to its death-containing transmembrane receptor [240]. As explained by differential expression of its receptors and the absence of TRAIL-R1 and TRAIL-R2 on normal cells, they are spared, while cancer cells including lymphoma cells are selectively killed. As demonstrated in in vitro studies, triggering death receptors with TRAIL or agonistic antibodies activates both extrinsic and intrinsic intracellular death signaling pathways [241–243]. Various TRAIL mAbs are developed which are discussed below.
8.13.3.1 Mapatumumab (HGS-ETR1, TRM-1)
Mapatumumab, an agonistic mAb directed against TRAIL-R1, is a fully human IgG1 mAb activating both intrinsic and extrinsic apoptotic pathways upon binding. By using a single-chain variable fragment (scFv) human antibody phage library, the domain TRAIL-R1-flag fusion protein is isolated, and its potent apoptosis induction in vitro and in vivo is established. In addition, it enhances the antitumor effect of chemotherapy in mouse models [241]. It exerts its antitumor effect on different lymphoma cell lines through apoptosis induction, ADCC, and CDC. Notably, significantly increased efficacy was demonstrated when combined with rituximab in mouse models [244]. Doses up to 40 mg/kg every 10 days for 6 months were well tolerated in chimpanzees [245]. During phase I/II trials on different solid tumors, mapatumumab was well tolerated, no patient required maximum tolerated dose, and a clinical response was reported in some cases (8 %) [245].
8.13.3.2 Lexatumumab (HGS-ETR2)
Lexatumumab, a human IgG1 anti-TRAIL-R2 mAb, is constructed under the same conditions as mapatumumab, by making use of a human phage display library [241]. Even though it was found efficient in apoptosis induction and growth inhibition in NHL cell lines after cross-linking, no survival gain was reached with lexatumumab or with the combination of lexatumumab plus rituximab in mice bearing human lymphoma [244]. It yielded no objective response in a phase Ib study in patients with solid tumors; however, stable disease was observed [246]. Overall, lexatumumab has proved less beneficial in the clinical setting, compared with mapatumumab. Further preclinical studies on different tumors need to be conducted to evaluate the effect of targeting TRAIL-R2 receptors [13].
8.13.3.3 Conatumumab (AMG 655)
Conatumumab, a fully human IgG1 monoclonal agonist antibody targeting human TRAIL-R5 upon binding, induces apoptosis via caspase activation [247]. The addition of recombinant TRAIL (rhApo2) to rituximab was found to induce a strong clinical effect; it was well tolerated in patients with low-grade lymphoma who previously failed therapy with rituximab, and a CR of 25 % and PR 13 % were obtained [248]. The combination of rhApo2L/TRAIL with rituximab led to increased survival rates in subcutaneous and disseminated tumors in a xenograft model [249].
8.14 Novel Immunotherapeutic Treatment Strategies
It has been postulated that eradication of human cancers may be accomplished by combining cancer treatment modalities [250]. The lack of specificity is acknowledged as the major shortcoming of conventional cancer therapies [251]. Promising results have been yielded by combining immunotherapy and conventional treatment procedures. Due to their different therapeutic mechanism, side effects differ, and toxicities are manageable [252]. In addition, the combination is proven to yield a synergic effect [54]. Furthermore, antibodies are considered ideal vehicles for drug and radionuclides delivery, due to their high specificity [54]. Multiple clinical trials have been conducted in this regard, yet their clinical applications need to be established.
8.14.1 Molecular Engineered Antibodies
Despite significant improvement in the survival of lymphoma patients by the development of first-generation mAbs, particularly rituximab, various limitations are encountered; hence, attempts have been made to overcome these constraints. The development of humanized or fully human next-generation antibodies demonstrated reduced immunogenicity, which made them more applicable in certain patient populations. More recently, novel technologies of antibody engineering have been developed, which offer the potential to tailor antibody effector functions. Peipp et al. demonstrated that glycoprotein engineering of the Fc region of the antibody yields promising activity in preclinical models. However, these novel molecules are still in their infancy, and further clinical studies are required to determine their efficacy in improvement lymphoma treatment [48].
Antibody engineering [10] is on the progress, and it is hoped to overcome some of these limitations. Various novel techniques are discussed below.
8.14.1.1 Target Antigen and Epitope Selection
Clonal idiotypes which have been developed recently ideally fulfill the abovementioned conditions, and striking results have been obtained by the application of idiotype-specific antibodies in clinical studies. Since each antibody employed in this technique needs to be patient specific, it poses particular challenges. Currently, B-cell idiotype is mainly employed for tumor vaccination strategies [48]. Notably, none of the available antigens fulfill all the requirements for an ideal target antigen. Results from recent preclinical studies demonstrated that the fine specificity of the targeted epitope may critically affect effector mechanisms of particular antibodies.
8.14.2 Radioimmunoconjugates
8.14.2.1 Radioimmunotherapy for Follicular Lymphoma
Complexing radioisotope to a monoclonal anti-CD20 has emerged as a promising treatment approach in patients with advanced FL. The two radioimmunoconjugates currently approved by the US FDA (Food and Drug Administration) are 90Y-ibritumomab tiuxetan and 131I-tositumomab which combine the antitumor activity of rituximab with the cell-killing activity of radioisotopes [253]. High response, OS, and PFS rates have been yielded. Hematologic toxicity (neutropenia, thrombocytopenia) has been observed as the most common adverse event [254]. The efficacy of 90Y-ibritumomab tiuxetan consolidation therapy with no further therapy in patients who at least achieved PR after different induction chemotherapy regimens was studied in phase III trial (FIT trial) which yielded a high PR-to-CR conversion rate and a significantly prolonged median PFS by 2 years. Interestingly, in the subgroup of patients who received rituximab-based induction chemotherapy, PFS was not different between treatment arms [255].
8.14.2.2 CD20-Directed Radioimmunotherapy
Significant antitumor activity has been observed by applying beta-emitting radioimmunoconjugates in patients with relapsed or refractory B-cell lymphoma [256, 257], comprising both patients refractory to mAbs [258, 259] and chemotherapy [260]. To reduce antibody binding to normal B cells by depleting peripheral blood B cells and lymph node B cells, radioimmunotherapy (RIT) is administered with large quantities of unlabeled “cold” antibodies to CD20, 1 week and 4 h prior to the administration of radiolabeled antibodies to CD20 [50, 261]. Therefore, sufficient amounts of radiolabeled antibody bypass these sites and eventually penetrate less accessible compartments including the lymph nodes and target tumor cells. Nonetheless, clinical and experimental studies in mice have revealed that even low blood rituximab concentrations lead to reduction in tumor cell targeting, followed by impairment in the clinical efficacy of CD20-directed RIT [262]. On the other hand, several cycles of “cold” rituximab may lower the effect of subsequent treatment [263, 264]. Due to the competition for the CD20 target, RICs targeting CD20 are not commonly used in medical practice.
CD20-directed RIT treatment in lymphoma patients is challenging in those previously treated with rituximab, as explained by the antigenic drift and possible blockage of the CD20 antigen. Therefore, RIT targeting other antigens seems intriguing.
131I-Tositumomab
131I-tositumomab (Bexxar) is a radioimmunoconjugate consisting of the radioisotope 131I and the murine CD20 mAb, tositumomab. 131I is both a beta and gamma emitter; therefore, it can be used for imaging and dosimetry. Initial clinical trials have been conducted by Kaminski et al. using either non-myeloablative or myeloablative doses [254]. An ORR of 50–70 %, with 20–40 % CR (in a pooled analysis of 250 patients with relapsed indolent or transformed lymphoma treated in five phase I/II trials), and a 5-year PFS of 17 % were achieved when used as monotherapy in the relapsed setting [257]. Surprisingly, durable responses are observed in complete responders. An overall of 32 % of complete responders including heavily pretreated patients with bone marrow infiltration, histologic transformation, and bulky disease yielded a PFS of 1 year or longer; in addition, approximately 17 % of the original treated population was still alive and disease-free in the 5-year follow-up [265]. Overall, 131I-tositumomab has proved effective in rituximab-refractory patients [257] and achieved significant results in terms of OR and CR rates in comparison with the unlabelled parent mAb tositumomab. In addition to experiences in refractory patients, 131I-tositumomab has also been applied in the front-line setting: An ORR of 95 %, CR of 75 %, and a median time to tumor progression (TTP) of >5 years were achieved in a clinical study on 76 previously untreated patients with FL. Nonetheless, the low tumor burden in the patient population might have biased the results [266].
90Y-Ibritumomab Tiuxetan Monotherapy
90Y-ibritumomab tiuxetan consists of the pure beta-emitter, yttrium-90 isotope. Due to its nature, it cannot be applied in imaging; in addition, densitometry is not routinely required, and dosing is done on the basis of body weight, with dose adjustment in patients with mild thrombocytopenia. It has the appeal of being administered on an outpatient basis, and no isolation measures are required. Promising results in the treatment of NHL, specifically FL, have been obtained, with an ORR of 50–80 % and CR of 20–30 %. It has been effective in rituximab-refractory patients [267]. In comparison with rituximab, 90Y-ibritumomab tiuxetan led to a higher ORR (80 vs. 56 %) and CR (30 vs. 16 %). However, no remarkable differences were observed in PFS or response duration [257]. A study demonstrated a long-term response exceeding 1 year, with a median duration of 21 months in 25 % of the treated population. Durable responses were mostly observed in complete responders, in patients with stage I/II or non-bulky disease [268].
8.14.2.3 CD37-Directed RIT
CD37-directed 177Lu-tetulomab demonstrated significantly enhanced inhibition of cell growth as compared with CD20-directed 177Lu-rituximab. 177Lu-tetulomab vs. 177Lu-rituximab revealed a growth delay factor of 1.6. 177Lu-tetulomab showed lower or similar uptake in lymphoma cells compared to 177Lu-rituximab. Notably, as explained by higher internalization of tetulomab compared to rituximab (almost ten times), the differences in cell growth inhibition were higher for 18-h than for 2-h incubation with the RICs. In SCID mice, the intravenously injection of Daudi cells was more effective when combined with 50 and 100 MBq/kg 177Lu-tetulomab vs. unlabeled tetulomab. CD37-targeted RIT has been previously studied with 131I-labeled murine monoclonal antibody (MB-1), both in mouse and human models [269–271]. In comparison with CD20, CD37 yielded a higher grade of internalization and de-halogenation of 131I-labeled RIC [269]. Despite clinical responses observed in that study, CD20 was chosen for further development. No subsequent efforts have been made to target CD37 with RICs. Initial studies with CD37 RIT used the chloramine-T method for 131I labeling [269]. However, the application of 131I-labeled Abs with the iodogen or the chloramine-T method is limited as they lack maintenance in the cells after internalization of the antigen–antibody complex [272]. Remarkably, metallic radionuclides labeled to antibodies with chelators are better preserved intracellularly after internalization [273]. Several metallic nuclides are applied for RIT against CD37. As indicated by clinical studies, NHLs are responsive to low linear energy transfer (LET) β-emitters [256, 274]; therefore, 177Lu has been chosen in clinical studies [55]. In addition, it is favored by its availability, suitable radiochemistry and half-life, and promising radiation properties. In another study conducted by Gethie et al. in [275] 177Lu-tetulomab demonstrated relatively high toxicity in SCID mice. It was postulated that the unusual biodistribution, as well as the high radiosensitivity of these DNA double-strand repair-defective mice (due to the SCID mutation), has led to high toxicity level. Yet, in line with the previous study, therapeutic effect of 177Lu-tetulomab was significantly greater than the unlabeled antibody [276]. 125I-labeled tetulomab and rituximab have also been compared which revealed similar antigen-binding properties for tetulomab and rituximab (Kd: 2.7–12.7 for tetulomab and 4.8–12 for rituximab, depending on the applied cell line). The variance in the obtained Kd for different cell lines could be explained by the possible bias caused by the curve-fitting method, as the parameters measured may influence each other. On the other hand, differences in antigen expression in various cell lines due to mutations or posttranslational changes could be involved [55]. Overall, tetulomab antibody was described as an appropriate candidate for RIT for CD37-expressing lymphoma cells. However, future clinical investigations are warranted [55]. Remarkable results have been obtained with anti-CD20 mAbs conjugated to radioisotopes. Patients refractory to rituximab who received Zevalin–rituximab combination achieved an ORR of 74 % with a duration of response of 8.7 months [257]. As demonstrated in the FIT trial, a single injection of Zevalin in first remission FL led to 3-year increase in PFS with reversible tolerable toxicity [255]. In conclusion, radioimmunotherapy has emerged as the most effective single agent in the treatment of FL; in addition it has proved beneficial in other lymphoma subtypes. Nonetheless, the need to logistic procedures has limited its application in the clinical setting [13].
8.14.3 Immunotherapy with Genetically Modified T Cells
Adoptive immunotherapy with genetically modified T cells expressing chimeric T-cell receptors, which target lymphoma-associated antigens, has become an interesting approach [38]. It is based on grafting cytotoxic T lymphocyte with chimeric antigen receptors consisting of a tumor-specific single-chain antibody (scFv) and a cellular activation intracellular signaling domain [277]. Evidence shows that genetically modified T cells with integral membrane scFv chimeric signaling receptors react with tumor-associated antigens in a non-MHC-restricted manner, thereby bypassing the MHC–peptide complex loss, which is a significant escape mechanism for most tumors [277–279]. The intracellular signaling domain, which induces cellular activation, is derived from the cytoplasmic part of a membrane-bound receptor and induces cellular activation. The CD3ζ chain has manifested as the most potent and sufficient T-cell activation mediator [280]. The introduction of a chimeric T-cell antigen receptor gene, consisting of an extracellular scFv and an intracellular part of a signaling molecule (CD3ζ), has led to the construction of tumor-specific cytotoxic T lymphocyte [281]. To elicit substantial lymphocyte activation, adequate co-stimulatory signals are required [38]. T cells modified with chimeric antigen receptors incorporating a CD28 signaling domain have been found much more active when tested in in vitro and in murine models [277, 279, 280].
8.14.3.1 Engineered CD20-Specific T Cells
Adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors has led to immense improvement in the treatment of lymphoma patients. However, modification of the cellular signaling pathways in target tumor cells by treatment with engineered CD20-specific T cells has yet to be fully elucidated [282]. Engineered T cells, expressing a single-chain anti-CD20 Ab, are fused to the T-cell receptor complex CD3ζ chain and MHC-unrestricted cytolysis of CD20-specific lymphoma cells [283]. The CD3ζ chain has been shown to result in sufficient T-cell activation signals [284]. In addition, CD3 and CD28 signals have revealed fundamental roles in cellular proliferation and antigen-induced IL-2 secretion of grafted T cells in an anti-CEA scFv-mediated T-cell adoptive immunotherapy study [280]. Therefore, both signals are elucidated by one recombinant receptor [280, 285]. NHL Raji cell lines were co-cultured with genetically modified T cells with anti-CD20scFvFc/CD28/CD3ζ or anti-CD20scFvFc gene, and the cytolytic activity of this engineered CD20-specific T cells was assessed. It was shown that treatment of Raji cells with T cells genetically modified with anti-CD20scFvFc/CD28/CD3ζ chimera (compared to anti-CD20scFvFc) yields a higher cytotoxicity against Raji cells. Additionally, engineered CD20-specific T cells led to a decrease in IL-10 secretion, as well as inhibition of phosphor-STAT3 and Bcl-2 expression in Raji cells, possibly through the downregulation of p38 MAPK and NF-κB activity. Thus, it was concluded that treatment of Raji cells with engineered CD20-specific T cells enhances its antitumor activities against CD20+ tumor cells through the inhibition of cellular p38 MAPK signaling pathways [282]. Furthermore, engineered CD20-specific T cells were shown to particularly lyse CD20+ target tumor cells and secrete IFN-γ and IL-2 after binding to their target cells. A recombinant anti-CD20scFvFc/CD28/CD3ζ gene has provided both primary and co-stimulatory signals to T cells through one chimera. It was revealed that engineered CD20-specific T cells specifically lysed CD20-positive target tumor cells and produced IFN-γ and IL-2 cytokines after binding to their target cells. Additionally, they significantly inhibited IL-10 secretion. Serum IL-10 is elevated in a number of patients with NHL, and a high IL-10 is associated with poor survival rate [286]. In addition exogenous IL-10 significantly increases NHL tumor cell proliferation [287]. It enhances growth progression and aids in the pathogenesis of NHL through autocrine–paracrine loops [287, 288]; hence, its inhibition seems crucial in the treatment of NHL. Engineered CD20-specific T cells were found to inhibit p-Lyn and p38 MAPK activities and decrease Sp1 and IL-10 levels in targeted Raji cells. In addition, genetically modified T cells reduced NF-κB DNA-binding activities and downregulated p-STAT3 and Bcl-2 expression levels.
It has been established that the downregulation of NF-κB activity induced by rituximab is mediated through the p38 MAPK signaling pathway and that phosphor-Lyn and p38 MAPK activities are inhibited by rituximab, resulting in the inhibition of IL-10 transcription via Sp1. Consequently, downregulation of the autocrine–paracrine loop of IL-10/IL-10R signaling leads to partial inhibition of p-STAT3 and Bcl-2 expression. Sp1 transcription factor is activated by p38 MAPK, and Sp1 is involved in the regulation of IL-10 expression in a number of cell lines [289]. Engineered T cells expressing anti-CD20scFvFc/CD28/CD3ζ have displayed stronger inhibition of p38 MAPK activity, downregulation of Bcl-2 expression, and IL-10 secretion, compared to the engineered T cells expressing anti-CD20scFvFc. It confers to increased cytotoxicity via inhibition of p38 MAPK activity and decrease in IL-10 secretion in the target tumor cells. Therefore, it is postulated that modifications of the cellular p38 MAPK signaling pathways in target cells hold potential in the antitumor effect of adoptive T-cell therapy [282].
8.14.4 Genetic Augmentation of Adoptive T Cells
Various challenges are encountered during the development of T-cell cancer immunotherapy; since T-cell therapy targets are mostly self-proteins, developing tolerance and weak antigenic properties, their cytotoxic activity is limited [290]. In addition, the immune system may be antagonized by the expression of inhibitory ligands and secreted factors. Thus, genetic modification of adoptively transferred T cells has been developed to overcome these evasive mechanisms. By redirecting T cells to tumor antigens through the expression of transgenic TCRs or chimeric antigen receptors (CARs), negative selection can be bypassed, and much higher levels of tumor-specific cells, with reduced dependence on co-stimulation and target cell MHC expression, are yielded. Transgenic expression of activating cytokines such as IL-2 and IL-15 can restore lymphocyte activity; in addition, suppressive factors can lead to T-cell resistance through overexpression of dominant-negative receptors [291]. Transgenic expression of receptors for tumor-secreted chemokines is believed to improve the localization of T cells at tumor site [292]. On the other hand, genetic modification may successfully result in T-cell resistance to immunosuppressive drugs [293].
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