Immunotherapy of Recurrent B-Cell Malignancies After Allo-SCT with Bi20 (FBTA05), a Trifunctional Anti-CD3 Â Anti-CD20 Antibody and Donor Lymphocyte Infusion

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ORIGINAL ARTICLE Immunotherapy of recurrent B-cell malignancies after allo-SCT with Bi20 (FBTA05), a trifunctional anti-CD3 Â anti-CD20 antibody and donor lymphocyte infusion

R Buhmann1,2,5, B Simoes1,2,5, M Stanglmaier3,5, T Yang2, M Faltin3, D Bund2, H Lindhofer4 and H-J Kolb1,2

1Department of Medicine III, University of Munich-Grosshadern, Munich, Germany; 2Helmholtz Center Munich, German Research Center for Environmental Health (GmbH)/Clinical Cooperative Group Haematopoietic Cell Transplantation (CCG-HCT), Munich, Germany; 3TRION Research GmbH, Martinsried, Germany and 4TRION Pharma GmbH, Munich, Germany

Donor lymphocyte infusions (DLIs) after allo-SCT Introduction displayed limited use in CLL and highly malignant non- Hodgkin’s lymphoma (NHL). Here we studied whether Evidence for a GVL effect of allo-SCT in highly malignant Bi20 (FBTA05), a novel trifunctional bispecific antibody lymphoma is limited1 and has been discussed controver- targeting CD20 on lymphoma cells and CD3 on T cells, sially.2–4 In a retrospective data analysis of the Interna- could induce GVL responses in combination with DLI or tional Bone Marrow Transplant Registry and European mobilized PBSCT after allogeneic transplantation in Group for Blood and Marrow Transplantation, allogeneic these diseases. Six patients (three cases with p53-mutated transplantation was not superior to syngeneic transplant- CLL and three with high-grade NHL (HG-NHL)) ation, indicating the absence of an allogeneic GVL effect.5 refractory to standard therapy were treated with escalat- However, several studies and observations report a GVL ing doses of Bi20 (range 10–2000 lg) followed by DLI or effect of donor lymphocyte infusions (DLIs).6–9 Further- SCT. Thereby, all CLL patients showed a prompt but more, cells of highly malignant lymphoma may escape transient clinical and hematological response. In one recognition and destruction by allogeneic lymphocytes by patient with HG-NHL, we observed a halt in progression various mechanisms. Downregulation of HLA antigens and for almost 4 months. Side effects (fever, chills and bone costimulatory molecules as well as secretion of inhibitory pain) were tolerable and appeared at antibody dose levels cytokines such as transforming growth factor-b might be between 40 and 200 lg. The cytokine profile was involved and disturb the generation of alloreactive T-cell characterized by transient increases of IL-6, IL-8 and immunity. Antibodies are, at least at first glance, unaffected IL-10. Neither human anti-mouse antibodies nor GVHD by these mechanisms and have been used with increasing developed, allowing repeated treatment courses. In success in the treatment of patients with indolent and summary, the trifunctional antibody Bi20 induced prompt aggressive lymphoma as well as CLL. Rituximab antitumor responses in extensively pretreated, p53-mu- (MabThera, Rituxan) is an IgG1 chimeric monoclonal tated alemtuzumab and rituximab refractory patients antibody directed against the CD20 antigen that improved indicating its therapeutic potential. response and survival of patients. The mechanism of Bone Marrow Transplantation (2009) 43, 383–397; activity is not entirely clear; antibody-dependent cellular doi:10.1038/bmt.2008.323; published online 13 October 2008 cytotoxicity, complement-dependent cytotoxicity and in- Keywords: adoptive immunotherapy; donor lymphocyte duction of apoptosis have been shown.10 The delayed infusion; allogeneic transplantation; B-cell malignancies; therapeutic effect has been discussed as evidence for bispecific antibody potential vaccination effect.10 In that context, bispecific antibodies might be a highly attractive therapeutic concept directing T cells efficiently toward tumor cells.11,12 Moreover, bispecific antibodies carrying a functional Fc part provide the additional capacity to recruit Fcg receptor-bearing accessory cells such as macrophages, DCs and natural killer cells. In this Correspondence: Dr R Buhmann, Department of Medicine III, way, an optimal vaccination might be achieved by inducing Helmholtz Center Munich, CCG-HCT, Marchioninistr. 25, 81377 T-cell cytotoxicity, phagocytosis and processing of relevant Munich, Germany. antigenic peptides, resulting in a lasting cellular immu- E-mail: [email protected] nity.13 Here we report on the use of Bi20 (FBTA05), 5These authors contributed equally to this work. Received 28 December 2007; revised 9 June 2008; accepted 29 July 2008; a trifunctional heterodimeric anti-CD3 (rat IgG2b) Â anti- published online 13 October 2008 CD20 (mouse IgG2a) antibody, already shown to kill Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 384 lymphoma cells very effectively even at very low expression levels in vitro.14 In a pilot study, we treated six patients with recurrent B-cell malignancies, CLL and highly malignant lymphoma

after allo-SCT. Escalating doses of Bi20 were followed by State ofdisease deletion p53 deletion p53 deletion p53 DLI or mobilized PBSCT. Here we show that Bi20 could induce a prompt tumor response in some patients with advanced disease, whereas the toxicity of treatment was tolerable. Previous antibody

Patients and methods 00 Rituximab0 Rituximab Refractory Rituximab Refractory Refractory DLI Patients male.

In a pilot trial, six patients with recurrent and refractory ¼ B-cell malignancies after allo-SCT were treated with escalating doses of Bi20 (anti-CD3 Â anti-CD20) followed by DLI or mobilized PBSCT. Three patients (nos. 1, 2 and 3) had been treated for refractory CLL with SCT from

HLA-identical sibling donors, and patient nos. 4, 5 and 6 lymph node; M had been treated for refractory high-grade lymphoma with ¼ SCT from HLA-haploidentical family donors. The internal

review board and ethics committee were informed about matched related allogeneic 3 Alemtuzumab Refractory, Â female; LN

the individual treatment plans. Informed consent of the Previous PBSCT2 Matched related allogeneic Previous Matched related allogeneicHaploidentical allogeneic, 0CD6-depleted Haploidentical allogeneic, 0CD6-depleted RituximabHaploidentical allogeneic, CD6-depleted Alemtuzumab Refractory, Refractory, patient was provided according to the Declaration of ¼ Helsinki. À À À The patient characteristics and earlier treatments are + + + Previous summarized in Table 1. In all patients, chimerism was radiation proved by the demonstration of sex chromosomes of the donor in blood and/or marrow, HLA typing and/or short tandem repeats; signs and symptoms of GVHD and infections were absent. Patient nos. 1, 2 and 3 had recurrent

B-CLL with p53 mutations. Patient no. 1 had already chemotherapy Extensive undergone re-transplantation and three treatments with diffuse large B-cell lymphoma; F

DLI. Patient nos. 4, 5 and 6 presented with recurrent high- ¼ grade lymphoma. The histology of patient no. 4 was Burkitt-like lymphoma and those of patient nos. 5 and 6 were diffuse large B-cell lymphoma (DLBCL). The high- grade lymphoma patients had received HLA-haploidentical transplantation after chemotherapy; radiation therapy or antibody (rituximab) treatment had failed. kidney, liver Expression of CD20 was conﬁrmed by immunohisto- chemistry or ﬂow cytometric analysis, and anti-CD20 treatment was not given 3 months before start of the treatment with Bi20. Testing for human anti-mouse central nervous system; DLBCL ¼ antibodies (HAMAs) was negative in all patients. The decision for treatment was taken as other treatment options were not available. Stage Affected organs Previous

Treatment schedule All patients were treated with escalating doses of Bi20 (anti- CD3 Â anti-CD20) followed by DLI or PBSCT of their (years) allogeneic stem cell donor (Table 2). Patient no. 1 with Burkitt-like lymphoma; CNS

advanced CLL received a test dose of 10 mg Bi20 on day 1, ¼ which was doubled every second day and increased up to 7 þ

600 mg on day 17, followed by DLI (1 Â 10 CD3 cells per Patient characteristics, diagnosis and previous treatments before combined treatment with Bi20 kg of recipient body weight) on day 18. In patient no. 2 (CLL), Bi20 was escalated up to 1000 mg followed on day 28 by DLI (1 Â 106 CD3 þ cells per kg of recipient body 7 þ Staging of CLL according to Binet classification; staging of B-cell lymphoma according to Ann Arber classification. weight). DLI was further escalated up to 1 Â 10 CD3 Table 1 Patient no. Diagnosis1 Sex/age 23 CLL4 CLL F/455 CLL M/586 Binet Classification BLL C F/52 Binet Blood, Classification LN, C DLBCL spleenAbbreviations: BLL M/30 Blood, LN, Binet spleen F/45 Classification DLBCL C Extensive IIIA Blood, LN, F/25 spleen IV A Extensive II A Extensive LN Mediastinal bulk, lung, Mediastinal bulk Extensive Extensive

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 385 Table 2 Treatment schedules

No.1 (CLL) DLI (1x107) Bi20 [µg] 10 10 20 40 80 160 320 600

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21 d22 d23 d24 d25 d26 d27 d28 d46 d47

No.2 (CLL) DLI DLI DLI (1x106) (1x107) (1x108)

Bi20 [µg] 10 20 50 100 200 500 500 500 5001000 1000 1000 1000500 1000

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10d11d12d13d14d15d16d17d18d19d20d21d22d23d24d25d26d27d28 d44 d52 d56 d57 d72 d76

No.3 (CLL) DLI (1x107) DLI DLI DLI (1x107) (1x107) (1x107) Bi20 [µg] 10 20 40 80 160 320 640 640 640

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10d11d12d13d14d15d16d17d18d19d20d21d22d23d24d25d26d27d28 d46 d47 d53 d71 d72

No.4 (BLL) SCT (1,6x107)

Bi20 [µg] 10 20 50 200 500 1000 1000

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21 d22 d23 d24 d25 d26 d27 d28 d29 d33 d43 d

No.5 (DLBCL) DLI (4,7x107)

Bi20 [µg] 10 100 200 500

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21 d22 d23 d24 d25 d26 d27 d28 d29 d33 d43 d

No.6 (DLBCL) SCT (1,4x107)

Bi20 [µg] 10 10 20 40 80 160 320 6001000 2000 2000

course I

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21 d22 d23 d24 d25 d26 d27 d28 d29 d33 d43 d51 d66 SCT DLI DLI 6 6 6 DLI (8x10 ) (1x10 ) (6x10 ) (1x106) Bi20 [µg] 20 150 200500 500 500 500 500 500 1000 1000 Chemo 1000 1000

course II d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21 d22 d23 d24 d25 d26 d27 d28 d29 d33 d43 d51 d66

Abbreviations: Chemo ¼ chemotherapy consisting of cytarabine, cisplatin and dexamethasone; DLI ¼ donor lymphocyte infusion. Application of Bi20 is shown in mg on a non-linear time scale; patient no. 6 received two treatment courses; treatment course II followed after an interval of 4 months; the corresponding numbers of transfused cells refer to cells per kg of recipient body weight.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 386 cells per kg on day 44 and 1 Â 108 CD3 þ cells per kg on day for i.v. infusion. The antibody was administered as a 57, preceded by two applications of 1000 mg Bi20 on days continuous 6–12 h i.v. infusion by a precision infusion 52 and 56, respectively, followed by 500 mg Bi20 on day 72 pump. Paracetamol (1000 mg orally) and antihistamines and 1000 mg on day 76. Patient no. 3 was escalated up to (for example, clemastine, 2 mg i.v.) were given as pre- 640 mg Bi20 on day 8, followed by DLI (1 Â 107 CD3 þ cells medication 30 min before antibody infusion. To avoid per kg of recipient body weight). DLI (1 Â 107 CD3 þ cells blood pressure instability and hypotension, 1000 ml of per kg) was repeated on days 47, 53 and 72 and preceded Ringer’s solution was given 15 min before treatment. with 640 mg Bi20 on days 46 and 71. In patient no. 4 with Intensive care monitoring was performed up to 24 h after high-grade lymphoblastic lymphoma, Bi20 was escalated the start of infusion. up to 1000 mg on day 17, followed by 16.3 Â 106 CD6- þ depleted CD34 cells, and in patient no. 5 with high-grade Toxicity and safety evaluation lymphoma stage IV, Bi20 was escalated up to 500 mg, Toxicity and adverse events were assessed by clinical 7 þ followed by 4.7 Â 10 CD3 cells per kg body weight on examination and laboratory tests, classified by their day 5. In patient no. 6 with high-grade non-Hodgkin’s relationship with study treatment and graded according lymphoma (HG-NHL), Bi20 was escalated up to 2000 mg to the National Cancer Institute (NCI) Common Toxicity 6 on days 24 and 27, followed by 14.2 Â 10 CD6-depleted Criteria (CTC). CD34 þ cells per kg body weight. After 4 months, a second treatment course was performed. Thereby, Bi20 was Clinical and experimental response evaluation escalated up to 1000 mg on days 14 and 16, followed by Patients were in the hospital when the decision for DLI (1 Â 106 CD3 þ cells per kg) on day 17. Four weeks treatment was taken. Baseline complete blood count, serum later, progressive disease was treated with 4 days of chemistries, electrocardiogram as well as appropriate chemotherapy (cytarabine, cisplatin and dexamethasone) radiological scans for tumor assessment were obtained followed by a PBSCT (8 Â 106 CD34 þ cells per kg) on day from each patient. Hematological and immunological 43 and DLI on day 51 (1 Â 106 CD3 þ cells per kg) and day parameters (for example, blood counts, serum chemistry, 66 (6 Â 106 CD3 þ cells per kg), each preceded with 1000 mg cytokine levels for IL-6, tumor necrosis factor-a and Bi20. lymphocyte subsets) were assessed before and 24 h after The treatment was considered as an individual trial in an each Bi20 infusion, respectively, DLI or SCT. Disease otherwise hopeless situation based on promising in vitro evaluation was performed according to the corresponding data. It allowed the investigation of safety and tolerability working group guidelines for CLL and NHL.15,16 In this of Bi20 (anti-CD3 Â anti-CD20) in combination with DLI study, clinical and laboratory evaluations were followed up as well as antitumor activity and monitoring of different weekly after treatment. Between 4 and 6 weeks after each immunologic variables (hematological cell populations, treatment, serum samples were tested for the presence of cytokine release and HAMAs). HAMAs and tumor response was studied by imaging investigations. Drug formulation and administration Bi20 was supplied by TRION Research (Munich, Ger- Flow cytometric analysis many) as a sterile, pyrogen-free, color-free and preserva- Lymphocyte subsets in the peripheral blood were quantified tive-free solution for infusion. The concentrate contained by flow cytometric analysis, including T cells (CD3, 0.2 mg/ml antibody per 100 mM sodium citrate buffer (pH CD4 and CD8), NK cells (CD16/CD56), B cells (CD19) 5.6), with 0.02% Tween 80. Depending on the dose level, and lymphoma cells (CD5/CD19). Moreover, before Bi20 was further diluted in 0.9% sodium chloride solution Bi20 treatment, the expression levels of CD20 were

Table 3 Summarized results of Bi20/DLI or SCT combination treatment

Patient no. No. of Total Dose No. of Clinical Hematological Side effects (CTC grade) Overall Bi20 dose (mg) range (mg) SCT/DLI response response survival (days) infusions

1 (CLL) 8 1240 10–600 1 Transient Transient Fever (III), chills (II), bone pain 77 granulocytopenia (II) thrombocytopenia (III) 2 (CLL) 15 7880 10–1000 3 Transient Transient Fever (III), chills (I) granulocytopenia (IV) 220 thrombocytopenia (III) 3 (CLL) 9 2550 10–640 4 Transient Transient Fever (III), chills (II), hypotension 189 granulocytopenia (III) thrombocytopenia (III) 4 (BLL) 7 2780 10–1000 1 Progression — Fever (III), chills (II) thrombocytopenia (III) 47 5 (DLBCL) 4 820 20–500 1 Progression — Fever (III) thrombocytopenia (I) 38 6 (DLBCL) 24 13610 10–2000 5 Stop of — Fever (I), prolonged granulocytopenia (IV) 486 progression thrombocytopenia (II) for 4 months

Abbreviations: BLL ¼ Burkitt-like lymphoma; CNS ¼ central nervous system; DLBCL ¼ diffuse large B-cell lymphoma; DLI ¼ donor lymphocyte infusion. CTC indicates common toxicity criteria according to the National Cancer Institute.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 387 CLL no.1 Bi20 4 10

DLI 8 3

6 2 4 CLL cells G/l Granulocytes G/l 1 2

0 0 7 9 1 3 5 9 1 d1 d3 d5 d d9 1 3 d11 d13 d15 d17 d d2 d2 d2 d27 d2 d Granulocytes CLL cells

CLL no.2 Bi20 Bi20 Bi20 2 0.5

1.6 DLI DLI DLI 0.4

1.2 0.3

0.8 0.2 CLL cells G/l Granulocyted G/l

0.4 0.1

0 0 1 6 1 6 1 6 1 6 d1 d6 d11 d16 d2 d2 d3 d36 d41 d4 d5 d5 d61 d66 d7 d7 Granulocytes CLL cells

5 5 CLL no.3 Bi20 Bi20 Bi20

4 4 DLI DLI DLI DLI

3 3

2 2 CLL cells G/l G/l CLL cells Granulocytes G/l

1 1

0 0 6 d1 d6 11 36 5 d d16 d21 d26 d31 d d41 d46 d51 d d61 d66 d71 d76 Granulocytes CLL cells

Figure 1 (a) Response to combined therapy of Bi20 and DLI in CLL. Escalating doses of Bi20 followed by DLI in patient nos. 1, 2 and 3 induced a prompt, but only transient, clearance of leukemic cells in the peripheral blood. Granulocytes were affected in a dose-dependent manner. (b) Response of lymphocyte subsets to treatment in CLL. T cells (CD3) and NK cells (CD16/CD56) showed a dose-dependent decrease, followed by a short peak and drop phase after DLI. DLI, donor lymphocyte infusion; NK, natural killer.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 388 CLL no.1 Bi20 1500

DLI 1250

1000 l µ 750 Cells/ 500

250

0 1 d d3 d5 d7 d9 d11 d13 d15 d17 d19 d21 d23 d25 d27 d29 d31

T cells NK cells

CLL no.2 Bi20 Bi20 Bi20

5000 DLI DLI DLI

4000

3000 l µ

Cells/ 2000

1000

d1 d6 d11 d16 d21 d26 d31 d36 d41 d46 d51 d56 d61 d66 d71 d76 T cells NK cells

CLL no.3 Bi20 Bi20 Bi20

2000

DLI DLI DLI DLI 1600

1200 l µ

800 Cells/

400

d0 d5 d10 d15 d20 d25 d30 d35 d40 d45 d50 d55 d60 d65 d70 d75

T cells NK cells

Figure 1 Continued.

measured and expressed as mean ﬂuorescence intensity Germany). Flow cytometry was performed on FACS (MFI). The respective monoclonal antibodies were Calibur (Becton Dickinson, Heidelberg, Germany) and conjugated with FITC, phycoerythrin or allophycocyanin; data were analyzed with CellQuest (BD Biosciences) they were purchased from BD Biosciences (Heidelberg, software.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 389 BLL no.4 DLBCL no.5 Bi20 Bi20

SCT 6 DLI 6 5 5

4 4

3 3 Cells G/l Cells G/l 2 2

1 1

0 0 d1 d3 d5 d7 d9 d11 d13 d15 d17 d19 d21 d23 d1 d2 d3 d4 d5 d6 d7 d8 Granulocytes T cells NK cells Granulocytes T cells NK cells

DLBCL no.6 DLBCL no.6 Course I Course II Bi20 Bi20Chemo Bi20 Bi20

5 4 SCT DLI SCT DLI DLI 4 3 3 2 Cells G/l 2 Cells G/l

1 1

0 0

d1 d4 d7 d0 d4 d8 d10 d13 d16 d19 d22 d25 d28 d31 d34 d37 d40 d43 d46 d49 d52 d55 d12 d16 d20 d24 d28 d32 d36 d40 d44 d48 d52 d56 d60 d64 Granulocytes T cells NK cells Granulocytes T cells NK cells Figure 2 Impact of combined therapy on granulocytes and lymphocyte subsets in HG-NHL. Granulocytes, T cells (CD3) and NK cells (CD16/CD56) showed a dose-dependent decrease, followed by a short peak and drop phase after DLI. T-cell levels in patient no. 5 were below the detection limit (shownas baseline). Chemo: chemotherapy consisting of cytarabine, cisplatin and dexamethasone. DLI, donor lymphocyte infusion; HG-NHL, high-grade non- Hodgkin’s lymphoma; NK, natural killer.

Cytokine measurement by FACS analysis. The total number of viable cells was Cytokines were determined on a routine basis in our determined by trypan blue exclusion counting. clinical laboratory (tumor necrosis factor-a and IL-6) or measured with a Bioplex array (BioRad, Munich, Ger- many) comprising INF-g, tumor necrosis factor-a, IL-2, Results IL-4, IL-6, IL-8, IL-10 and IL-12. Data acquisition and analysis were performed according to the manufacturer’s Promising in vitro studies14 encouraged us to treat patients instructions. with otherwise resistant B-lymphocyte malignancies that had relapsed after allo-SCT treatment. Three patients with B-cell depletion assays refractory CLL and three patients with refractory HG-NHL Bi20-mediated cytotoxicity was determined using a bioas- were treated with escalating doses of Bi20 (CD3 Â CD20) say described earlier by Stanglmaier et al.14 PBMCs followed by DLI or G-CSF-mobilized PBSCT. In two cases (1 Â 106 per ml) obtained from the stem cell donor, as well of CLL (patient nos. 2 and 3) and one case of aggressive as patient-derived cells (2 Â 105 per ml), were incubated NHL (patient no. 6), repeated courses of Bi20 and DLI were with different concentrations of Bi20 (CD3 Â CD20), given without unexpected effects. Thereby, patient no. 6 catumaxomab (CD3 Â EpCAM) (TRION Pharma, Mu- received a total of 24 infusions with a cumulative dose of nich, Germany) or rituximab (Roche, Basel, Switzerland). 13.610 mg of Bi20 and DLI from her HLA-haploidentical After 3 days, cells were collected and washed, and the father. The overall survival ranged from 38 days (patient no. percentage of viable CLL cells (CD5 þ /CD19 þ ) determined 5) to 486 days (patient no. 6).

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 390 CLL no.1 Bi20 500 25 DLI 400 20

300 15

200 10 CRP mg/dl IL-6 pg/ml

100 5

0 0 2 6 8 d0 d2 d4 d6 d8 1 1 1 d10 d d14 d d d20 d22 d24 d26 d28 d30 d32

IL-6 CRP

Bi20 Bi20 Bi20 CLL no.2

1500 DLI DLI DLI 25

1200 20

900 15

600 10 CRP mg/dl IL-6 pg/ml

300 5

0 0 6 1 1 6 6 1 1 6 1 6 d1 d 1 2 3 5 6 d d16 d d2 d31 d d4 d46 d d56 d61 d d7 d7

IL -6 CRP

CLL no.3 Bi20 Bi20 Bi20 1500 25

DLI DLI DLI DLI 1200 20

900 15

600 10 CRP mg/dl IL-6 pg/ml

300 5

0 0

d0 d5 d10 d15 d20 d25 d30 d35 d40 d45 d50 d55 d60 d65 d70 d75

IL-6 CRP Figure 3 (a) Release of IL-6 and CRP in CLL. Escalated treatment induced measurable but transient release of inﬂammatory cytokines. (b) Assessment of the Th1/Th2 cytokine proﬁle in CLL. Increasing concentrations of Bi20 favored the release of IL-8 and IL-10. The cytokine release is measured in pg/ml. CRP, C-reactive protein.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 391 CLL no.1 Bi20 3000

2500 DLI 2000

pg/ml 1500

1000

500

0 2 c d0 d d5 d8 9b 11 b 14 16 17 26 29 32 d3 c d5 d d 2 d d d d20 d23 d d d d1 d18 c IL-8 IL-10

CLL no.2 Bi20 Bi20 Bi20 500

DLI 400 DLI DLI

300 pg/ml 200

100

0 4 d0 d 0 5 d7b d11 d15 d20 d25 d30 d35 d40 d45 d5 d55 d60 d65 d70 d7

IL -8 IL -10

CLL no.3 Bi20 Bi20 Bi20

500 DLI DLI DLI DLI 400

300 pg/ml 200

100

0 0 4 9 4 8 3 8 d d3 d6 d9 14 19 2 2 3 49 53 5 6 6 78 d d d d d d39 d44 d d d d d d73 d

IL-8 IL-10 Figure 3 Continued.

Response to treatment lymph nodes and B symptoms disappeared, and cough In four of the six patients, we observed response to associated with a mediastinal tumor mass improved treatment. Two patients with HG-NHL (patient nos. 4 and signiﬁcantly for almost 4 months. In patients with CLL, a 5) did not respond (Table 3). In patient no. 6, enlarged transient clinical (B symptoms, enlarged lymph nodes and

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 392 spleen) and hematological response was observed. Leuke- To anticipate the cytotoxic capacity of Bi20, CLL cells of mia B cells in the peripheral blood disappeared with patient nos. 1 (MFI 20), 2 (MFI 120) and 3 (MFI 30) were increasing doses of Bi20 (Figure 1a), but reappeared within incubated for 72 h with PBMCs of the corresponding stem weeks after cessation of Bi20 despite DLI. Similarly, T cells cell donor in the presence of different concentrations of (CD3/CD4; CD3/CD8) and NK cells (CD16/CD56) Bi20 (CD3 Â CD20), catumaxomab (CD3 Â EpCAM) and decreased dose-dependently during Bi20 treatment and rituximab (Figure 5). In all cases, effective CLL depletion recovered in part after DLI or SCT (Figures 1b and 2). The with Bi20 could be shown, in line with the clinical findings. CD4/CD8 ratio was unaffected and did not favor a shift toward a CD4 or CD8 sub-population (data not shown). Moreover, measurement of the cytokine profile revealed in Adverse events cases of CLL and HG-NHL a transient but significant Fever (CTCpgrade 3) and chills (CTCpgrade 2) were increase of IL-6, IL-8 and IL-10 at increasing concentra- observed in all patients as treatment-related toxicities. tions of Bi20 accompanied by an increase of the C-reactive Transient bone pain (CTCpgrade 3) was observed in protein (Figures 3a,b and 4). Interestingly, in the case of patient no. 1 and a singular episode of hypotension (CTC patient no. 6 (DLBCL), the release of IL-10 was observable grade 2) was observed in patient no. 3 (Table 3). In case of only during the first treatment course, whereas it was not CLL, adverse effects appeared at Bi20 dose levels of 40– detectable during the second treatment course. For patient 160 mg and in case of NHL not before dose levels 4200 mg, no. 5 (DLBCL), no cytokine release data are available and were not more severe at higher concentrations. In regarding IL-8 and IL-10. general, adverse reactions could be controlled effectively by prolongation of the infusion time and decreased within hours after cessation of antibody application. In patient no. Experimental evaluation 6, a prolonged period (3 weeks) of leukopenia (CTC grade Although all patients were stained positive for CD20, the 4) was observed during the first treatment course after dose expression level as expressed in MFI differed significantly. escalation of Bi20, up to 2000 mg, and SCT, which did not

BLL no.4 Bi20 DLBCL no.5 Bi20

1500 SCT 25 500 DLI 25

1200 20 400 20

900 15 300 15

600 10 IL-6 pg/ml CRP mg/dl

200 10 CRP mg/dl IL-6 pg/ml

300 5 100 5

0 0 0 0 4 0 2 6 6 8 0 6 7 8 9 0 d0 d2 d d6 d8 14 20 d d1 d2 d3 d4 d5 d d d d 1 d1 d1 d d1 d18 d d22 d24 d2 d2 d IL-6 CRP IL-6 CRP

DLBCL no.6 DLBCL no.6 Course I Course II Bi20 Bi20Chemo Bi20 Bi20

500 25 1500 25 SCT DLI SCT DLI DLI 400 20 1200 20

300 15 900 15 IL-6 pg/ml 200 10 CRP mg/dl 600 10 IL-6 pg/ml CRP mg/dl

100 5 300 5

0 0 0 0 3 6 9 5 1 4 0 9 2 5 8 2 6 0 2 6 2 d0 d d d 12 1 18 2 3 33 36 4 4 51 d0 d4 d8 24 28 40 44 48 60 64 d d d d2 d d27 d d d d3 d d4 d d d54 d1 d1 d2 d d d3 d3 d d d d5 d56 d d d68 IL-6 CRP IL-6 CRP

Figure 4 (a) Release of IL-6 and CRP in HG-NHL. Escalated treatment induced measurable but transient release of inflammatory cytokines. (b) Assessment of the Th1/Th2 cytokine profile in HG-NHL. Increasing concentrations of Bi20 favored the release of IL-8 and IL-10 in patient no. 4 and during the first treatment course of patient no. 6. During the second course, no significant cytokine release was detectable (data not shown). For patient no. 5,no cytokine release data are available. The cytokine release is measured in pg/ml. CRP, C-reactive protein; HG-NHL, high-grade non-Hodgkin’s lymphoma.

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 393 BLL no.4 Bi20

500

SCT 400

300

pg/ml 200

100

d1 d2 d4 d6 d7 1b 3 8 0 d8b d10 1 d1 d15 d1 d2 d IL-8 IL-10

DLBCL no.6 Course I Bi20

3000

SCT 2500

2000

1500 pg/ml 1000

500

0 0 b 3 6 2 9 3 7 1 5 3 d d4 d9 1 1 2 2 3 4 5 d6 d d d19 d d25 d d3 d d4 d d49 d Il-8 Il-10 Figure 4 Continued. reappear in context with Bi20 application during the second GGT from sixfold up to eightfold of upper limit of normal treatment course. In CLL patient nos. 1, 2 and 3, was detected. No other organ toxicities (renal, cardiac application of Bi20 induced a transient decrease of and nervous system) were observed in any of the granulocytes, which was most pronounced (CTCpgrade patients. HAMAs were not detectable, even after repeated 4) in patient no. 2 (Table 3). In patient nos. 4 and 5, a slight applications of Bi20 (patient nos. 2, 3 and 6). Moreover, increase of granulocytes was detected after Bi20 applica- GVHD was not observed in any patient, although escalated tion. A temporary thrombocytopenia (CTCpgrade 3) applications of DLI and SCT were performed (patient nos. (Table 3) occurred in all cases without requirement of 2, 3 and 6). transfusion. Moreover, laboratory abnormalities com- prised a transient elevation of g-glutamyl transferase (GGT), not exceeding twofold of the upper limit of normal Discussion in patient nos. 1, 3, 4 and 6 (data not shown). The transaminases (alanine aminotransferase and aspartate CD20 antibody treatment regimens have been proven to be aminotransferase), as well as bilirubin, were unaffected. highly effective in patients with B-cell NHL.17 However, In patient no. 2, after dose-escalated application of Bi20 most patients ultimately relapse despite continued antibody (2 Â 1000 mg) and DLI (1 Â 108 CD3 þ cells per kg), a treatment.18 Mechanisms of resistance are not fully under- maximal but transient increase of GGT and bilirubin, up to stood; some might be related to the tumor cells and some to eightfold, and fourfold of upper limit of normal, respec- host factors. Lymphoma cells may become resistant by tively, was observed. In patient no. 5, presenting with decreased expression of CD20 antigen, or they may also hepatic manifestation of lymphoma, a slight increase of activate antiapoptotic signal transduction pathways, which

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 394 CLL no.1 (CD20 expression: MFI 20) 120

100

60 % CLL cells 40

0 neg 0.5 ng/ml 5 ng/ml 50 ng/ml 250 ng/ml

CLL no.2 (CD20 expression: MFI 120) 120

100

60 % CLL cells 40

0 neg 0.5 ng/ml 5 ng/ml 50 ng/ml 250 ng/ml

120 CLL no.3 (CD20 expression: MFI 30)

100

60 % CLL cells

0 neg 0.5 ng/ml 5 ng/ml 50 ng/ml 250 ng/ml

Bi20 Catumaxomab Rituximab

Figure 5 Bi20-mediated depletion of CLL cells. CLL cells of patient nos. 1, 2 and 3 were incubated in the presence of the corresponding donor-derived PBMCs with different concentrations of Bi20 (CD3 Â CD20), catumaxomab (CD3 Â EpCAM) and rituximab (range: 0.5–250 ng/ml). After 72 h, CLL cell depletion (CD5 þ /CD19 þ ) was determined by FACS analysis and trypan blue exclusion. CD20 expression level is deﬁned by MFI. The absolute CLL cell numbers in the assays without antibody (neg) were set as 100%. MFI, mean ﬂuorescence intensity.

renders these cells resistant to chemotherapy.19,20 Host variants of the FcgIII receptor have an impact on the factors involve effector cells of antibody-dependent cellular response to rituximab treatment.21 cytotoxicity and the reticulo-endothelial system removing Bispeciﬁc antibodies could overcome the problem of antibody-loaded cells.18 These functions may be defective; resistance and insufﬁcient stimulation of effector cells by

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 395 directly binding with one arm to the tumor cell and the consequences are not well understood and need to be other arm to a T cell. An earlier study from our group investigated further. showed effective stimulation of T cells with the secretion of Without any doubt, antibody concentrations in the range tumor necrosis factor-a by CD20 Â CD3 and of micrograms (Table 3) caused a significant clinical and/or 22 CD20 Â CD28 bispecific F(ab)2 fragments. CD20 Â CD28 hematological response in patients previously refractory to induced reaction of allogeneic T cells directed against B- treatment with alemtuzumab or rituximab. Thereby, even lymphoma cells. However, in a patient with recurrent B- low expression levels of CD20 seemed to be sufficient for ALL after allo-SCT, responses to bispecific CD20 Â CD28 effective tumor cell depletion, although it might be assumed

F(ab)2 antibodies and DLIs were of short duration. T-cell that higher expression levels probably correlate with a more immunity against ALL cells could not be shown. Therefore, favorable response pattern.14 Strikingly, in the two cases of we studied the effects of a complete bispecific antibody treatment failure, both patients had already presented against CD20 Â CD3 (Bi20). This antibody has a functional before treatment initiation with low (patient no. 4) or even Fc part and is therefore called ‘trifunctional.’ This new undetectable T-cell levels (patient no. 5), indicating that class of antibodies activates monocytes and DCs by binding response to treatment might be strictly dependent on the to their Fc receptors and subsequent phagocytosis.23 The presence of sufficient T cells during antibody application. additional property of activating accessory cells not only Thus, although the induction of lymphopenia seems to be contributed to an effective cytotoxicity but it was also treatment associated using trifunctional antibodies,29–31 a found to be a prerequisite for the induction of long-lasting certain threshold of T cells seems to be absolutely necessary antitumor immunity.13 to mediate tumor-cell depletion. In general, the observed In a pilot trial, three patients with refractory, p53- lymphopenic state resolved within 2–3 days after antibody mutated CLL and three patients with refractory HG-NHL application or could, at least in part, be replenished by (Burkitt-like lymphoma and DLBCL) were treated with the DLI, as shown in the present trial, and should be combination of the trifunctional antibody Bi20 and donor anticipated in future clinical studies. cellular transfusions. Thereby, in all patients suffering from Treatment-related adverse effects were restricted to fever CLL (patient nos. 1, 2 and 3), as well as in one patient with and chills and seemed to occur in high-grade lymphoma at DLBCL (patient no. 6), we could observe a response to higher dose ranges of Bi20 (4200 mg) than in CLL (dose treatment. Two patients with HG-NHL (patient nos. 4 and range 40–160 mg). In our CLL patients, the circulating 5) did not respond. In cases of response, we observed a tumor load was very low, and a hypothesized correlation of significant improvement of clinical symptoms. Enlarged adverse effects and tumor mass might also have to be lymph nodes and spleen decreased in size and B symptoms assumed and anticipated in future clinical studies. In disappeared almost completely. In patient no. 6, the patient no. 1 presenting with bone pain, a subsequent respiratory symptoms (cough), caused by a large mediast- analysis of bone marrow revealed a significantly reduced inal tumor mass, improved markedly and remained fraction of mutated (p53 deletion) lymphoma cells (data asymptomatic for 4 months. In cases of CLL, a dose- not shown). In general, adverse effects did not increase dependent decrease of leukemia cells in the peripheral significantly at higher concentrations and could be con- blood could be observed. Unfortunately, responses were trolled effectively by prolongation of the infusion time and only transient and the disease relapsed in all cases. supportive medication. The release of the inflammatory The absence of antidrug antibodies (for example, cytokine IL-6 followed the application of Bi20 and HAMAs) allowed repeated treatment cycles. In two cases preceded the appearance of C-reactive protein at higher of CLL (patient nos. 2 and 3) and one case of HG-NHL Bi20 concentrations. In all cases, the adverse effects (patient no. 6), repeated clinical and hematological disappeared after cessation of therapy. responses could be observed. Thereby, the induction of A temporary thrombocytopenia (CTCpgrade 3) without HAMAs might be prevented by collateral targeting of requirement of transfusions was observed in all cases after CD20-bearing B cells, as the trifunctional antibodies Bi20 application. With the exception of the non-responders catumaxomab (CD3 Â EpCAM) and ertumaxomab (patient nos. 4 and 5), granulocytopenia was observed in all (CD3 Â HER2/neu) targeting tumor cells of epithelial other patients. In CLL, granulocytopenia resolved within origin did induce HAMAs.24 Although we escalated the days after Bi20 application. In patient no. 6 (DLBCL), a doses of donor cells, GVHD was observed neither in the prolonged (3 weeks) granulocytopenia (CTC grade 4) was HLA-identical nor in the haploidentical setting. This might observed in the first treatment course, receiving the highest go along with recently published data by Morecki et al.25 in single dose of Bi20 (2000 mg) and the highest cumulative which, the presence of target cells, bispecific antibodies dose (6240 mg) per treatment course of all patients treated. divert potentially tissue-damaging alloreactive cells from After dose limitation up to 1000 mg per singular antibody host tissues, susceptible to GVHD, to tumor cells. More- application, during the second course, granulocytopenia, at over, escalated doses of Bi20 induced increased serum levels least in the context of Bi20 application, did not reappear. A of IL-10, a Th1-antagonizing cytokine, which might dose-dependent mechanism might therefore be assumed, additionally contribute to the prevention of the develop- although the precise mechanism is still unclear, and has to ment of GVHD.26,27 Interestingly, a significant release of be considered in future clinical studies. Interestingly, in no IL-10 was already observed in context with in vitro studies case was any reactivation or infectious problem caused by using the trifunctional antibodies catumaxomab (anti- CMV or EBV observed. Moreover, the hepatic toxicity EpCAM Â anti-CD3), TRBs02/07 (anti-GD2/GD3 Â anti- profile appeared to be more favorable as compared with the CD3) and Bi20.14,28 However, so far, the therapeutic other bispecific trifunctional antibodies, ertumaxomab

Bone Marrow Transplantation Immunotherapy of NHL with Bi20 and DLI R Buhmann et al 396 (anti-CD3 Â anti-HER2/neu) or catumaxomab (anti- lymphoma with allogeneic peripheral blood stem cell trans- CD3 Â EpCAM),24,31,32 even though our patients received plantation followed by interferon-alpha and donor lymphocyte higher singular as well as cumulative doses of Bi20. In only infusion]. Rinsho Ketsueki 2004; 45: 155–160. one case (patient no. 2), a significant Bi20/DLI-induced 8 Porter DL, Levine BL, Bunin N, Stadtmauer EA, Luger SM, CTC grade 3 increase of GGT was observed in contrast Goldstein S et al. A phase 1 trial of donor lymphocyte ex vivo to 8 of 17 (47%) cases with ertumaxomab and metastatic infusions expanded and activated via CD3/CD28 31 costimulation. Blood 2006; 107: 1325–1331. breast cancer. The slight increase of GGT observed in 9 Ritgen M, Stilgenbauer S, von Neuhoff N, Humpe A, patient no. 5 presenting with hepatic manifestation of Bruggemann M, Pott C et al. Graft-versus-leukemia activity lymphoma and non-responsiveness to therapy might at may overcome therapeutic resistance of chronic lymphocytic best be associated with disease progression rather than leukemia with unmutated immunoglobulin variable heavy-chain treatment induced. gene status: implications of minimal residual disease measure- Taken together, the combined treatment with Bi20 and ment with quantitative PCR. Blood 2004; 104: 2600–2602. adoptive cellular transfer could induce in part a prompt 10 Cartron G, Watier H, Golay J, Solal-Celigny P. From the antitumor response in extensively pretreated, p53-mutated, bench to the bedside: ways to improve rituximab efficacy. and alemtuzumab and rituximab refractory patients, Blood 2004; 104: 2635–2642. indicating its therapeutic potential. However, until now, 11 Segal DM, Weiner GJ, Weiner LM. Bispecific antibodies in cancer therapy. Curr Opin Immunol 1999; 11: 558–562. the responses are of short duration and further studies are 12 Kufer P, Lutterbuse R, Baeuerle PA. A revival of bispecific necessary to optimize the clinical outcome. antibodies. Trends Biotechnol 2004; 22: 238–244. 13 Ruf P, Lindhofer H. Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood 2001; Acknowledgements 98: 2526–2534. 14 Stanglmaier S, Faltin M, Ruf P, Bodenhausen A, Schro¨ der P, This study is supported by grants of the Deutsche Jose´ Carreras Lindhofer H. Bi20 (FBTA05) a novel trifunctional bispecific Leuka¨ mie-Stiftung eV (DJCLS R 06/23) to Raymund Buhmann antibody (anti-CD20 Â anti-CD3), mediates efficient killing of and Hans-Jochem Kolb; B Simoes is supported by grant 01/ lymphoma B cells even with very low CD20 expression levels. 04492-8 FAPESP (Fundacao de Amparo a Pesquisa do Estado Int J Cancer 2008; 123: 1181–1189. de Sao Paulo), Brasil; T Yang is supported by a fellowship 15 Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, of the German Academic Exchange Service (DAAD). We are O’Brien S et al. National Cancer Institute-sponsored Working indebted to many nurses and physicians for unconditional Group guidelines for chronic lymphocytic leukemia: revised assistance in patient care, referral of patient material and data guidelines for diagnosis and treatment. Blood 1996; 87: 4990–4997. collection. 16 Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM et al. 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