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Immunotoxin therapy for hematologic malignancies: where are we heading? Q1
Jayaprakasam Madhumathi, Sithambaram Devilakshmi, Surapally Sridevi and Rama S. Verma Stem Cell and Molecular Biology Laboratory, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India
The identification of numerous unique targets in recent years has led to the development of various immunotoxins (ITs) for treating hematological malignancies. Some of these ITs have advanced to clinical trials and have resulted in a high response rate against leukemia. Newer targets with improve specificity are also being identified for targeting several leukemias. Currently, several modified versions of ITs with increased efficacy are being constructed and evaluated for cytotoxicity in vitro as well as in vivo. Here, we summarize recent advances in preclinical and clinical studies of recombinant ITs targeting diverse surface receptors.
Introduction Q2 Innumerable therapeutic strategies for different types of cancer have emerged with improved specificity and potency while minimizing toxicity. ITs represent fusion proteins carrying a target molecule conjugated to a toxin molecule. The target moieties can be either antibody based, such as monoclonal antibodies (Mabs), genetically engineered single chain/double chain antibody fragments or ligands, cytokines, and growth factors targeting cell surface receptors [1,2]. After the initial success of monoclonal therapy for cancer, Mabs were linked to toxin molecules, which were more specific in targeting and more potent in destroying cancer cells. The careful design of target moiety and toxin is the key factor of a successful therapy because each type of cancer cell expresses a different set of surface antigens and responds differently to each toxin. Some ITs have been approved by the US Food and Drug Administration (FDA), such as denileukin diftitox (ONTAK1) for the treatment of cutaneous T cell lymphoma (CTCL). Here, we discuss the advances in antibody–toxin and ligand–toxin conjugates for targeting maligQ3 nancies of myeloid and lymphoid lineages (Figs 1 and 2).
Antibody–toxin conjugates In antibody–toxin conjugates, antibodies or their fragments are fused to a range of toxins, such as bacterial, plant, and fungal Corresponding author: Verma, R.S. ([email protected]) 1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.05.002
toxins or human apoptotic proteins, which become internalized in the target cell and induce apoptosis. Mabs or single-chain variable fragments (ScFvs) against several target antigens have been developed and fused to toxins such as diphtheria toxin (DT), Pseudomonas exotoxin (PE), ricin, saporin, and gelonin, to construct ITs [3]. These target surface molecules are lineage specific and are being utilized for targeting specific types of leukemia or lymphoma, as discussed below (Table 1).
CD19 CD19 is expressed on the surface of B cells and acts as a B cell coreceptor in conjunction with CD21 and CD81. It is used to diagnose and target B cell lymphomas. The anti-CD19 Mab, B4, was conjugated to blocked ricin (anti-B4-bR) and exhibits cytotoxic activity in patients with lymphoid malignancies. In a phase I trial using antiB4-bR IT, 11 out of 12 patients with B cell non-Hodgkin’s lymphoma (B-NHL) remained in complete response (CR) to the treatment. HD37-dgA was effective in patients with B cell lymphoma in a phase I study. In a phase II study using adjuvant therapy in relapsed B cell NHL with anti-B4-bR, 26 out of 49 patients remained in CR [4]. A CD19-specific ScFv fused to a 38-kDa fragment of PE prolonged the survival of nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice transplanted with Nalm-6 cells [5]. The conjugate showed synergistic toxicity along with valproic acid and cyclosporine, inducing apoptosis in 12 patients with B cell chronic
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Dt2219 RFB4-dgA, LL2-PE HB22.7-saporin. RFB4-PE38
Anti-B4-bR,anti-CD19-RTA, HD37-dgA CD19ScFv-PE CD7scFv-ETA ScFvCD7:sTRAIL ScFvCD7:sFASL
CD19
CD22 Anti-CD20-saporin S6
CD7
CD30
Ki-4(scFv)ETA, Ki-4-dgA Anti-CD30 Fv-PE
CD30 CD3
CD3 ScFv-DT390(Bi3) A-dmDT390-bisFv(UCHT1)
CD20
Anti-JL1-gelonin
E9(Fv)-PE38
JL-1
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FCRL
B Lymphocytic leukemia
T Lymphocytic leukemia
CCR4
IL2R
CCL17-PE
DAB389IL2
RORI
IL2Rα
Anti-RORI-PE (BT-1)
Anti-Tac(Fv)-PE38 RFT5(scFv)-ETA RFT5-dgA RFT5-SMPT-dgA Drug Discovery Today
FIGURE 1
Surface receptors and their respective immunotoxins being used to target lymphocytes for hematological malignancies, such acute lymphoblastic leukemia (ALL), chronic LL (CLL), cutaneous T cell lymphoma (CTCL), Hodgkin’s disease (HD), non-Hodgkin’s lymphoma (NHL), and other lymphomas. For additional definitions, please see the main text.
lymphocytic leukemia (B-CLL). An anti-CD19 Mab conjugated with ricin A toxin (rRTA) was cytotoxic in pre-B acute lymphoblastic leukemia (pre-B-ALL) and Burkitt’s lymphoma cells [6]. The combination of two ITs, known as Combotox, contains anti-CD19 (HD37dgRTA) and anti-CD22 (RFB4-dgRTA) conjugated to deglycosylated H22(scFv)-ETA H22(scFV)RNase angiogenin Gb-H22(scFv) Anti-My9-bR HUM-195/rGel CD33ScFv-ETA
Anti-JL1-gelonin
CD64 CD33
JL-1
Myeloid leukemia
DTIL3 DT388-IL3 IL3-Bax 26292(Fv)-PE38
IL38
CD20
C-Kit DT-SCF
GM-CSF
RTA (dgRTA). Combotox showed improved survival in SCID mice injected with a NALM-6-UM1 cell line [4]. A phase 1 study of Combotox in pediatric patients with refractory B-ALL resulted in three CR and seven partial responses (PRs) out of a total of 17 children [7]. A recent multi-institutional Phase I trial in refractory B-ALL showed decreased blast counts in 17 patients in the study and partial remission in an additional patient [8]. A combination of Q4 Combotox and cytarabine (Ara-C) was used in an advanced ALL murine xenograft model using a NALM/6 cell line and exhibited longer median survival [9]. Anti-CD19 IT BU12-Saporin showed higher activity and, along with anti-CD20 Mab (rituximab) and its F(ab)2 derivative, induced apoptosis in Ramos cells and was effective in SCID-Ramos mice [10].
GMCSF-DT DT388-GMCSF GMCSF-PE38KDEL hGMCSF-Bad GMCSF-DFF40
CD20 is a 35-kDa protein expressed on normal B cells and abundantly expressed in hairy cell leukemia (HCL). Rituximab was developed to carry mouse variable and human constant regions targeting CD20, and was highly effective in patients with relapsed B-NHL and mantle cell lymphoma (MCL) [11]. A rituximabsaporin-S6 conjugate showed cytotoxicity in Raji and D430B cell lines, enhanced toxicity along with fludarabine, and induced apoptosis in 80% of lymphoma cells from patients with NHL [12].
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FIGURE 2
Surface receptors and their respective immunotoxins being used to target myeloid cells in leukemic conditions, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and other myeloid leukemias. For additional definitions, please see the main text. 2
CD22 CD22 is expressed on B cells in most B cell leukemias and lymphomas. Initially, chemical conjugates with dgA using Mabs H6 and RFB4 or Mabs HD6 and HD39 linked to saporin were used. RFB4-dgA resulted in two CRs and ten PRs out of 41 patients with
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TABLE 1
Antibody–toxin conjugates used in hematological malignancies Target antigen
Immunotoxin
Toxin moiety
Study
Disease
Refs
CD19
Anti-B4-bR
Blocked ricin
Phase I and II
[4]
Anti-CD19-RTA
Ricin A toxin
[6]
HD37-dgA
Deglycosylated ricin A chain Pseudomonas exotoxin (PE)
Pre-B-ALL and Burkitt’s lymphoma cells Phase I
Lymphoid malignancies; relapsed B-NHL Pre B-ALL and Burkitt’s lymphoma B cell lymphoma
SCID mice-Nalm-6 cell line
B-CLL
[5]
CD20
Anti-CD20-saporin-S6
Saporin
Raji and D430B cell lines and cells from patients with NHL
B-NHL
[12]
CD19 + CD20
BU12-Saporin + Anti-CD20
Saporin
SCID mice; Ramos cells
B cell lymphoma
[10]
CD22
RFB4-dgA
Deglycosylated ricin A chain PE Saporin
Phase I
B-cell lymphoma and/or leukemia B cell lymphoma Pre-B-ALL
[4]
HCL, NHL, and CLL
[15–17]
LL2-PE HB22.7- saporin RFB4 (dsFv)-PE38 [BL22/CAT-3888] HA22 or CAT-8015 (moxetumomab pasudotox) CD19 + CD22
HD37-dgRTA and RFB4-dgRTA (Combotox) DT2219 DT2219ARL
PE38
Xenograft models NOD/SCID xenograft mice model Phase I and II
[4] [13]
PE38
Phase I
Relapsed HCL, ALL, or NHL
[18]
Deglycosylated ricin A chain
Phase I
Refractory B-ALL
[4,7,8,19]
Diphtheria toxin (DT389) Diphtheria toxin (DT389)
SCID model
B cell leukemia
[27]
Xenograft imaging model of human Raji Burkitt’s lymphoma
Burkitt’s lymphoma
[28]
HL-60 and U937 cell lines; U937/SCID mouse xenograft model CD64+ cells
AML
[29]
AML
[30]
H22 (scFv)-ETA
PE A
H22(scFv)- RNase angiogenin. Gb-H22 (scFv)
RNAse Granzyme B
U937 cell line and CD64+ AML cells
AML
[31]
Anti-MY9-bR HUM-195/rGEL
Blocked-Ricin Gelonin
CD33 +ve AML cells Phase I
[2] [33,34]
CD33ScFv-ETA
PE A
U937, HL-60, and THP-1
AML Refractory myeloid leukemias AML
Ki-4(scFv)-ETA0
PE A Ricin A PE
Hodgkin lymphoma (HD) HD HD and NHL Lymphoma
[36]
Ki-4.dgA Anti-CD30 Fv-PE
L540Cy cell line SCID mice Phase I CD30 + ve cell lines and CD30-transfected A431 cells
CD7 scFV-ETA
PE A
T-ALL
[4,32]
ScFvCD7:sTRAIL
TRAIL
CEM and Jurkat cells; patients with CD7 + ve T-ALL Leukemic T cell lines and cells from patients with T-ALL
T-ALL
[32]
scFvCD7:sFasL
FasL
T-ALL, PTCL, and CD7+ AML cells
[37]
FCRL
E9(Fv)-PE38
PE
Cell lines
CLL, FL, HCL, and MCL
[38]
CD3
CD3 ScFv-DT390 (Bic3) A-dmDT390-bisFv (UCHT1)
Diphtheria toxin (DT390) Diphtheria toxin (DT390)
HPB-MLT.UM T and SCID mice
T cell leukemia
[39]
Phase I
CTCL
[40]
Anti-JL1-gelonin
Gelonin
Leukemia
Leukemia
[41]
CD64
CD33
CD30
CD7
JL-1
[35]
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ScFv-PE
[4]
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B-cell lymphoma/leukemia. Later, a modified mutant of RTA was prepared (RFB4-N87A) that reduced toxicity. Another anti-CD22 IT with PE using Mab LL2 induced complete regression in human xenograft models [4]. The anti-CD22 monoclonal antibody, HB22.7 conjugated with saporin was effective in increasing median survival time from 20 to 50 days in a NOD/SCID xenograft model of pre-B ALL [13]. PEbased anti-CD22 ITs for B cell malignancies were reviewed by Lechleider and Pastan [14]. Based on the promising results of an anti-CD22 Mab LL2-PE conjugate, scFv was made and tested [4]. However, because it was unstable, the variable domain from Mab RFB4 was fused to PE [RFB4(Fv)-PE38], which was cytotoxic in CD22+ cell lines [15]. RFB4(Fv)-PE38 was further improved by phage display and hot-spot mutagenesis. The IT with Thr-HisTrp (THW) in place of the Ser-Ser-Tyr (SSY) of Fv had improved affinity and was approximately 50 times more cytotoxic in cells derived from patients with CLL or HCL patients [4]. A disulfide stabilized form of IT RFB4 (dsFv)-PE38 (BL22, CAT-3888) had improved stability and showed significant cytotoxicity in vitro and in vivo [14]. In phase I and II trials, BL22 was found phase I Q5 study of XXXX among patients with refractory and/or resistant HCL, NHL, and CLL, there was an 81% overall response rate (ORR), Q6 with 61% CR and 19% PR [16]. A phase II trial of XXXX showed a 72% ORR with 47% CR and 25% PR in retreated patients with best Q7 responses after cladribine failure [15]. It was promising in pediatric Q8 ALL and NHL in another preclinical study in vitro and showed transient clinical activity in another phase I trial [17]. Genetic modification by three point mutations in the heavychain domain of BL22, named HA22 or CAT-8015, or generically, moxetumomab pasudotox, showed higher binding affinity and a tenfold higher cytotoxicity in vitro in B cell leukemia [14]. A doseescalation phase I study of HA22 in adult patients with relapsed and/or refractory HCL showed 81% ORR with 37.5% CR and 43.8% PR. In another phase I study in pediatric patients with refractory or relapsed ALL or NHL, HA22 showed 25% CR (three out of 12 Q9 patients) and hematologic activity in 42% of patients (XXX out of 12 patients) [18]. In a phase I trial of HCL using BL22, 46% of patients showed CR [19]. The ORR was 86%, with responses at all dose levels. Approximately 38% of patients showed immunogenicity and 25–64% were reported to exhibit drug-related toxicities. A mutant of HA22 (HA22LR) was developed by eliminating eight epitopes (HA22-8X), which greatly reduced immunogenicity in mice [20]. Another version of HA22, HA22(R490A), was developed by mutating Arg490 of HA22 to alanine. HA22(R490A) was more cytotoxic in CD22+ cell lines and had potent antitumor activity against CA46 tumors in mice [21]. A single-chain version of HA22-LR (scdsFv-HA22-LR) with peptide linker did not show any difference. However, mutation of asparagine34 in VLCDR1 to alanine (N34A) showed a tenfold increase in affinity in SUDHL-6 and HAL-1 cells [22]. Onda et al. [23] developed HA22-LR8M with no immunogenicity in mice and later constructed HA22-LR-LO10 without human epitopes; this showed cytotoxicity in CD22+ lymphoma cell lines and mice [24]. Similarly, the T helper epitopes of PE toxin were identified and silenced in another construct, LMB-T18 [25]. Recently, Bera et al. [26] developed a less immunogenic version of HA22 (LMB11), which was more effective than HA22 in vitro and showed complete remission in vivo with a longer half-life in circulation. 4
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A bispecific IT targeting CD19 and CD22 using two repeating scFv subunits fused to DT390 (DT2219) significantly inhibited tumor growth in a SCID model of B cell leukemia [27]. It showed greater affinity in Daudi cells compared with ITs with anti-CD19 or anti-CD22 alone. An improved version, DT2219ARL, showed superior antileukemic activity with long-term survival in vivo [28]. This construct was more potent in a bioluminescent xenograft-imaging model of human Raji Burkitt’s lymphoma.
CD64 The high-affinity receptor for immunoglobulin (Ig)-G, CD64 (FcgRI), is a surface antigen abundantly expressed on activated cells of the myeloid lineage and on monocytoid-differentiated AML subtypes. Anti-CD64 Mab linked to ricin A showed promising results in animal models. The IT h22 (scFv)-ETA, comprising truncated PE and humanized scFv, was found to be efficient in vitro [4] and in a U937/SCID mouse xenograft model, where it prolonged the overall survival of AML xenograft animals [29]. The humanized h22(scFv) fused to human RNase angiogenin carrying an engineered adapter with a synthetic translocation domain, showed a 20-fold increase in cytotoxicity and higher serum stability in CD64+ cells [30]. In another study, humanized scFv was linked to granzyme B (Gb), a human serine protease from cytotoxic granules. Gb-H22 scFv showed specific cytotoxicity in a CD64+ cell line, U937 and in primary CD64+ AML cells [31]. The advances in humanized ITs were reviewed in detail by Mathew and Verma [32].
CD33 CD33 is another potential target expressed on myeloid, erythroid, megakaryocyte, and multipotent progenitor cells. Almost 90% of patients with AML are CD33+, with higher numbers of these molecules compared with normal samples. The CD33-targeting IT, anti-MY9 blocked-Ricin (Anti-MY9-bR), was effective against CD33+ AML cells. The humanized anti-CD33 Mab, M195, conjugated to gelonin (HUM-195/rGEL) was used in CD33+ leukemic cell lines, xenografts in nude mice [2] and in a phase I trial of refractory myeloid leukemias [33]. It induced a 50% reduction in peripheral blood blasts in four out of 28 patients and a 38–50% reduction in bone marrow blasts in another three patients [34]. ScFv of anti-CD33 Mab conjugated with ETA and carrying the KDEL peptide was developed to improve retrograde transport to the endoplasmic reticulum. It induced apoptosis in myeloid cell lines U937, HL-60, and THP-1 as well as in primary AML cells [35].
CD30 CD30 belongs to the tumor necrosis factor (TNF) receptor family and is expressed only in activated but not resting T and B cells. It is used as a marker for Hodgkin’s and Reed–Sternberg cells in HL and NHL. Anti-CD30 scFv fused to ETA [Ki-4(scFv)-ETA0 ] showed cytotoxicity in the HL-derived cell line L540Cy and SCID mice. AntiCD30 scFv conjugated to ricin A (Ki-4.dgA) showed one PR and one minor response (MR) in 15 patients in a phase I study of patients with HL or NHL [36].
CD7 CD7 is found on thymocytes and mature T cells and is involved in T cell–B cell interactions during early lymphoid development. An IT targeting CD7 was constructed by fusing the svFv of anti-CD7
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FCRL1 Fc receptor-like (FCRL) proteins are type I transmembrane glycoproteins belonging to the FCRL family and have been reported to be expressed in multiple myeloma, CLL, MCL, HCL, mucosaassociated lymphoid tissue lymphoma, and diffuse large B cell lymphoma. FCRL1 is highly expressed in B cells and at a higher level compared with other FCRL proteins in CLL. Two anti-FCRL1 ITs, E3(Fv)-PE38 and E9(Fv)-PE38, showed specific cytotoxicity in CLL, follicular lymphoma (FL), HCL, and MCL [38].
CD3 CD3 is overexpressed in T cell malignancies and is a potential target for treating these diseases. A bivalent protein carrying two repeating scFv linked to DT390, named Bic3, was cytotoxic in HPBMLT.UM T leukemia cells and prolonged survival of SCID mice [39]. Similarly, an anti-CD3e immunotoxin A-dmDT390-bisFv (UCHT1) resulted in two PRs in five patients with CTCL in a phase I trial [40].
JL-1 A unique thymocyte-specific surface molecule called JL-1, expressed in 75.6% of leukemic cases, was identified as a leukemic marker. Anti-JL1 Mab conjugated with gelonin, showed toxicity in JL-1+ leukemic cells [41].
CD74 Anti-CD74 fused to ranpirnase (Rap) has shown antitumor activity in lymphoma xenograft models. Mab (NIMR7) against lymphocytic glycoprotein p58, conjugated to ricin, showed cytotoxicity in BCL1 lymphoma cells. Other targets being tested include CD72, CD79b, and CD180 for NHL. Currently, leukemic stem cells (LSC) are being targeted using specific LSC markers, such as CXCR4, b1-integrin or VLA-4, CD123, and CD44 [3]. CD2, CD5, and co-stimulatory antigens CD80, CD86, CD38, and CTLA-4 (CTLA-4 homolog) are other potential targets being tested, along with saporin toxin [42].
Ligand–toxin conjugates Receptors of cytokines and growth factors are overexpressed generally in all types of cancer. In leukemia, receptors for interleukin (IL)-2, IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) are overexpressed compared
with normal blood cells and have been targeted successfully by several research groups (Table 2).
IL2R IL-2 receptor (IL2R) has three subunits [alpha (CD25), beta (CD122), and gamma (CD132)] and binds the cytokine IL-2 with high affinity. IL2R is highly expressed in leukemias such as CTCL, adult T cell leukemia (ATL), HD, and other B and T cell leukemias and lymphomas. Given that only a small percentage of normal T cells are IL2R+, this receptor has been widely used to target leukemias and lymphomas. A fusion construct of IL-2 and DT containing the first 485 amino acids of IL-2 (DAB486IL2) showed promise in clinical trials. An- Q10 other improved toxin that showed higher cytotoxicity and a longer half-life was constructed by eliminating amino acids 389–485; it was named DAB389IL-2, but is also known as denileukin diftitox or ONTAK (see above). A multicenter, dose-escalation phase I trial using DAB389IL-2 in relapsed patients with CTCL, NHL, or HD resulted in five CR and eight PR in 35 patients with CTCL, and one CR and two PR out of 17 patients with NHL. In a pivotal phase III trial of 71 patients with CTCL, a response rate of 20% PR and 10% CR was observed. ONTAK was used successfully in patients with relapsed, refractory peripheral T cell lymphomas (PTCL) who failed to respond to 13 standard single and multiple chemotherapy regimens. In patients with CTCL, the objective response rate ranged from 38% to 49% [43], which increased to 67% in a phase I trial of patients who also received retinoid bexarotene because of higher IL2R levels [44]. In another phase II trial of 12 patients with fludarabine-refractory CLL, treatment with ONTAK resulted in two PRs (17%) and seven minimal responses (58%) [4]. In 29 patients with indolent B cell NHL, ONTAK resulted in three PRs (10%) in a phase II study. Another Phase II study of ONTAK for relapsed/refractory B Cell NHL showed 24.5% ORR with 6.7% CR and 17.8% PR. The activity was increased further in another phase II trial, when it was combined with rituximab, with an ORR of 55% (four CRs and two PRs) in 11 out of 14 patients with rituximab-refractory FL and 100% in three patients with rituximab-sensitive B-NHL tumors. Phase II clinical studies in patients with recurrent or refractory CLL showed CR in one (4%) and partial remission in five (23%) out of 22 patients [44]. ITs conjugating DT (390) with IL-2 (69) have been constructed Q11 to selectively target the IL2Ra subunit that exhibited specific cytotoxicity in CD25+ leukemic cell lines [45]. A humanized IT carrying IL-2 linked to human Bcl-2-associated X protein (Bax), was reported to induce apoptosis in T cell lines and activated lymphocytes [32]. In a recent trial, patients with human T cell lymphotropic virus type-1 (HTLV-1)-associated ATL showed improvement in scleritis using ONTAK [46]. Given that CD25 is upregulated in leukemia, ScFv from the anti-CD25 Mab was fused to truncated PE to construct anti-Tac (Fv) PE40 and anti-Tac (Fv)PE38 or LMB-2. The anti-Tac(Fv)-PE40KDEL showed cytotoxic effects in patients with ATL and in the human T cell lines KUT1 and KUT-2 when used along with other agents PAK-200, FK-506, quinidine, cepharanthine, and cyclosporine A (CsA) [47]. LMB-2 showed selective cytotoxicity in preclinical studies in mice CD25+ human xenografts in vivo, where it showed synergy with chemotherapy [48]. It resulted in one CR out of four patients
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Mab TH-69 to ETA. It induced apoptosis in T cell leukemic cell lines, CEM, and Jurkat cells, and in 20% of CD7+ cells in vitro from a patient with acute T cell leukemia [4]. Human TNF-related apoptosis-inducing ligand (TRAIL) linked to scFv against CD7 (ScFvCD7:sTRAIL) induced apoptosis in leukemic T cell lines and cells from T-ALL patients. Its activity was stronger than that of scFvCD7:ETA and was augmented by vincristine [32]. Similarly, another humanized IT targeting CD7, scFvCD7:sFasL, was made using the Fas ligand (FasL) to target T cell leukemia. FasL (CD95L) belongs to the TNF family that induces apoptosis by binding to Fas and is involved in the progression of cancer. It induced apoptosis in T-ALL cell lines and patient-derived T-ALL, peripheral T cell lymphoma (PTCL), and CD7+ AML cells, which was augmented by farnesyl transferase inhibitor L-744832 and the proteasome inhibitor bortezomib [37].
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TABLE 2
Ligand–toxin conjugates used in hematological malignancies
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Target antigen
Immunotoxin
Toxin moiety
Study
Disease
Refs
IL2R
DAB389IL-2
Diphtheria toxin (DT389)
Phase I
CTCL, NHL, or HD; refractory PTCL
[44]
Phase II
Fludarabine-refractory CLL; indolent B cell NHL; relapsed B cell NHL; refractory CLL CTCL
Phase III IL2Ra
Anti-Tac(Fv)-PE40KDEL
PE
Anti-Tac (Fv)-PE38 [LMB-2]
PE
RFT5(scFv)-ETA0
PE A
RFT5-dgA
Deglycosylated ricin A chain Deglycosylated ricin A chain
RFT5-SMPT-dgA
KUT-1 and KUT-2 cell lines Phase I Hodgkin-derived cell lines; HL in SCID mice Phase I
ATL
[47]
HCL, CTCL, HCL, CLL, HD, and ATL HD
[44,48] [4]
HD
Phase I
HD
GM-CSFR
GMCSF–DT DT388–GM-CSF GM-CSF–PE38KDEL hGM-CSF–Bad GMCSF–DFF40
Diphtheria toxin Diphtheria toxin PE Human Bad Human DFF40
AML cell lines Phase I AML cell lines HL cell lines AML cell lines
AML AML AML AML AML
[49] [36] [50] [52] [53]
IL3R
DTIL3
Diphtheria toxin Diphtheria toxin (DTT388)
AML CML AML
[4]
DT388-IL3
AML cell lines and patient cells SCID mice Phase I
Chemo-refractory AML MDS AML
[54]
[4] [55]
IL3-Bax
Bax
FBL-3 myeloid leukemia
IL3Ra
26292(Fv)-PE38
PE
TF-1, Molm-13, and Molm-14 cell lines
Leukemic blasts
[56]
c-Kit
DT-SCF
Diphtheria toxin
K562 and MOLT-4 cell lines
AML blasts
[57]
ROR1
Anti-ROR1-PE (BT-1)
PE
CLL cells and MCL cell lines
CLL and MCL
[58]
CCR4
CCL17-PE
PE
NOD-SCID mice
CTCL
[59]
with HCL resistant to standard therapies, whereas three patients showed a 98–99.8% reduction in malignant circulating cells in a phase I trial. LMB-2 also resulted in one CR in HCL and a total of seven PRs, which included patients with CTCL (one), HCL (three), CLL (one), HD (one), and ATL (one), in a Phase I trial. A disulfidestabilized form of LMB-2 was designed [anti-Tac(dsFv)-PE38KDEL] that showed complete regression of tumors in mice with subcutaneous tumor xenografts of human IL2R-bearing cells [44]. The anti-CD25 Mab RFT5 conjugated to ETA0 [RFT5(scFv)-ETA0 ] showed cytotoxicity in Hodgkin-derived cell lines and eliminated disseminated human HD in SCID mice [4]. Anti-CD25 Mab was conjugated to dgA (RFT5-dgA) and resulted in two PRs out of 18 patients with HD. Another study also showed two PRs and 1 MR out of 18 patients with refractory HL, who were administered with bolus infusions of RFT5.dgA. In a phase I study, 17 patients with refractory HD treated with RFT5.dgA resulted in two PRs and one MR. The phase I study using RFT5 linked to dgA via sterically hindered disulfide linker (SMPT) (RFT5-SMPT-dgA) in 15 patients with refractory HD resulted in two PRs and one MR. A recombinant IT targeting CD122, Mik-b 1(Fv)-PE40 was also reported to co-internalize with LMB-2 into cells expressing both CD25 and CD122 subunits of the IL2R and was cytotoxic in NK leukemia cells that expressed more CD122 than CD25 [4]. 6
GM-CSF receptors GM-CSF is a cytokine responsible for the growth and differentiation of granulocytes and macrophages. GM-CSF receptors are overexpressed in leukemic cells and have been targeted in leukemia. GM-CSF–saporin killed mouse cells transfected with human receptor [32]. GM-CSF–DT showed cytotoxicity in vitro in multi- Q12 drug-resistant and radiation-resistant AML cell lines and cells from patients with AML patients [49]. The ITs GM-CSF–PE38KDEL and DT388–GM-CSF showed specific cytotoxicity in leukemic cell lines and patients. DT388–GM-CSF was more cytotoxic than GM-CSF– PE38KDEL [50] and showed synergistic toxicity with Ara-C [51]. DT388–GM-CSF was used in refractory AML in a phase I trial that resulted in one CR and two PR out of 31 patients [36]. Humanized toxin hGM-CSF–Bad induced apoptosis of human leukemia cells [52]. In a recent study, a fully humanized toxin was constructed by fusing GM-CSF with DNA fragmentation factor 40 (DFF40), which induced apoptosis in leukemic cell lines and patients with AML [53].
IL3R IL-3 is a cytokine that supports the proliferation and differentiation of myeloid and lymphoid progenitors, but is absent from mature myeloid cells. It is overexpressed in myeloid leukemic
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CD117 The tyrosine kinase proto-oncogene c-kit receptor (CD117) is overexpressed in several cancers, including hematological malignancies. SCF is a cytokine that binds CD117 and is involved in
hematopoiesis. A DT-SCF construct was made that targeted c-Kit and induced apoptosis in vitro in K562 and MOLT4 cell lines [57].
ROR1 Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed during the development of the nervous system. It is a survival factor for various malignancies and is expressed selectively in B cell CLL and MCL. In a recent study, ROR1 immunotoxin (BT-1) conjugated to PE38 showed specific cytotoxicity in primary CLL cells and MCL cell lines, while sparing ROR1-negative cell lines [58]. CCR4 was targeted using its ligand TARC/CCL17 conjugated with PE38 and RNase for CTCL in NOD-SCID mice [59].
Concluding remarks The success rate of immunotoxin therapy in clinical trials of leukemia has driven efforts towards the investigation of newer and enhanced versions of these anticancer drugs. Although toxicity and immunogenicity remain as major concerns, they have been overcome partly by strategies such as the genetic modification of toxic domains, removal of epitopes, and development of humanized forms of ITs. However, there is a need to integrate two or three strategies as combination therapy, instead of a unidirectional approach, in future studies of how to combat leukemia.
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16 Kreitman, R.J. et al. (2005) Phase I trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with B-cell malignancies. J. Clin. Oncol. 23, 6719–6729 17 Wayne, A.S. et al. (2010) Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial. Clin. Cancer Res. 16, 1894–1903 18 Wayne, A. et al. (2010) Complete remissions in 3 of 12 patients with pediatric acute lymphoblastic leukemia (ALL) during phase 1 testing of the anti-CD22 immunotoxin moxetumomab pasudotox. Blood ASH Annu. Meet. Abstr. 116, 3246 19 Kreitman, R.J. et al. (2012) Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J. Clin. Oncol. 30, 1822–1828 20 Onda, M. (2008) An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 105, 11311–11316 21 Bang, S. et al. (2005) HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin. Cancer Res. 11, 1545–1550 22 Kawa, S. et al. (2011) The improvement of an anti-CD22 immunotoxin: conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan. MAbs 3, 479–486 23 Onda, M. et al. (2011) Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 108, 5742–5747 24 Liu, W. et al. (2012) Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 109, 11782–11787 25 Mazor, R. et al. (2014) Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 111, 8571–8576 26 Bera, T.K. et al. (2014) An improved recombinant Fab-immunotoxin targeting CD22 expressing malignancies. Leuk. Res. 38, 1224–1229 27 Vallera, D.A. et al. (2005) A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin. Cancer Res. 11, 3879–3888 28 Vallera, D.A. et al. (2009) Genetic alteration of a bispecific ligand directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy. Leuk. Res. 33, 1233–1242
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progenitors and, thus, is used as a therapeutic target. DTIL3 showed cytotoxicity in AML cell lines expressing the IL-3 receptor (IL3R) and patient-derived AML blasts. DTIL3 with a (G4S)2 linker was toxic to leukemic progenitors from several patients with acutephase CML. DT388IL3, fused with a Met-His linker, showed antileukemic effects in a SCID model of differentiated AML [4]. A human retroviral IT using IL-3 fused to Bax, induced apoptosis in FBL-3, a lethal myeloid leukemia [54]. A phase I trial with DT388IL3 showed one CR and one PR out of 40 evaluable patients with chemorefractory AML and one PR out of five patients with myelodysplasia (MDS) [55]. The alpha subunit of IL3R (IL-3Ralpha, CD123) is highly expressed in leukemic blasts and leukemic stem cells. ScFv from anti-CD123 Mab 26292 was conjugated to PE to construct the IT 26292(Fv)-PE38, which was further improved by mutation to make 26292(Fv)-PE38-KDEL. It was cytotoxic to the CD123+ cell lines TF-1, Molm-13 and Molm-14, but not in those with low or no expression of CD123 [56].
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29 Tur, M.K. et al. (2011) In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)-ETA0 in a human acute myeloid leukemia xenograft tumor model. Int. J. Cancer 129, 1277–1282 30 Hetzel, C. et al. (2008) Small cleavable adapters enhance the specific cytotoxicity of a humanized immunotoxin directed against CD64-positive cells. J. Immunother. 31, 370–376 31 Stahnke, B. et al. (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol. Cancer Ther. 7, 2924–2932 32 Mathew, M. and Verma, R.S. (2009) Humanized immunotoxins: a new generation of immunotoxins for targeted cancer therapy. Cancer Sci. 100, 1359–1365 33 Dean, A. et al. (2010) Phase I clinical trial of the anti-CD33 immunotoxin HuM195/rgel in patients (pts) with advanced myeloid malignancies. J. Clin. Oncol. 28 (Suppl.), 15 34 Borthakur, G. et al. (2013) Phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with advanced myeloid malignancies. Haematologica 98, 217–221 35 Schwemmlein, M. et al. (2006) A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells. Br. J. Haematol. 133, 141–151 36 Pastan, I. et al. (2007) Immunotoxin treatment of cancer. Annu. Rev. Med. 58, 221–237 37 Bremer, E. et al. (2006) CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia. Blood 107, 2863–2870 38 Du, X. et al. (2008) FCRL1 on chronic lymphocytic leukemia, Hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins. Blood 111, 338–343 39 Vallera, D.A. et al. (2005) Molecular modification of a recombinant, bivalent antihuman CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice. Leuk. Res. 29, 331–341 40 Frankel, A.E. et al. (2009) Anti-CD3 recombinant diphtheria immunotoxin therapy of cutaneous T cell lymphoma. Curr. Drug Targets 10, 104–109 41 Shin, Y.K. et al. (2003) Targeted cytotoxic effect of anti-JL1 immunotoxin against a human leukemic cell line and its clinical implications. Cancer Immunol. Immunother. 52, 506–512 42 Polito, L. et al. (2011) Immunotoxins and other conjugates containing saporin-s6 for cancer therapy. Toxins 3, 697–720 43 FitzGerald, D.J. et al. (2011) Treatment of hematologic malignancies with immunotoxins and antibody-drug conjugates. Cancer Res. 71, 6300–6309 44 Kreitman, R.J. (2009) Recombinant immunotoxins containing truncated bacterial toxins for the treatment of hematologic malignancies. BioDrugs 23, 1–13
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45 Potala, S. and Verma, R.S. (2010) Modified DT-IL2 fusion toxin targeting uniquely IL2Ralpha expressing leukemia cell lines – construction and characterization. J. Biotechnol. 148, 147–155 46 Larson, T.A. et al. (2012) Interleukin-2 receptor targeted therapy of ocular disease of HTLV-1-associated adult T-cell leukemia. Ocul. Immunol. Inflamm. 20, 312–314 47 Ohno, N. et al. (2002) Augmentation of the activity of an immunotoxin, antiTac(Fv)-PE40KDEL, in T cell lines infected with human T cell leukemia virus type-I. Leuk. Lymphoma 4, 885–888 48 Singh, R. et al. (2012) Synergistic antitumor activity of anti-CD25 recombinant immunotoxin LMB-2 with chemotherapy. Clin. Cancer Res. 18, 152–160 49 Perentesis, J.P. et al. (1997) Granulocyte-macrophage colony-stimulating factor receptor-targeted therapy of chemotherapy- and radiation-resistant human myeloid leukemias. Leuk. Lymphoma 25, 247–256 50 Kreitman, R.J. and Pastan, I. (1997) Recombinant toxins containing human granulocyte-macrophage colony-stimulating factor and either pseudomonas exotoxin or diphtheria toxin kill gastrointestinal cancer and leukemia cells. Blood 90, 252–259 51 Kim, C.N. et al. (1999) Diphtheria toxin fused to granulocyte-macrophage colonystimulating factor and Ara-C exert synergistic toxicity against human AML HL-60 cells. Leuk. Res. 23, 527–538 52 Antignani, A. and Youle, R.J. (2005) A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad. Biochemistry 44, 4074–4082 53 Mathew, M. et al. (2013) GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells. Apoptosis 8, 882–895 54 Vallera, D.A. et al. (2003) Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene. Hum. Gene Ther. 14, 1787–1798 55 Frankel, A. et al. (2008) Phase I clinical study of diphtheria toxin-interleukin 3 fusion protein in patients with acute myeloid leukemia and myelodysplasia. Leuk. Lymphoma 49, 543–553 56 Du, X. et al. (2007) New immunotoxins targeting CD123, a stem cell antigen on acute myeloid leukemia cells. J. Immunother. 30, 607–613 57 Potala, S. and Verma, R.S. (2010) A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization. Appl. Biochem. Biotechnol. 162, 1258–1269 58 Baskar, S. et al. (2012) Targeting malignant B cells with an immunotoxin against ROR1. MAbs 4, 349–361 59 Baatar, D. et al. (2007) CCR4-expressing T cell tumors can be specifically controlled via delivery of toxins to chemokine receptors. J. Immunol. 179, 1996–2004
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Immunotoxin therapy for hematologic malignancies: where are we heading? Q1
Jayaprakasam Madhumathi, Sithambaram Devilakshmi, Surapally Sridevi and Rama S. Verma Stem Cell and Molecular Biology Laboratory, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India
The identification of numerous unique targets in recent years has led to the development of various immunotoxins (ITs) for treating hematological malignancies. Some of these ITs have advanced to clinical trials and have resulted in a high response rate against leukemia. Newer targets with improve specificity are also being identified for targeting several leukemias. Currently, several modified versions of ITs with increased efficacy are being constructed and evaluated for cytotoxicity in vitro as well as in vivo. Here, we summarize recent advances in preclinical and clinical studies of recombinant ITs targeting diverse surface receptors.
Introduction Q2 Innumerable therapeutic strategies for different types of cancer have emerged with improved specificity and potency while minimizing toxicity. ITs represent fusion proteins carrying a target molecule conjugated to a toxin molecule. The target moieties can be either antibody based, such as monoclonal antibodies (Mabs), genetically engineered single chain/double chain antibody fragments or ligands, cytokines, and growth factors targeting cell surface receptors [1,2]. After the initial success of monoclonal therapy for cancer, Mabs were linked to toxin molecules, which were more specific in targeting and more potent in destroying cancer cells. The careful design of target moiety and toxin is the key factor of a successful therapy because each type of cancer cell expresses a different set of surface antigens and responds differently to each toxin. Some ITs have been approved by the US Food and Drug Administration (FDA), such as denileukin diftitox (ONTAK1) for the treatment of cutaneous T cell lymphoma (CTCL). Here, we discuss the advances in antibody–toxin and ligand–toxin conjugates for targeting maligQ3 nancies of myeloid and lymphoid lineages (Figs 1 and 2).
Antibody–toxin conjugates In antibody–toxin conjugates, antibodies or their fragments are fused to a range of toxins, such as bacterial, plant, and fungal Corresponding author: Verma, R.S. ([email protected]) 1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.05.002
toxins or human apoptotic proteins, which become internalized in the target cell and induce apoptosis. Mabs or single-chain variable fragments (ScFvs) against several target antigens have been developed and fused to toxins such as diphtheria toxin (DT), Pseudomonas exotoxin (PE), ricin, saporin, and gelonin, to construct ITs [3]. These target surface molecules are lineage specific and are being utilized for targeting specific types of leukemia or lymphoma, as discussed below (Table 1).
CD19 CD19 is expressed on the surface of B cells and acts as a B cell coreceptor in conjunction with CD21 and CD81. It is used to diagnose and target B cell lymphomas. The anti-CD19 Mab, B4, was conjugated to blocked ricin (anti-B4-bR) and exhibits cytotoxic activity in patients with lymphoid malignancies. In a phase I trial using antiB4-bR IT, 11 out of 12 patients with B cell non-Hodgkin’s lymphoma (B-NHL) remained in complete response (CR) to the treatment. HD37-dgA was effective in patients with B cell lymphoma in a phase I study. In a phase II study using adjuvant therapy in relapsed B cell NHL with anti-B4-bR, 26 out of 49 patients remained in CR [4]. A CD19-specific ScFv fused to a 38-kDa fragment of PE prolonged the survival of nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice transplanted with Nalm-6 cells [5]. The conjugate showed synergistic toxicity along with valproic acid and cyclosporine, inducing apoptosis in 12 patients with B cell chronic
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Dt2219 RFB4-dgA, LL2-PE HB22.7-saporin. RFB4-PE38
Anti-B4-bR,anti-CD19-RTA, HD37-dgA CD19ScFv-PE CD7scFv-ETA ScFvCD7:sTRAIL ScFvCD7:sFASL
CD19
CD22 Anti-CD20-saporin S6
CD7
CD30
Ki-4(scFv)ETA, Ki-4-dgA Anti-CD30 Fv-PE
CD30 CD3
CD3 ScFv-DT390(Bi3) A-dmDT390-bisFv(UCHT1)
CD20
Anti-JL1-gelonin
E9(Fv)-PE38
JL-1
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FCRL
B Lymphocytic leukemia
T Lymphocytic leukemia
CCR4
IL2R
CCL17-PE
DAB389IL2
RORI
IL2Rα
Anti-RORI-PE (BT-1)
Anti-Tac(Fv)-PE38 RFT5(scFv)-ETA RFT5-dgA RFT5-SMPT-dgA Drug Discovery Today
FIGURE 1
Surface receptors and their respective immunotoxins being used to target lymphocytes for hematological malignancies, such acute lymphoblastic leukemia (ALL), chronic LL (CLL), cutaneous T cell lymphoma (CTCL), Hodgkin’s disease (HD), non-Hodgkin’s lymphoma (NHL), and other lymphomas. For additional definitions, please see the main text.
lymphocytic leukemia (B-CLL). An anti-CD19 Mab conjugated with ricin A toxin (rRTA) was cytotoxic in pre-B acute lymphoblastic leukemia (pre-B-ALL) and Burkitt’s lymphoma cells [6]. The combination of two ITs, known as Combotox, contains anti-CD19 (HD37dgRTA) and anti-CD22 (RFB4-dgRTA) conjugated to deglycosylated H22(scFv)-ETA H22(scFV)RNase angiogenin Gb-H22(scFv) Anti-My9-bR HUM-195/rGel CD33ScFv-ETA
Anti-JL1-gelonin
CD64 CD33
JL-1
Myeloid leukemia
DTIL3 DT388-IL3 IL3-Bax 26292(Fv)-PE38
IL38
CD20
C-Kit DT-SCF
GM-CSF
RTA (dgRTA). Combotox showed improved survival in SCID mice injected with a NALM-6-UM1 cell line [4]. A phase 1 study of Combotox in pediatric patients with refractory B-ALL resulted in three CR and seven partial responses (PRs) out of a total of 17 children [7]. A recent multi-institutional Phase I trial in refractory B-ALL showed decreased blast counts in 17 patients in the study and partial remission in an additional patient [8]. A combination of Q4 Combotox and cytarabine (Ara-C) was used in an advanced ALL murine xenograft model using a NALM/6 cell line and exhibited longer median survival [9]. Anti-CD19 IT BU12-Saporin showed higher activity and, along with anti-CD20 Mab (rituximab) and its F(ab)2 derivative, induced apoptosis in Ramos cells and was effective in SCID-Ramos mice [10].
GMCSF-DT DT388-GMCSF GMCSF-PE38KDEL hGMCSF-Bad GMCSF-DFF40
CD20 is a 35-kDa protein expressed on normal B cells and abundantly expressed in hairy cell leukemia (HCL). Rituximab was developed to carry mouse variable and human constant regions targeting CD20, and was highly effective in patients with relapsed B-NHL and mantle cell lymphoma (MCL) [11]. A rituximabsaporin-S6 conjugate showed cytotoxicity in Raji and D430B cell lines, enhanced toxicity along with fludarabine, and induced apoptosis in 80% of lymphoma cells from patients with NHL [12].
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FIGURE 2
Surface receptors and their respective immunotoxins being used to target myeloid cells in leukemic conditions, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and other myeloid leukemias. For additional definitions, please see the main text. 2
CD22 CD22 is expressed on B cells in most B cell leukemias and lymphomas. Initially, chemical conjugates with dgA using Mabs H6 and RFB4 or Mabs HD6 and HD39 linked to saporin were used. RFB4-dgA resulted in two CRs and ten PRs out of 41 patients with
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TABLE 1
Antibody–toxin conjugates used in hematological malignancies Target antigen
Immunotoxin
Toxin moiety
Study
Disease
Refs
CD19
Anti-B4-bR
Blocked ricin
Phase I and II
[4]
Anti-CD19-RTA
Ricin A toxin
[6]
HD37-dgA
Deglycosylated ricin A chain Pseudomonas exotoxin (PE)
Pre-B-ALL and Burkitt’s lymphoma cells Phase I
Lymphoid malignancies; relapsed B-NHL Pre B-ALL and Burkitt’s lymphoma B cell lymphoma
SCID mice-Nalm-6 cell line
B-CLL
[5]
CD20
Anti-CD20-saporin-S6
Saporin
Raji and D430B cell lines and cells from patients with NHL
B-NHL
[12]
CD19 + CD20
BU12-Saporin + Anti-CD20
Saporin
SCID mice; Ramos cells
B cell lymphoma
[10]
CD22
RFB4-dgA
Deglycosylated ricin A chain PE Saporin
Phase I
B-cell lymphoma and/or leukemia B cell lymphoma Pre-B-ALL
[4]
HCL, NHL, and CLL
[15–17]
LL2-PE HB22.7- saporin RFB4 (dsFv)-PE38 [BL22/CAT-3888] HA22 or CAT-8015 (moxetumomab pasudotox) CD19 + CD22
HD37-dgRTA and RFB4-dgRTA (Combotox) DT2219 DT2219ARL
PE38
Xenograft models NOD/SCID xenograft mice model Phase I and II
[4] [13]
PE38
Phase I
Relapsed HCL, ALL, or NHL
[18]
Deglycosylated ricin A chain
Phase I
Refractory B-ALL
[4,7,8,19]
Diphtheria toxin (DT389) Diphtheria toxin (DT389)
SCID model
B cell leukemia
[27]
Xenograft imaging model of human Raji Burkitt’s lymphoma
Burkitt’s lymphoma
[28]
HL-60 and U937 cell lines; U937/SCID mouse xenograft model CD64+ cells
AML
[29]
AML
[30]
H22 (scFv)-ETA
PE A
H22(scFv)- RNase angiogenin. Gb-H22 (scFv)
RNAse Granzyme B
U937 cell line and CD64+ AML cells
AML
[31]
Anti-MY9-bR HUM-195/rGEL
Blocked-Ricin Gelonin
CD33 +ve AML cells Phase I
[2] [33,34]
CD33ScFv-ETA
PE A
U937, HL-60, and THP-1
AML Refractory myeloid leukemias AML
Ki-4(scFv)-ETA0
PE A Ricin A PE
Hodgkin lymphoma (HD) HD HD and NHL Lymphoma
[36]
Ki-4.dgA Anti-CD30 Fv-PE
L540Cy cell line SCID mice Phase I CD30 + ve cell lines and CD30-transfected A431 cells
CD7 scFV-ETA
PE A
T-ALL
[4,32]
ScFvCD7:sTRAIL
TRAIL
CEM and Jurkat cells; patients with CD7 + ve T-ALL Leukemic T cell lines and cells from patients with T-ALL
T-ALL
[32]
scFvCD7:sFasL
FasL
T-ALL, PTCL, and CD7+ AML cells
[37]
FCRL
E9(Fv)-PE38
PE
Cell lines
CLL, FL, HCL, and MCL
[38]
CD3
CD3 ScFv-DT390 (Bic3) A-dmDT390-bisFv (UCHT1)
Diphtheria toxin (DT390) Diphtheria toxin (DT390)
HPB-MLT.UM T and SCID mice
T cell leukemia
[39]
Phase I
CTCL
[40]
Anti-JL1-gelonin
Gelonin
Leukemia
Leukemia
[41]
CD64
CD33
CD30
CD7
JL-1
[35]
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ScFv-PE
[4]
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B-cell lymphoma/leukemia. Later, a modified mutant of RTA was prepared (RFB4-N87A) that reduced toxicity. Another anti-CD22 IT with PE using Mab LL2 induced complete regression in human xenograft models [4]. The anti-CD22 monoclonal antibody, HB22.7 conjugated with saporin was effective in increasing median survival time from 20 to 50 days in a NOD/SCID xenograft model of pre-B ALL [13]. PEbased anti-CD22 ITs for B cell malignancies were reviewed by Lechleider and Pastan [14]. Based on the promising results of an anti-CD22 Mab LL2-PE conjugate, scFv was made and tested [4]. However, because it was unstable, the variable domain from Mab RFB4 was fused to PE [RFB4(Fv)-PE38], which was cytotoxic in CD22+ cell lines [15]. RFB4(Fv)-PE38 was further improved by phage display and hot-spot mutagenesis. The IT with Thr-HisTrp (THW) in place of the Ser-Ser-Tyr (SSY) of Fv had improved affinity and was approximately 50 times more cytotoxic in cells derived from patients with CLL or HCL patients [4]. A disulfide stabilized form of IT RFB4 (dsFv)-PE38 (BL22, CAT-3888) had improved stability and showed significant cytotoxicity in vitro and in vivo [14]. In phase I and II trials, BL22 was found phase I Q5 study of XXXX among patients with refractory and/or resistant HCL, NHL, and CLL, there was an 81% overall response rate (ORR), Q6 with 61% CR and 19% PR [16]. A phase II trial of XXXX showed a 72% ORR with 47% CR and 25% PR in retreated patients with best Q7 responses after cladribine failure [15]. It was promising in pediatric Q8 ALL and NHL in another preclinical study in vitro and showed transient clinical activity in another phase I trial [17]. Genetic modification by three point mutations in the heavychain domain of BL22, named HA22 or CAT-8015, or generically, moxetumomab pasudotox, showed higher binding affinity and a tenfold higher cytotoxicity in vitro in B cell leukemia [14]. A doseescalation phase I study of HA22 in adult patients with relapsed and/or refractory HCL showed 81% ORR with 37.5% CR and 43.8% PR. In another phase I study in pediatric patients with refractory or relapsed ALL or NHL, HA22 showed 25% CR (three out of 12 Q9 patients) and hematologic activity in 42% of patients (XXX out of 12 patients) [18]. In a phase I trial of HCL using BL22, 46% of patients showed CR [19]. The ORR was 86%, with responses at all dose levels. Approximately 38% of patients showed immunogenicity and 25–64% were reported to exhibit drug-related toxicities. A mutant of HA22 (HA22LR) was developed by eliminating eight epitopes (HA22-8X), which greatly reduced immunogenicity in mice [20]. Another version of HA22, HA22(R490A), was developed by mutating Arg490 of HA22 to alanine. HA22(R490A) was more cytotoxic in CD22+ cell lines and had potent antitumor activity against CA46 tumors in mice [21]. A single-chain version of HA22-LR (scdsFv-HA22-LR) with peptide linker did not show any difference. However, mutation of asparagine34 in VLCDR1 to alanine (N34A) showed a tenfold increase in affinity in SUDHL-6 and HAL-1 cells [22]. Onda et al. [23] developed HA22-LR8M with no immunogenicity in mice and later constructed HA22-LR-LO10 without human epitopes; this showed cytotoxicity in CD22+ lymphoma cell lines and mice [24]. Similarly, the T helper epitopes of PE toxin were identified and silenced in another construct, LMB-T18 [25]. Recently, Bera et al. [26] developed a less immunogenic version of HA22 (LMB11), which was more effective than HA22 in vitro and showed complete remission in vivo with a longer half-life in circulation. 4
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A bispecific IT targeting CD19 and CD22 using two repeating scFv subunits fused to DT390 (DT2219) significantly inhibited tumor growth in a SCID model of B cell leukemia [27]. It showed greater affinity in Daudi cells compared with ITs with anti-CD19 or anti-CD22 alone. An improved version, DT2219ARL, showed superior antileukemic activity with long-term survival in vivo [28]. This construct was more potent in a bioluminescent xenograft-imaging model of human Raji Burkitt’s lymphoma.
CD64 The high-affinity receptor for immunoglobulin (Ig)-G, CD64 (FcgRI), is a surface antigen abundantly expressed on activated cells of the myeloid lineage and on monocytoid-differentiated AML subtypes. Anti-CD64 Mab linked to ricin A showed promising results in animal models. The IT h22 (scFv)-ETA, comprising truncated PE and humanized scFv, was found to be efficient in vitro [4] and in a U937/SCID mouse xenograft model, where it prolonged the overall survival of AML xenograft animals [29]. The humanized h22(scFv) fused to human RNase angiogenin carrying an engineered adapter with a synthetic translocation domain, showed a 20-fold increase in cytotoxicity and higher serum stability in CD64+ cells [30]. In another study, humanized scFv was linked to granzyme B (Gb), a human serine protease from cytotoxic granules. Gb-H22 scFv showed specific cytotoxicity in a CD64+ cell line, U937 and in primary CD64+ AML cells [31]. The advances in humanized ITs were reviewed in detail by Mathew and Verma [32].
CD33 CD33 is another potential target expressed on myeloid, erythroid, megakaryocyte, and multipotent progenitor cells. Almost 90% of patients with AML are CD33+, with higher numbers of these molecules compared with normal samples. The CD33-targeting IT, anti-MY9 blocked-Ricin (Anti-MY9-bR), was effective against CD33+ AML cells. The humanized anti-CD33 Mab, M195, conjugated to gelonin (HUM-195/rGEL) was used in CD33+ leukemic cell lines, xenografts in nude mice [2] and in a phase I trial of refractory myeloid leukemias [33]. It induced a 50% reduction in peripheral blood blasts in four out of 28 patients and a 38–50% reduction in bone marrow blasts in another three patients [34]. ScFv of anti-CD33 Mab conjugated with ETA and carrying the KDEL peptide was developed to improve retrograde transport to the endoplasmic reticulum. It induced apoptosis in myeloid cell lines U937, HL-60, and THP-1 as well as in primary AML cells [35].
CD30 CD30 belongs to the tumor necrosis factor (TNF) receptor family and is expressed only in activated but not resting T and B cells. It is used as a marker for Hodgkin’s and Reed–Sternberg cells in HL and NHL. Anti-CD30 scFv fused to ETA [Ki-4(scFv)-ETA0 ] showed cytotoxicity in the HL-derived cell line L540Cy and SCID mice. AntiCD30 scFv conjugated to ricin A (Ki-4.dgA) showed one PR and one minor response (MR) in 15 patients in a phase I study of patients with HL or NHL [36].
CD7 CD7 is found on thymocytes and mature T cells and is involved in T cell–B cell interactions during early lymphoid development. An IT targeting CD7 was constructed by fusing the svFv of anti-CD7
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FCRL1 Fc receptor-like (FCRL) proteins are type I transmembrane glycoproteins belonging to the FCRL family and have been reported to be expressed in multiple myeloma, CLL, MCL, HCL, mucosaassociated lymphoid tissue lymphoma, and diffuse large B cell lymphoma. FCRL1 is highly expressed in B cells and at a higher level compared with other FCRL proteins in CLL. Two anti-FCRL1 ITs, E3(Fv)-PE38 and E9(Fv)-PE38, showed specific cytotoxicity in CLL, follicular lymphoma (FL), HCL, and MCL [38].
CD3 CD3 is overexpressed in T cell malignancies and is a potential target for treating these diseases. A bivalent protein carrying two repeating scFv linked to DT390, named Bic3, was cytotoxic in HPBMLT.UM T leukemia cells and prolonged survival of SCID mice [39]. Similarly, an anti-CD3e immunotoxin A-dmDT390-bisFv (UCHT1) resulted in two PRs in five patients with CTCL in a phase I trial [40].
JL-1 A unique thymocyte-specific surface molecule called JL-1, expressed in 75.6% of leukemic cases, was identified as a leukemic marker. Anti-JL1 Mab conjugated with gelonin, showed toxicity in JL-1+ leukemic cells [41].
CD74 Anti-CD74 fused to ranpirnase (Rap) has shown antitumor activity in lymphoma xenograft models. Mab (NIMR7) against lymphocytic glycoprotein p58, conjugated to ricin, showed cytotoxicity in BCL1 lymphoma cells. Other targets being tested include CD72, CD79b, and CD180 for NHL. Currently, leukemic stem cells (LSC) are being targeted using specific LSC markers, such as CXCR4, b1-integrin or VLA-4, CD123, and CD44 [3]. CD2, CD5, and co-stimulatory antigens CD80, CD86, CD38, and CTLA-4 (CTLA-4 homolog) are other potential targets being tested, along with saporin toxin [42].
Ligand–toxin conjugates Receptors of cytokines and growth factors are overexpressed generally in all types of cancer. In leukemia, receptors for interleukin (IL)-2, IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) are overexpressed compared
with normal blood cells and have been targeted successfully by several research groups (Table 2).
IL2R IL-2 receptor (IL2R) has three subunits [alpha (CD25), beta (CD122), and gamma (CD132)] and binds the cytokine IL-2 with high affinity. IL2R is highly expressed in leukemias such as CTCL, adult T cell leukemia (ATL), HD, and other B and T cell leukemias and lymphomas. Given that only a small percentage of normal T cells are IL2R+, this receptor has been widely used to target leukemias and lymphomas. A fusion construct of IL-2 and DT containing the first 485 amino acids of IL-2 (DAB486IL2) showed promise in clinical trials. An- Q10 other improved toxin that showed higher cytotoxicity and a longer half-life was constructed by eliminating amino acids 389–485; it was named DAB389IL-2, but is also known as denileukin diftitox or ONTAK (see above). A multicenter, dose-escalation phase I trial using DAB389IL-2 in relapsed patients with CTCL, NHL, or HD resulted in five CR and eight PR in 35 patients with CTCL, and one CR and two PR out of 17 patients with NHL. In a pivotal phase III trial of 71 patients with CTCL, a response rate of 20% PR and 10% CR was observed. ONTAK was used successfully in patients with relapsed, refractory peripheral T cell lymphomas (PTCL) who failed to respond to 13 standard single and multiple chemotherapy regimens. In patients with CTCL, the objective response rate ranged from 38% to 49% [43], which increased to 67% in a phase I trial of patients who also received retinoid bexarotene because of higher IL2R levels [44]. In another phase II trial of 12 patients with fludarabine-refractory CLL, treatment with ONTAK resulted in two PRs (17%) and seven minimal responses (58%) [4]. In 29 patients with indolent B cell NHL, ONTAK resulted in three PRs (10%) in a phase II study. Another Phase II study of ONTAK for relapsed/refractory B Cell NHL showed 24.5% ORR with 6.7% CR and 17.8% PR. The activity was increased further in another phase II trial, when it was combined with rituximab, with an ORR of 55% (four CRs and two PRs) in 11 out of 14 patients with rituximab-refractory FL and 100% in three patients with rituximab-sensitive B-NHL tumors. Phase II clinical studies in patients with recurrent or refractory CLL showed CR in one (4%) and partial remission in five (23%) out of 22 patients [44]. ITs conjugating DT (390) with IL-2 (69) have been constructed Q11 to selectively target the IL2Ra subunit that exhibited specific cytotoxicity in CD25+ leukemic cell lines [45]. A humanized IT carrying IL-2 linked to human Bcl-2-associated X protein (Bax), was reported to induce apoptosis in T cell lines and activated lymphocytes [32]. In a recent trial, patients with human T cell lymphotropic virus type-1 (HTLV-1)-associated ATL showed improvement in scleritis using ONTAK [46]. Given that CD25 is upregulated in leukemia, ScFv from the anti-CD25 Mab was fused to truncated PE to construct anti-Tac (Fv) PE40 and anti-Tac (Fv)PE38 or LMB-2. The anti-Tac(Fv)-PE40KDEL showed cytotoxic effects in patients with ATL and in the human T cell lines KUT1 and KUT-2 when used along with other agents PAK-200, FK-506, quinidine, cepharanthine, and cyclosporine A (CsA) [47]. LMB-2 showed selective cytotoxicity in preclinical studies in mice CD25+ human xenografts in vivo, where it showed synergy with chemotherapy [48]. It resulted in one CR out of four patients
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Mab TH-69 to ETA. It induced apoptosis in T cell leukemic cell lines, CEM, and Jurkat cells, and in 20% of CD7+ cells in vitro from a patient with acute T cell leukemia [4]. Human TNF-related apoptosis-inducing ligand (TRAIL) linked to scFv against CD7 (ScFvCD7:sTRAIL) induced apoptosis in leukemic T cell lines and cells from T-ALL patients. Its activity was stronger than that of scFvCD7:ETA and was augmented by vincristine [32]. Similarly, another humanized IT targeting CD7, scFvCD7:sFasL, was made using the Fas ligand (FasL) to target T cell leukemia. FasL (CD95L) belongs to the TNF family that induces apoptosis by binding to Fas and is involved in the progression of cancer. It induced apoptosis in T-ALL cell lines and patient-derived T-ALL, peripheral T cell lymphoma (PTCL), and CD7+ AML cells, which was augmented by farnesyl transferase inhibitor L-744832 and the proteasome inhibitor bortezomib [37].
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TABLE 2
Ligand–toxin conjugates used in hematological malignancies
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Target antigen
Immunotoxin
Toxin moiety
Study
Disease
Refs
IL2R
DAB389IL-2
Diphtheria toxin (DT389)
Phase I
CTCL, NHL, or HD; refractory PTCL
[44]
Phase II
Fludarabine-refractory CLL; indolent B cell NHL; relapsed B cell NHL; refractory CLL CTCL
Phase III IL2Ra
Anti-Tac(Fv)-PE40KDEL
PE
Anti-Tac (Fv)-PE38 [LMB-2]
PE
RFT5(scFv)-ETA0
PE A
RFT5-dgA
Deglycosylated ricin A chain Deglycosylated ricin A chain
RFT5-SMPT-dgA
KUT-1 and KUT-2 cell lines Phase I Hodgkin-derived cell lines; HL in SCID mice Phase I
ATL
[47]
HCL, CTCL, HCL, CLL, HD, and ATL HD
[44,48] [4]
HD
Phase I
HD
GM-CSFR
GMCSF–DT DT388–GM-CSF GM-CSF–PE38KDEL hGM-CSF–Bad GMCSF–DFF40
Diphtheria toxin Diphtheria toxin PE Human Bad Human DFF40
AML cell lines Phase I AML cell lines HL cell lines AML cell lines
AML AML AML AML AML
[49] [36] [50] [52] [53]
IL3R
DTIL3
Diphtheria toxin Diphtheria toxin (DTT388)
AML CML AML
[4]
DT388-IL3
AML cell lines and patient cells SCID mice Phase I
Chemo-refractory AML MDS AML
[54]
[4] [55]
IL3-Bax
Bax
FBL-3 myeloid leukemia
IL3Ra
26292(Fv)-PE38
PE
TF-1, Molm-13, and Molm-14 cell lines
Leukemic blasts
[56]
c-Kit
DT-SCF
Diphtheria toxin
K562 and MOLT-4 cell lines
AML blasts
[57]
ROR1
Anti-ROR1-PE (BT-1)
PE
CLL cells and MCL cell lines
CLL and MCL
[58]
CCR4
CCL17-PE
PE
NOD-SCID mice
CTCL
[59]
with HCL resistant to standard therapies, whereas three patients showed a 98–99.8% reduction in malignant circulating cells in a phase I trial. LMB-2 also resulted in one CR in HCL and a total of seven PRs, which included patients with CTCL (one), HCL (three), CLL (one), HD (one), and ATL (one), in a Phase I trial. A disulfidestabilized form of LMB-2 was designed [anti-Tac(dsFv)-PE38KDEL] that showed complete regression of tumors in mice with subcutaneous tumor xenografts of human IL2R-bearing cells [44]. The anti-CD25 Mab RFT5 conjugated to ETA0 [RFT5(scFv)-ETA0 ] showed cytotoxicity in Hodgkin-derived cell lines and eliminated disseminated human HD in SCID mice [4]. Anti-CD25 Mab was conjugated to dgA (RFT5-dgA) and resulted in two PRs out of 18 patients with HD. Another study also showed two PRs and 1 MR out of 18 patients with refractory HL, who were administered with bolus infusions of RFT5.dgA. In a phase I study, 17 patients with refractory HD treated with RFT5.dgA resulted in two PRs and one MR. The phase I study using RFT5 linked to dgA via sterically hindered disulfide linker (SMPT) (RFT5-SMPT-dgA) in 15 patients with refractory HD resulted in two PRs and one MR. A recombinant IT targeting CD122, Mik-b 1(Fv)-PE40 was also reported to co-internalize with LMB-2 into cells expressing both CD25 and CD122 subunits of the IL2R and was cytotoxic in NK leukemia cells that expressed more CD122 than CD25 [4]. 6
GM-CSF receptors GM-CSF is a cytokine responsible for the growth and differentiation of granulocytes and macrophages. GM-CSF receptors are overexpressed in leukemic cells and have been targeted in leukemia. GM-CSF–saporin killed mouse cells transfected with human receptor [32]. GM-CSF–DT showed cytotoxicity in vitro in multi- Q12 drug-resistant and radiation-resistant AML cell lines and cells from patients with AML patients [49]. The ITs GM-CSF–PE38KDEL and DT388–GM-CSF showed specific cytotoxicity in leukemic cell lines and patients. DT388–GM-CSF was more cytotoxic than GM-CSF– PE38KDEL [50] and showed synergistic toxicity with Ara-C [51]. DT388–GM-CSF was used in refractory AML in a phase I trial that resulted in one CR and two PR out of 31 patients [36]. Humanized toxin hGM-CSF–Bad induced apoptosis of human leukemia cells [52]. In a recent study, a fully humanized toxin was constructed by fusing GM-CSF with DNA fragmentation factor 40 (DFF40), which induced apoptosis in leukemic cell lines and patients with AML [53].
IL3R IL-3 is a cytokine that supports the proliferation and differentiation of myeloid and lymphoid progenitors, but is absent from mature myeloid cells. It is overexpressed in myeloid leukemic
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CD117 The tyrosine kinase proto-oncogene c-kit receptor (CD117) is overexpressed in several cancers, including hematological malignancies. SCF is a cytokine that binds CD117 and is involved in
hematopoiesis. A DT-SCF construct was made that targeted c-Kit and induced apoptosis in vitro in K562 and MOLT4 cell lines [57].
ROR1 Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed during the development of the nervous system. It is a survival factor for various malignancies and is expressed selectively in B cell CLL and MCL. In a recent study, ROR1 immunotoxin (BT-1) conjugated to PE38 showed specific cytotoxicity in primary CLL cells and MCL cell lines, while sparing ROR1-negative cell lines [58]. CCR4 was targeted using its ligand TARC/CCL17 conjugated with PE38 and RNase for CTCL in NOD-SCID mice [59].
Concluding remarks The success rate of immunotoxin therapy in clinical trials of leukemia has driven efforts towards the investigation of newer and enhanced versions of these anticancer drugs. Although toxicity and immunogenicity remain as major concerns, they have been overcome partly by strategies such as the genetic modification of toxic domains, removal of epitopes, and development of humanized forms of ITs. However, there is a need to integrate two or three strategies as combination therapy, instead of a unidirectional approach, in future studies of how to combat leukemia.
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16 Kreitman, R.J. et al. (2005) Phase I trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with B-cell malignancies. J. Clin. Oncol. 23, 6719–6729 17 Wayne, A.S. et al. (2010) Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial. Clin. Cancer Res. 16, 1894–1903 18 Wayne, A. et al. (2010) Complete remissions in 3 of 12 patients with pediatric acute lymphoblastic leukemia (ALL) during phase 1 testing of the anti-CD22 immunotoxin moxetumomab pasudotox. Blood ASH Annu. Meet. Abstr. 116, 3246 19 Kreitman, R.J. et al. (2012) Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J. Clin. Oncol. 30, 1822–1828 20 Onda, M. (2008) An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 105, 11311–11316 21 Bang, S. et al. (2005) HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin. Cancer Res. 11, 1545–1550 22 Kawa, S. et al. (2011) The improvement of an anti-CD22 immunotoxin: conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan. MAbs 3, 479–486 23 Onda, M. et al. (2011) Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 108, 5742–5747 24 Liu, W. et al. (2012) Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 109, 11782–11787 25 Mazor, R. et al. (2014) Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 111, 8571–8576 26 Bera, T.K. et al. (2014) An improved recombinant Fab-immunotoxin targeting CD22 expressing malignancies. Leuk. Res. 38, 1224–1229 27 Vallera, D.A. et al. (2005) A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin. Cancer Res. 11, 3879–3888 28 Vallera, D.A. et al. (2009) Genetic alteration of a bispecific ligand directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy. Leuk. Res. 33, 1233–1242
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progenitors and, thus, is used as a therapeutic target. DTIL3 showed cytotoxicity in AML cell lines expressing the IL-3 receptor (IL3R) and patient-derived AML blasts. DTIL3 with a (G4S)2 linker was toxic to leukemic progenitors from several patients with acutephase CML. DT388IL3, fused with a Met-His linker, showed antileukemic effects in a SCID model of differentiated AML [4]. A human retroviral IT using IL-3 fused to Bax, induced apoptosis in FBL-3, a lethal myeloid leukemia [54]. A phase I trial with DT388IL3 showed one CR and one PR out of 40 evaluable patients with chemorefractory AML and one PR out of five patients with myelodysplasia (MDS) [55]. The alpha subunit of IL3R (IL-3Ralpha, CD123) is highly expressed in leukemic blasts and leukemic stem cells. ScFv from anti-CD123 Mab 26292 was conjugated to PE to construct the IT 26292(Fv)-PE38, which was further improved by mutation to make 26292(Fv)-PE38-KDEL. It was cytotoxic to the CD123+ cell lines TF-1, Molm-13 and Molm-14, but not in those with low or no expression of CD123 [56].
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29 Tur, M.K. et al. (2011) In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)-ETA0 in a human acute myeloid leukemia xenograft tumor model. Int. J. Cancer 129, 1277–1282 30 Hetzel, C. et al. (2008) Small cleavable adapters enhance the specific cytotoxicity of a humanized immunotoxin directed against CD64-positive cells. J. Immunother. 31, 370–376 31 Stahnke, B. et al. (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol. Cancer Ther. 7, 2924–2932 32 Mathew, M. and Verma, R.S. (2009) Humanized immunotoxins: a new generation of immunotoxins for targeted cancer therapy. Cancer Sci. 100, 1359–1365 33 Dean, A. et al. (2010) Phase I clinical trial of the anti-CD33 immunotoxin HuM195/rgel in patients (pts) with advanced myeloid malignancies. J. Clin. Oncol. 28 (Suppl.), 15 34 Borthakur, G. et al. (2013) Phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with advanced myeloid malignancies. Haematologica 98, 217–221 35 Schwemmlein, M. et al. (2006) A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells. Br. J. Haematol. 133, 141–151 36 Pastan, I. et al. (2007) Immunotoxin treatment of cancer. Annu. Rev. Med. 58, 221–237 37 Bremer, E. et al. (2006) CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia. Blood 107, 2863–2870 38 Du, X. et al. (2008) FCRL1 on chronic lymphocytic leukemia, Hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins. Blood 111, 338–343 39 Vallera, D.A. et al. (2005) Molecular modification of a recombinant, bivalent antihuman CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice. Leuk. Res. 29, 331–341 40 Frankel, A.E. et al. (2009) Anti-CD3 recombinant diphtheria immunotoxin therapy of cutaneous T cell lymphoma. Curr. Drug Targets 10, 104–109 41 Shin, Y.K. et al. (2003) Targeted cytotoxic effect of anti-JL1 immunotoxin against a human leukemic cell line and its clinical implications. Cancer Immunol. Immunother. 52, 506–512 42 Polito, L. et al. (2011) Immunotoxins and other conjugates containing saporin-s6 for cancer therapy. Toxins 3, 697–720 43 FitzGerald, D.J. et al. (2011) Treatment of hematologic malignancies with immunotoxins and antibody-drug conjugates. Cancer Res. 71, 6300–6309 44 Kreitman, R.J. (2009) Recombinant immunotoxins containing truncated bacterial toxins for the treatment of hematologic malignancies. BioDrugs 23, 1–13
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www.drugdiscoverytoday.com Please cite this article in press as: Madhumathi, J. et al. Immunotoxin therapy for hematologic malignancies: where are we heading?, Drug Discov Today (2015), http://dx.doi.org/10.1016/ j.drudis.2015.05.002
