Leukemia & Lymphoma
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations Marius Stiefelhagen, Carola Gigel, Elena Vasyutina, Michael Möllmann, Alexandra Breuer, Petra Mayer, Jan Dürig & Marco Herling To cite this article: Marius Stiefelhagen, Carola Gigel, Elena Vasyutina, Michael Möllmann, Alexandra Breuer, Petra Mayer, Jan Dürig & Marco Herling (2016) Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations, Leukemia & Lymphoma, 57:9, 2235-2238, DOI: 10.3109/10428194.2015.1135433 To link to this article: http://dx.doi.org/10.3109/10428194.2015.1135433
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Date: 08 October 2016, At: 18:55
LEUKEMIA & LYMPHOMA, 2016 VOL. 57, NO. 9, 2235–2238 http://dx.doi.org/10.3109/10428194.2015.1135433
LETTER TO THE EDITOR
Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations Marius Stiefelhagena*, Carola Gigela*, Elena Vasyutinaa, Michael Mo¨llmannb, Alexandra Breuera, Petra Mayera, Jan Du¨rigb and Marco Herlinga a
Department of Medicine I, Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Germany; bClinic of Hematology, University Hospital of Essen, Germany
ARTICLE HISTORY Received 17 August 2015; Revised 13 November 2015; Accepted 17 December 2015
With great interest, we read the report by Byrd et al. [1] in this journal about lucatumumab (HCD122, formerly CHIR-12.12) in patients with relapsed chronic lymphocytic leukemia (CLL). This phase-I trial [1] concluded an acceptable tolerability and a limited single-agent activity of this CD40 monoclonal antibody (mAb). Two subsequent early-phase studies in relapsed/refractory (r/r) lymphoma [2] and multiple myeloma (MM) [3] confirmed selective response inductions as well as the encouraging safety profile and pharmacodynamics of this agent. We noted the interesting clinical response patterns of nodal debulking versus unaffected peripheral blood (PB) burden reported in CLL and specific lymphoma subsets, i.e., follicular lymphoma (FL). It prompted us to communicate here our novel preclinical observations in CLL and to review reported data on HCD122. Postulating a particular milieu-dictated activity and potential synergies in combined applications of HCD122, we addressed here the effects of lucatumumab at the regenerative niche of CLL and in drug combinations. In a first set of experiments, we employed our established NOD/SCID CLL-xenograft model.[4] The human disease engrafts in spleen without PB lymphocytosis and only when cotransplanted with T-/NK-cells and monocytes. This allows selective therapeutic and analytic access to CLL at its sites of active crosstalk with the CD40L-providing micromilieu while preserving prerequisites for antibody-dependent cellular cytotoxicity (ADCC). Freshly isolated PBMC from four patients with CLL were incubated ex vivo for 15 min with HCD122 (1 mg/kg and 10 mg/kg) or controls (PBS or hu-IgG1 isotype) followed by intravenous injection (100 106 cells) into 6-week-old female hosts (3 animals/patient). CONTACT Marco Herling [email protected] 50937 Cologne, Germany *These authors contributed equally to this work. ß 2016 Informa UK Limited, trading as Taylor & Francis Group
After 24 h, we observed a significant (paired t-tests) reduction of human leukocytes (CD45 flow cytometry) and CLL cells (CD5+/19+ gate) in the splenic suspensions at both HCD122 doses (CD45: HCD122 1 mg/kg: 0.29 ± 0.10, p ¼ 0.03; 10 mg/kg: 0.30 ± 0.19, p ¼ 0.03 (not shown); CLL cells: HCD122 1 mg/kg: 0.28 ± 0.07, p ¼ 0.02; 10 mg/kg: 0.28 ± 0.14, p ¼ 0.02 [Figure 1A]; all means ± SEMs). No reductions in splenic T-cell counts or (normal level) PB leukocytes (not shown) were observed. In a modification of this system, HCD122 treatment (days +14 and +21) was commenced after CLL cells had engrafted. Analysis of splenic infiltration at day +28 showed a significant depletion of human CLL cells in both HCD122 dosage groups (1 mg/kg: 0.72 ± 0.34, p ¼ 0.04; 10 mg/kg: 0.44 ± 0.19, p ¼ 0.03; means ± SEMs [Figure1B]). Although mainly attributable to leukemia cell reduction, the accompanying drop in infiltrating pan-leukocytes (CD45) was paralleled by decreases in infiltrating CD3+ T-cells as well (not shown). A series of immunohistochemistries (IHC) on paraffin-embedded portions of these spleens confirmed the results semiquantitatively. The perivascular CLL infiltrates tended to be smaller in the HCD122-groups (as per H&E, CD45, CD19 [Figure 1C], and TCL1 stains), which was associated with slightly less-intense nuclear signals for NFkB-p65. There was no difference in CD38 (flow cytometry) as well as Ki67 and bcl2 signals (IHC). In another modification of the latter xenograft set-up in two additional patients (6 mice), the onset of splenic CLL cell depletion was as early as 2-h post-injection with peaks between 6–24 h. The next set of in vitro experiments on freshly isolated CLL patient cells showed that HCD122 reverses the CD40L-induced resistance to fludarabine cytotoxicity. An
Cologne University, Dept. of Medicine I, Building 15, Level 1, Room 1.010, Kerpener Straße 62,
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M. STIEFELHAGEN ET AL.
1.5
12 mice (4 CLL pts. in 3 animals each)
(B) hCLL-NOD/SCID xenografts, HCD122 post-engraftment P = 0.03
P = 0.02
CD19+/CD5+ cells in spleen (ratio to PBS)
CD19+/CD5+ cells in spleen (ratio to PBS)
(A) hCLL-NOD/SCID xenografts, co-transplant with HCD122 P = 0.02
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Titrated Fludarabine EC50‘s (nM)
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Fludarabine titrations (representative pt. from top) 20000 hu-IgG1+CD40L hu-IgG1 + sCD40L Untreated cells Fludarabine only HCD122+CD40L HCD122 + sCD40L
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Figure 1. HCD122 administered simultaneously with human CLL cells (A) into NOD-SCID recipients reduced engrafted CLL content in murine spleens at 24 h. HCD122, given +14 and +21 days post-transplantation of CLL (B), decreases murine splenic leukemia cell burden (analysis on day +28), which was confirmed by immunohistochemistry (C; shown HCD122 at 10 mg/kg). HCD122 reverses the CD40L-induced resistance to fludarabine-mediated cytotoxicity based on cell viability (D; MTT assay, 7 pts.) and titrated half-maximal effective dosages (EC50’s) for fludarabine (E; 6 pts., incubation for 72 h in each condition). Western blots (selected experiment from 6 CLL) indicate the targeted effect of anti-CD40 HCD122 on pro-survival molecules, NFB components, and cell-death executioners (F; at 24 h).
ACTIVITY OF LUCATUMUMAB, A CD40 ANTIBODY
illustrative read-out of MTT-based viability is given in [Figure 1D]. Titrations of half-maximal effective dosages (EC50’s) for fludarabine [Figure 1E] and densitometry (6 CLL) of immunoblot-based PARP cleavage (apoptosis; not shown) confirmed this observation. There was no synergism of HCD122 with fludarabine. Biochemical profiling (6 CLL) of CD40-signaling revealed the preceding (24 h) targeted effect of HCD122 on NFkB components and distal cell-death executioners [Figure 1F], but less markedly on MAPK or PI3K/AKT cascades (not shown). The costimulatory receptor CD40 is a member of the tumor necrosis factor receptor (TNFR) family. It is widely expressed in antigen-presenting cells (APC), including B cells, but also in endothelia, smooth muscles, fibroblasts, and epithelial cells. Binding of CD40 to its natural ligand CD40L (CD154) recruits raft-associated TRAF adapters and promotes critical regulatory signals of B-cell survival and differentiation. Biologically active CD40L is expressed either as a soluble cytokine or as a homotrimeric transmembrane protein. The major source of CD40L are activated TH-cells, in which it can provide coactivating input. An adequate germinal center reaction of T-celldependent B-cell differentiation and expansion critically relies on the CD40-CD40L axis. Biochemically, ligation of CD40 by CD40L triggers major cascades, predominantly IKKa/b-NFkB, MAPK (p38 and MEK/ERK), and PI3K/AKT. Signals through CD40 are implicated as a major milieuderived driver of clonal sustenance by promoting apoptosis resistance in CLL [5] and other B-cell lymphomas, e.g. FL.[6] CLL cells of the disease-characteristic proliferation centers, where robust NFkB activation is induced, show a marked CD40 expression.[7] Fittingly, lymph-node-derived CLL cells distinguish themselves from their recirculating PB clones by a prominent CD40 gene expression signature.[8] Although biased by analyses mainly from PB-isolated tumor cells, CD40 expression and in vitro responses to CD40L in CLL are also characterized by considerable intertumoral heterogeneity. Moreover, the tumor-protective immune dysregulations in CLL (likely also in other cancers) include an expression of CD154 by the tumor cells themselves in a subset of cases paralleled by a deficiency of bystander Tcells to upregulate this CD40L upon stimulation.[9,10] Together, this implicates a T-cell independent constitutive autocrine character of the CD40–CD40L interaction in CLL, at least in a subset of cases or cells defined by the intensity of their milieu-interactions. Given its pivotal role in immune (dys)regulation, interception in the CD40-CD40L interaction has been explored as a therapeutic strategy.[11,12] The blocking of CD40-CD40L engagement is tolerogenic in settings of tissue transplantation and autoimmunity. Reconstitution
2237
of an adequate anti-tumor response of APC or T-cells has been accomplished by systemic application of soluble (s) recombinant CD40L in solid cancers and by adenoviral gene therapy introducing CD40L or CD40-binding proteins into CLL cells.[11,13] There are also humanized or fully human mAb against CD40.[12,13] The agonistic IgG CP-870,893 (Pfizer Inc.) induced objective partial responses (PR) in melanoma and other solid tumors presumably by reactivation of immunity. The weak agonist dacetuzumab (SGN-40; Seattle Genetics Inc., Bothell, WA), believed to trigger apoptosis, showed some activity in r/r MM and other lymphomas. The fully human lucatumumab (HCD122; Novartis, Basel, Switzerland) acts CD40-antagonistic and is the currently best-studied CD40-impeding mAb. Preclinical data demonstrate a dual mode of action of HCD122. In the absence of effector cells, its specific binding to CD40 on CLL cells blocks CD40L-mediated proliferative/survival signaling and cytokine release.[14] Without competitive CD40L input, engagement of CD40 by HCD122 appears functionally inert.[14] In contrast to rituximab, HCD122 is not internalized and has low offrates. As many IgG1’s, HCD122 also efficiently mediates ADCC and clearance of CLL cells by cocultured NK-cells; to a greater extent than rituximab.[14] Similar observations have been made in experimental systems of MM.[15] There are reports on three early-phase clinical trials with lucatumumab as monotherapy in categories of r/r B-cell lymphomas.[1–3] Given intravenously, maximally tolerated doses (MTD) were 3–4.5 mg/kg in a schedule of once weekly for 4 weeks of an 8-week cycle. At the MTD, antibody accumulations allowed extrapolations of serum half-lives of &2 days after the first administration and &5 days or more after the fourth infusion (each of the first cycle). There was sustained CD40 saturation by lucatumumab on circulating normal B-cells at the MTD; on average 87%, up to 5 weeks after the last infusion.[3] All studies demonstrated a low frequency and severity of toxicity. Common adverse events were mild-to-moderate infusion reactions (&40%) including chills and pyrexia within the first 4 h as well as elevated, but subclinical and self-limiting, serum aminotransferases and lipases (up to 27%).[1–3] The B-cell nonexclusive CD40 expression (above) can explain such particular side effects. Major hematotoxicities as cytopenias were rare. Despite the limitations of such phase-I designs, the clinical efficacy of lucatumumab was at best moderate in specific subsets. Of the 28 patients with r/r MM, 12 patients (43%) had a stable disease (SD) and one patient (4%) maintained a PR for 8 months.[3] Among 24 r/r CLL, there was only one patient (3.8%) with nonbulky lymphadenopathy lacking lymphocytosis who attained a nodal PR that lasted 230 days; SD in 17 more patients
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lasted at mean 76 days (range 29–504 days).[1] Most interestingly, there was hardly any CLL cell clearance of PB at one week after the 4th infusions. A larger cohort of Hodgkin (HL) and non-Hodgkin lymphomas (NHL) included a phase-IIA part.[2] Complete responses (by CT-scan) occurred one each in FL, diffuse large B-cell lymphoma (62.9 weeks), and mucosa-associated tissue (MALT) lymphoma. The overall response rates (ORR) in FL and MALT-lymphoma were 7/21 (33.3%, with 6 PR) and (3/7) 42.9%, respectively. Among FL, 11 additional patients demonstrated an SD as best response and a total of 15/21 (71%) of FL showed a reduction in tumor size. Among the lymphoma subsets, the highest partial metabolic response rates (per FDG-PET in 62%) and the most durable responses (7.9–31 weeks) were observed in FL.[2] These distinct patterns of response point to a relative importance of CD40-CD40L engagement across different lymphoma subsets. Moreover, it suggests a differential relevance of CD40 signaling and hence interventional sensitivity between site-defined milieus in CLL. In conjunction with our experimental data, we conclude that HCD122 (lucatumumab) is active at sites of relevant CD40–CD40L interaction; in CLL mostly nodal sites of residing regenerative centers. A yet insufficient single-agent clinical activity calls for explorations of more suitable combinations than with fludarabine, such as other mAb or small molecules. In extension, the success of novel milieu-modulatory strategies, including the one described here, likely depends on their capacity to also sufficiently restore anti-tumor (T cell) immunity in addition to perturbation of dependance signals.
Acknowledgements Novartis provided HCD122, however, had no role in study design, data collection, and data analysis as well as in the decision to publish or in the preparation of the manuscript.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at http:// dx.doi.org/10.3109/10428194.2015.1135433.
References [1] Byrd JC, Kipps TJ, Flinn IW, et al. Phase I study of the antiCD40 humanized monoclonal antibody lucatumumab (HCD122) in relapsed chronic lymphocytic leukemia. Leuk Lymphoma. 2012;53:2136–2142.
[2] Fanale M, Assouline S, Kuruvilla J, et al. Phase IA/II, multicentre, open-label study of the CD40 antagonistic monoclonal antibody lucatumumab in adult patients with advanced non-Hodgkin or Hodgkin lymphoma. Br J Haematol. 2014;164:258–265. [3] Bensinger W, Maziarz RT, Jagannath S, et al. A phase 1 study of lucatumumab, a fully human anti-CD40 antagonist monoclonal antibody administered intravenously to patients with relapsed or refractory multiple myeloma. Br J Haematol. 2012;159:58–66. [4] Du¨rig J, Ebeling P, Grabellus F, et al. A novel nonobese diabetic/severe combined immunodeficient xenograft model for chronic lymphocytic leukemia reflects important clinical characteristics of the disease. Cancer Res. 2007;67:8653–8661. [5] Girbl T, Hinterseer E, Gro¨ssinger EM, et al. CD40-mediated activation of chronic lymphocytic leukemia cells promotes their CD44-dependent adhesion to hyaluronan and restricts CCL21-induced motility. Cancer Res. 2013;73:561–570. [6] Travert M, Ame-Thomas P, Pangault C, et al. CD40 ligand protects from TRAIL-induced apoptosis in follicular lymphomas through NF-kappaB activation and up-regulation of c-FLIP and Bcl-xL. J Immunol. 2008;181:1001– 1011. [7] Herreros B, Rodrı´guez-Pinilla SM, Pajares R, et al. Proliferation centers in chronic lymphocytic leukemia: the niche where NF-kappaB activation takes place. Leukemia. 2010;24:872–876. [8] Mittal AK, Chaturvedi NK, Rai KJ, et al. Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease. Mol Med Camb Mass. 2014;20:290–301. [9] Schattner EJ, Mascarenhas J, Reyfman I, et al. Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity. Blood. 1998;91:2689–2697. [10] Cantwell M, Hua T, Pappas J, et al. Acquired CD40-ligand deficiency in chronic lymphocytic leukemia. Nat Med. 1997;3:984–989. [11] Ferrajoli A. Interfering with CD40 ligation: a sensitive matter in chronic lymphocytic leukemia. Leuk Lymphoma. 2012;53:2093–2094. [12] Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev. 2009;229:152–172. [13] Hassan SB, Sørensen JF, Olsen BN, et al. Anti-CD40mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol. 2014;36:96–104. [14] Luqman M, Klabunde S, Lin K, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells. Blood. 2008;112:711–720. [15] Tai YT, Li X, Tong X, et al. Human anti-CD40 antagonist antibody triggers significant antitumor activity against human multiple myeloma. Cancer Res. 2005;65:5898–5906.
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations Marius Stiefelhagen, Carola Gigel, Elena Vasyutina, Michael Möllmann, Alexandra Breuer, Petra Mayer, Jan Dürig & Marco Herling To cite this article: Marius Stiefelhagen, Carola Gigel, Elena Vasyutina, Michael Möllmann, Alexandra Breuer, Petra Mayer, Jan Dürig & Marco Herling (2016) Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations, Leukemia & Lymphoma, 57:9, 2235-2238, DOI: 10.3109/10428194.2015.1135433 To link to this article: http://dx.doi.org/10.3109/10428194.2015.1135433
View supplementary material
Published online: 12 Jan 2016.
Submit your article to this journal
Article views: 118
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ilal20 Download by: [Cornell University Library]
Date: 08 October 2016, At: 18:55
LEUKEMIA & LYMPHOMA, 2016 VOL. 57, NO. 9, 2235–2238 http://dx.doi.org/10.3109/10428194.2015.1135433
LETTER TO THE EDITOR
Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations Marius Stiefelhagena*, Carola Gigela*, Elena Vasyutinaa, Michael Mo¨llmannb, Alexandra Breuera, Petra Mayera, Jan Du¨rigb and Marco Herlinga a
Department of Medicine I, Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Germany; bClinic of Hematology, University Hospital of Essen, Germany
ARTICLE HISTORY Received 17 August 2015; Revised 13 November 2015; Accepted 17 December 2015
With great interest, we read the report by Byrd et al. [1] in this journal about lucatumumab (HCD122, formerly CHIR-12.12) in patients with relapsed chronic lymphocytic leukemia (CLL). This phase-I trial [1] concluded an acceptable tolerability and a limited single-agent activity of this CD40 monoclonal antibody (mAb). Two subsequent early-phase studies in relapsed/refractory (r/r) lymphoma [2] and multiple myeloma (MM) [3] confirmed selective response inductions as well as the encouraging safety profile and pharmacodynamics of this agent. We noted the interesting clinical response patterns of nodal debulking versus unaffected peripheral blood (PB) burden reported in CLL and specific lymphoma subsets, i.e., follicular lymphoma (FL). It prompted us to communicate here our novel preclinical observations in CLL and to review reported data on HCD122. Postulating a particular milieu-dictated activity and potential synergies in combined applications of HCD122, we addressed here the effects of lucatumumab at the regenerative niche of CLL and in drug combinations. In a first set of experiments, we employed our established NOD/SCID CLL-xenograft model.[4] The human disease engrafts in spleen without PB lymphocytosis and only when cotransplanted with T-/NK-cells and monocytes. This allows selective therapeutic and analytic access to CLL at its sites of active crosstalk with the CD40L-providing micromilieu while preserving prerequisites for antibody-dependent cellular cytotoxicity (ADCC). Freshly isolated PBMC from four patients with CLL were incubated ex vivo for 15 min with HCD122 (1 mg/kg and 10 mg/kg) or controls (PBS or hu-IgG1 isotype) followed by intravenous injection (100 106 cells) into 6-week-old female hosts (3 animals/patient). CONTACT Marco Herling [email protected] 50937 Cologne, Germany *These authors contributed equally to this work. ß 2016 Informa UK Limited, trading as Taylor & Francis Group
After 24 h, we observed a significant (paired t-tests) reduction of human leukocytes (CD45 flow cytometry) and CLL cells (CD5+/19+ gate) in the splenic suspensions at both HCD122 doses (CD45: HCD122 1 mg/kg: 0.29 ± 0.10, p ¼ 0.03; 10 mg/kg: 0.30 ± 0.19, p ¼ 0.03 (not shown); CLL cells: HCD122 1 mg/kg: 0.28 ± 0.07, p ¼ 0.02; 10 mg/kg: 0.28 ± 0.14, p ¼ 0.02 [Figure 1A]; all means ± SEMs). No reductions in splenic T-cell counts or (normal level) PB leukocytes (not shown) were observed. In a modification of this system, HCD122 treatment (days +14 and +21) was commenced after CLL cells had engrafted. Analysis of splenic infiltration at day +28 showed a significant depletion of human CLL cells in both HCD122 dosage groups (1 mg/kg: 0.72 ± 0.34, p ¼ 0.04; 10 mg/kg: 0.44 ± 0.19, p ¼ 0.03; means ± SEMs [Figure1B]). Although mainly attributable to leukemia cell reduction, the accompanying drop in infiltrating pan-leukocytes (CD45) was paralleled by decreases in infiltrating CD3+ T-cells as well (not shown). A series of immunohistochemistries (IHC) on paraffin-embedded portions of these spleens confirmed the results semiquantitatively. The perivascular CLL infiltrates tended to be smaller in the HCD122-groups (as per H&E, CD45, CD19 [Figure 1C], and TCL1 stains), which was associated with slightly less-intense nuclear signals for NFkB-p65. There was no difference in CD38 (flow cytometry) as well as Ki67 and bcl2 signals (IHC). In another modification of the latter xenograft set-up in two additional patients (6 mice), the onset of splenic CLL cell depletion was as early as 2-h post-injection with peaks between 6–24 h. The next set of in vitro experiments on freshly isolated CLL patient cells showed that HCD122 reverses the CD40L-induced resistance to fludarabine cytotoxicity. An
Cologne University, Dept. of Medicine I, Building 15, Level 1, Room 1.010, Kerpener Straße 62,
2236
M. STIEFELHAGEN ET AL.
1.5
12 mice (4 CLL pts. in 3 animals each)
(B) hCLL-NOD/SCID xenografts, HCD122 post-engraftment P = 0.03
P = 0.02
CD19+/CD5+ cells in spleen (ratio to PBS)
CD19+/CD5+ cells in spleen (ratio to PBS)
(A) hCLL-NOD/SCID xenografts, co-transplant with HCD122 P = 0.02
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Figure 1. HCD122 administered simultaneously with human CLL cells (A) into NOD-SCID recipients reduced engrafted CLL content in murine spleens at 24 h. HCD122, given +14 and +21 days post-transplantation of CLL (B), decreases murine splenic leukemia cell burden (analysis on day +28), which was confirmed by immunohistochemistry (C; shown HCD122 at 10 mg/kg). HCD122 reverses the CD40L-induced resistance to fludarabine-mediated cytotoxicity based on cell viability (D; MTT assay, 7 pts.) and titrated half-maximal effective dosages (EC50’s) for fludarabine (E; 6 pts., incubation for 72 h in each condition). Western blots (selected experiment from 6 CLL) indicate the targeted effect of anti-CD40 HCD122 on pro-survival molecules, NFB components, and cell-death executioners (F; at 24 h).
ACTIVITY OF LUCATUMUMAB, A CD40 ANTIBODY
illustrative read-out of MTT-based viability is given in [Figure 1D]. Titrations of half-maximal effective dosages (EC50’s) for fludarabine [Figure 1E] and densitometry (6 CLL) of immunoblot-based PARP cleavage (apoptosis; not shown) confirmed this observation. There was no synergism of HCD122 with fludarabine. Biochemical profiling (6 CLL) of CD40-signaling revealed the preceding (24 h) targeted effect of HCD122 on NFkB components and distal cell-death executioners [Figure 1F], but less markedly on MAPK or PI3K/AKT cascades (not shown). The costimulatory receptor CD40 is a member of the tumor necrosis factor receptor (TNFR) family. It is widely expressed in antigen-presenting cells (APC), including B cells, but also in endothelia, smooth muscles, fibroblasts, and epithelial cells. Binding of CD40 to its natural ligand CD40L (CD154) recruits raft-associated TRAF adapters and promotes critical regulatory signals of B-cell survival and differentiation. Biologically active CD40L is expressed either as a soluble cytokine or as a homotrimeric transmembrane protein. The major source of CD40L are activated TH-cells, in which it can provide coactivating input. An adequate germinal center reaction of T-celldependent B-cell differentiation and expansion critically relies on the CD40-CD40L axis. Biochemically, ligation of CD40 by CD40L triggers major cascades, predominantly IKKa/b-NFkB, MAPK (p38 and MEK/ERK), and PI3K/AKT. Signals through CD40 are implicated as a major milieuderived driver of clonal sustenance by promoting apoptosis resistance in CLL [5] and other B-cell lymphomas, e.g. FL.[6] CLL cells of the disease-characteristic proliferation centers, where robust NFkB activation is induced, show a marked CD40 expression.[7] Fittingly, lymph-node-derived CLL cells distinguish themselves from their recirculating PB clones by a prominent CD40 gene expression signature.[8] Although biased by analyses mainly from PB-isolated tumor cells, CD40 expression and in vitro responses to CD40L in CLL are also characterized by considerable intertumoral heterogeneity. Moreover, the tumor-protective immune dysregulations in CLL (likely also in other cancers) include an expression of CD154 by the tumor cells themselves in a subset of cases paralleled by a deficiency of bystander Tcells to upregulate this CD40L upon stimulation.[9,10] Together, this implicates a T-cell independent constitutive autocrine character of the CD40–CD40L interaction in CLL, at least in a subset of cases or cells defined by the intensity of their milieu-interactions. Given its pivotal role in immune (dys)regulation, interception in the CD40-CD40L interaction has been explored as a therapeutic strategy.[11,12] The blocking of CD40-CD40L engagement is tolerogenic in settings of tissue transplantation and autoimmunity. Reconstitution
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of an adequate anti-tumor response of APC or T-cells has been accomplished by systemic application of soluble (s) recombinant CD40L in solid cancers and by adenoviral gene therapy introducing CD40L or CD40-binding proteins into CLL cells.[11,13] There are also humanized or fully human mAb against CD40.[12,13] The agonistic IgG CP-870,893 (Pfizer Inc.) induced objective partial responses (PR) in melanoma and other solid tumors presumably by reactivation of immunity. The weak agonist dacetuzumab (SGN-40; Seattle Genetics Inc., Bothell, WA), believed to trigger apoptosis, showed some activity in r/r MM and other lymphomas. The fully human lucatumumab (HCD122; Novartis, Basel, Switzerland) acts CD40-antagonistic and is the currently best-studied CD40-impeding mAb. Preclinical data demonstrate a dual mode of action of HCD122. In the absence of effector cells, its specific binding to CD40 on CLL cells blocks CD40L-mediated proliferative/survival signaling and cytokine release.[14] Without competitive CD40L input, engagement of CD40 by HCD122 appears functionally inert.[14] In contrast to rituximab, HCD122 is not internalized and has low offrates. As many IgG1’s, HCD122 also efficiently mediates ADCC and clearance of CLL cells by cocultured NK-cells; to a greater extent than rituximab.[14] Similar observations have been made in experimental systems of MM.[15] There are reports on three early-phase clinical trials with lucatumumab as monotherapy in categories of r/r B-cell lymphomas.[1–3] Given intravenously, maximally tolerated doses (MTD) were 3–4.5 mg/kg in a schedule of once weekly for 4 weeks of an 8-week cycle. At the MTD, antibody accumulations allowed extrapolations of serum half-lives of &2 days after the first administration and &5 days or more after the fourth infusion (each of the first cycle). There was sustained CD40 saturation by lucatumumab on circulating normal B-cells at the MTD; on average 87%, up to 5 weeks after the last infusion.[3] All studies demonstrated a low frequency and severity of toxicity. Common adverse events were mild-to-moderate infusion reactions (&40%) including chills and pyrexia within the first 4 h as well as elevated, but subclinical and self-limiting, serum aminotransferases and lipases (up to 27%).[1–3] The B-cell nonexclusive CD40 expression (above) can explain such particular side effects. Major hematotoxicities as cytopenias were rare. Despite the limitations of such phase-I designs, the clinical efficacy of lucatumumab was at best moderate in specific subsets. Of the 28 patients with r/r MM, 12 patients (43%) had a stable disease (SD) and one patient (4%) maintained a PR for 8 months.[3] Among 24 r/r CLL, there was only one patient (3.8%) with nonbulky lymphadenopathy lacking lymphocytosis who attained a nodal PR that lasted 230 days; SD in 17 more patients
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lasted at mean 76 days (range 29–504 days).[1] Most interestingly, there was hardly any CLL cell clearance of PB at one week after the 4th infusions. A larger cohort of Hodgkin (HL) and non-Hodgkin lymphomas (NHL) included a phase-IIA part.[2] Complete responses (by CT-scan) occurred one each in FL, diffuse large B-cell lymphoma (62.9 weeks), and mucosa-associated tissue (MALT) lymphoma. The overall response rates (ORR) in FL and MALT-lymphoma were 7/21 (33.3%, with 6 PR) and (3/7) 42.9%, respectively. Among FL, 11 additional patients demonstrated an SD as best response and a total of 15/21 (71%) of FL showed a reduction in tumor size. Among the lymphoma subsets, the highest partial metabolic response rates (per FDG-PET in 62%) and the most durable responses (7.9–31 weeks) were observed in FL.[2] These distinct patterns of response point to a relative importance of CD40-CD40L engagement across different lymphoma subsets. Moreover, it suggests a differential relevance of CD40 signaling and hence interventional sensitivity between site-defined milieus in CLL. In conjunction with our experimental data, we conclude that HCD122 (lucatumumab) is active at sites of relevant CD40–CD40L interaction; in CLL mostly nodal sites of residing regenerative centers. A yet insufficient single-agent clinical activity calls for explorations of more suitable combinations than with fludarabine, such as other mAb or small molecules. In extension, the success of novel milieu-modulatory strategies, including the one described here, likely depends on their capacity to also sufficiently restore anti-tumor (T cell) immunity in addition to perturbation of dependance signals.
Acknowledgements Novartis provided HCD122, however, had no role in study design, data collection, and data analysis as well as in the decision to publish or in the preparation of the manuscript.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at http:// dx.doi.org/10.3109/10428194.2015.1135433.
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[2] Fanale M, Assouline S, Kuruvilla J, et al. Phase IA/II, multicentre, open-label study of the CD40 antagonistic monoclonal antibody lucatumumab in adult patients with advanced non-Hodgkin or Hodgkin lymphoma. Br J Haematol. 2014;164:258–265. [3] Bensinger W, Maziarz RT, Jagannath S, et al. A phase 1 study of lucatumumab, a fully human anti-CD40 antagonist monoclonal antibody administered intravenously to patients with relapsed or refractory multiple myeloma. Br J Haematol. 2012;159:58–66. [4] Du¨rig J, Ebeling P, Grabellus F, et al. A novel nonobese diabetic/severe combined immunodeficient xenograft model for chronic lymphocytic leukemia reflects important clinical characteristics of the disease. Cancer Res. 2007;67:8653–8661. [5] Girbl T, Hinterseer E, Gro¨ssinger EM, et al. CD40-mediated activation of chronic lymphocytic leukemia cells promotes their CD44-dependent adhesion to hyaluronan and restricts CCL21-induced motility. Cancer Res. 2013;73:561–570. [6] Travert M, Ame-Thomas P, Pangault C, et al. CD40 ligand protects from TRAIL-induced apoptosis in follicular lymphomas through NF-kappaB activation and up-regulation of c-FLIP and Bcl-xL. J Immunol. 2008;181:1001– 1011. [7] Herreros B, Rodrı´guez-Pinilla SM, Pajares R, et al. Proliferation centers in chronic lymphocytic leukemia: the niche where NF-kappaB activation takes place. Leukemia. 2010;24:872–876. [8] Mittal AK, Chaturvedi NK, Rai KJ, et al. Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease. Mol Med Camb Mass. 2014;20:290–301. [9] Schattner EJ, Mascarenhas J, Reyfman I, et al. Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity. Blood. 1998;91:2689–2697. [10] Cantwell M, Hua T, Pappas J, et al. Acquired CD40-ligand deficiency in chronic lymphocytic leukemia. Nat Med. 1997;3:984–989. [11] Ferrajoli A. Interfering with CD40 ligation: a sensitive matter in chronic lymphocytic leukemia. Leuk Lymphoma. 2012;53:2093–2094. [12] Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev. 2009;229:152–172. [13] Hassan SB, Sørensen JF, Olsen BN, et al. Anti-CD40mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol. 2014;36:96–104. [14] Luqman M, Klabunde S, Lin K, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells. Blood. 2008;112:711–720. [15] Tai YT, Li X, Tong X, et al. Human anti-CD40 antagonist antibody triggers significant antitumor activity against human multiple myeloma. Cancer Res. 2005;65:5898–5906.
