Elsevier

Leukemia Research

Volume 38, Issue 8, August 2014, Pages 948-954
Leukemia Research

Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers

https://doi.org/10.1016/j.leukres.2014.05.010Get rights and content

Highlights

  • Anti-CD40 plus anti-CD137 (BiMab) induced regression of subcutaneous MC38 tumors.

  • BiMab treatment of Eμ-Myc lymphoma led to significant increase in survival of mice.

  • Majority of mice bearing spontaneous Vk*MYC myeloma survived long-term after BiMab.

Abstract

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eμ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.

Introduction

We wished to determine if the combined delivery of two monoclonal murine antibodies, anti-CD40 and anti-CD137 (hereafter called BiMab), could impact on hematological malignancies. There is evidence that both antibodies with the addition of anti-DR5 can impact on a variety of subcutaneous tumors [1]. However, to simplify translation of this approach, we wished to see if reducing this to just the two antibodies in BiMab could be effectual. CD40 is expressed on antigen presenting cells and leads to their activation, generation of CTL and eradication of BCL1 lymphoma in mice [2]. Agonist antibodies specific for CD40 have been demonstrated to be effective against malignancies in mice and in the clinic [3]. CD137 (4-1BB) is expressed on activated T cells, and ligation leads to T cell proliferation and activation. Anti-CD137 is an agonistic antibody and has been shown to inhibit tumor growth in a model of multiple myeloma in mice [4]. We decided to use these two therapies in combination to target hematological malignancies with elevated expression of the oncogene MYC, which is over expressed in a large proportion of tumors [5]. Deregulated expression of MYC induces B-cell neoplasm development when expression of the MYC locus is driven by the immunoglobulin promoters [6]. We utilized two different transplantable hematological cancers, Eμ-Myc [7], [8] B-cell lymphoma and Vk*MYC [9] multiple myeloma, each derived from transgenic mice, injected intravenously to test the combination therapy. We also treated spontaneously generated cancers in Vk*MYC transgenic mice.

The Vk*MYC multiple myeloma model of disease in mice [9] mimics human multiple myeloma by generating a clinically similar disease in mice: indolent course of disease, with organ damage including renal dysfunction, bone disease and anemia, and elevated paraprotein levels in the blood. This disease results from the MYC oncogene activation, under the kappa light chain (Vk) promoter 1, conditionally expressed in maturing B cells expressing adenosine deaminase. In the Eμ-Myc lymphoma model, on the other hand, the MYC gene is driven by the Ig heavy chain enhancer and causes enhanced proliferation of precursor and maturing B lymphoid cells, causing enlarged lymph nodes, spleen, and thymus, thoracic vertebral column tumors, and disseminated lymphoma.

Section snippets

Cell line

The mouse (C57BL/6) MC38 colon carcinoma tumor cell line (ATCC, Manassas, VA, USA) was maintained in complete medium consisting of RPMI (Gibco, Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal calf serum (FCS; MultiSer, Thermo Trace, Melbourne) and additives (2 mM glutamine (Gibco)), 100 μg/ml streptomycin (Sigma–Aldrich, St Louis, MO) and 100 U/ml penicillin (Sigma–Aldrich) in a humidified incubator at 37 °C with 5% CO2.

Mouse tumor models

Wild type female C57BL/6 mice were purchased from either

Treatment with αCD40 in combination with αCD137 enhanced the survival of mice bearing MC38 subcutaneous tumors

C57BL/6 mice injected SC with MC38 colon carcinoma tumor cells were treated with either αCD40 IT or IP either as a monotherapy on days 10, 13, 15 and 17 or combined with αCD137 IP (injected on days 10, 13 and 17). Fig. 1A shows that treatment with αCD40 alone did not significantly enhance the survival of mice above the control (isotype control antibody) group (P = 0.3346 for IT, and P = 0.2401 for IP). Anti-CD137 as a monotherapy was nearly statistically significant in improving survival (P = 

Discussion

In this study, we determined the effect of two immune-agonist antibodies in several mouse cancer models. Anti-CD40 has been used previously against lymphoma, which demonstrated eradication of CD40+ lymphomas (BCL1, A31 and A20), and partial effects in a CD40− T cell lymphoma (Ten1) in a CD4T helper cell independent manner [2]. We have previously demonstrated the potential of three agonist antibodies to inhibit tumor growth in mice. In the present study, we treated a hematological cancer model

Conflict of interest statement

No competing interests were disclosed.

Grant information

MK, PD and JW funded by National Health & Medical Research Council of Australia (NHMRC) Project Grant. MK and PD are supported by Senior Research Fellowships from NHMRC and Grant #1066554 from the Cancer Council of Victoria. Some of the work was funded by Pfizer, San Diego, CA.

Acknowledgments

We wish to thank the Peter MacCallum Cancer Centre Animal Facility staff for their care and maintenance of mice used in this study.

Author contributions: MK conceived the study. MK and JW designed and analyzed the experiments. JW, HJP and MK carried out the research. GMM, RWJ and JS derived the Vk*MYC tumor cell clone 4929, Em-Myc clone 4242 and provided the Vk*MYC transgenic mice. DF analyzed the sera for paraprotein (M spike). MC and PLB generated the Vk*MYC mouse model. PD, LLS and RDH

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