Synergistic Antitumor Effect of Bispecific CD19 × CD3 and CD19 × CD16 Diabodies in a Preclinical Model of Non-Hodgkin's Lymphoma This information is current as of September 28, 2021. Sergey M. Kipriyanov, Björn Cochlovius, Holger J. Schäfer, Gerhard Moldenhauer, Alexandra Bähre, Fabrice Le Gall, Stefan Knackmuss and Melvyn Little J Immunol 2002; 169:137-144; ; doi: 10.4049/jimmunol.169.1.137 Downloaded from http://www.jimmunol.org/content/169/1/137 References This article cites 50 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/169/1/137.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Synergistic Antitumor Effect of Bispecific CD19 ؋ CD3 and CD19 ؋ CD16 Diabodies in a Preclinical Model of Non-Hodgkin’s Lymphoma1 Sergey M. Kipriyanov,2,3* Bjo¨rn Cochlovius,2† Holger J. Scha¨fer,2* Gerhard Moldenhauer,‡ Alexandra Ba¨hre,† Fabrice Le Gall,* Stefan Knackmuss,* and Melvyn Little* To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and Fc␥RIII (CD16). Bacterially produced CD19 ؋ CD16 BsDb specifically interacted with both CD19؉ and CD16؉ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combi- nation of the CD19 ؋ CD16 BsDb with a previously described CD19 ؋ CD3 BsDb and CD28 costimulation significantly increased Downloaded from the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 ؋ CD16 BsDb, CD19 ؋ CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target. The Journal of Immunology, 2002, 169: 137–144. http://www.jimmunol.org/ on-Hodgkin’s lymphoma (NHL)4 encompasses a heter- typically express one or more B cell markers, e.g., CD19 or CD20, ogeneous group of hematological malignancies of B and these markers can be used to redirect effector cells toward malig- N T cell origin occurring in blood, lymph nodes, and bone nant B cells. Although normal B cells will also be destroyed, they marrow, which frequently disseminate throughout the body (1). are repopulated from stem cells lacking the targeted Ags. To me- NHL is one of the few malignancies that has increased in fre- diate redirected lysis, a BsAb must bind a target cell directly to a quency more than the increase in population, with ϳ53,000 new triggering molecule on the effector cell (4). The best-studied cy- cases occurring annually in the United States (2). The most com- totoxic triggering receptors are multichain signaling complexes mon forms of NHL are derived from the B cell lineage. While such as TCR/CD3 on T cells, Fc␥RIIIa (CD16) on NK cells, and NHL can be treated with reasonable success at early and interme- Fc␥RI (CD64) and Fc␣RI (CD89) expressed by monocytes, mac- by guest on September 28, 2021 diate stages, the results of conventional chemotherapy and radia- rophages, and granulocytes (3, 5). BsAbs directed to the TCR/CD3 tion in advanced stages remain disappointing. This particularly complex have the potential to target all T cells regardless of their holds true for the prevalent low grade lymphomas. A fairly large natural MHC specificity. To date, different forms of the CD19 ϫ number of patients relapse, and most remissions cannot be ex- CD3 BsAb have been generated and used in a number of in vitro tended beyond the minimal residual disease. This discouraging and in vivo therapeutic studies (6–17). These BsAbs have been situation has stimulated the search for alternative therapeutic strat- mainly produced using rodent hybrid hybridomas (7–9) or by egies, such as activation of host immune mechanisms using bispe- chemical cross-linking of two mAbs (6). However, the human anti- cific Abs (BsAbs) (3). The BsAb makes a bridge between the murine Ab response and release of inflammatory cytokines are the tumor cell and the immune effector cell, followed by triggering the major drawbacks of BsAb derived from rodent mAbs in clinical cytotoxic responses that include perforin and granzyme release, use (18, 19). Recent advances in recombinant Ab technology have Fas-mediated apoptosis, and cytokine production. Since NHLs provided alternative methods for constructing and producing BsAb molecules (20, 21). For example, CD19 ϫ CD3 single-chain vari- able fragment of Ab (scFv)-scFv tandems have been produced in *Affimed Therapeutics, Heidelberg, Germany; and †Recombinant Ab Research Group mammalian cells (15). Alternatively, recombinant BsAbs can be and ‡Department of Molecular Immunology, German Cancer Research Center, Hei- formed by noncovalent association of two single-chain fusion delberg, Germany products consisting of the VH and VL domains of different speci- Received for publication October 22, 2001. Accepted for publication May 1, 2002. ficity in an orientation preventing intramolecular pairing with the The costs of publication of this article were defrayed in part by the payment of page formation of a four-domain heterodimer diabody (12, 22) or an charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. eight-domain homodimer tandem diabody (17, 23). The two Ag binding domains have been shown by crystallographic analysis to 1 This work was supported by the German BioRegio Program (Grant BEO32/AZ12389). be on opposite sides of the diabody such that they are able to 2 S.M.K., B.C., and H.J.S. contributed equally to this work. cross-link two cells (24). Bispecific diabodies (BsDbs) are poten- 3 Address correspondence and reprint requests to Dr. Sergey M. Kipriyanov, Affirmed tially less immunogenic than quadroma-derived BsAb and can be Therapeutics, Im Neuenheimer Feld 582, D-69120 Heidelberg, Germany. E-mail ad- easily produced in bacteria in relatively high yield (16, 25). We dress: s.kipriyanov@affimed.com have previously shown that CD3 ϫ CD19 BsDbs are more effec- 4 Abbreviations used in this paper: NHL, non-Hodgkin’s lymphoma; BsAb, bispecific tive than quadroma-derived BsAb in mediating T cell cytotoxicity Ab; BsDb, bispecific diabody; ECD, extracellular domain; IMAC, immobilized metal affinity chromatography; PMN, polymorphonuclear granulocyte; scFv, single-chain in vitro against tumor cells (12, 16) and that they had similar variable fragment of Ab; SPR, surface plasmon resonance. antitumor activities in vivo (16, 17). Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 138 DIABODY THERAPY OF HUMAN LYMPHOMA The aim of the present study was to target another subset of Cell binding measurements lymphocyte effectors, NK cells, against CD19-positive tumor cells. The human CD19ϩ B cell line JOK-1 and 293-CD16 cells were used for NK cells are one component of innate immunity and have the flow cytometry experiments performed as previously described (12). In ability to both lyse target cells and provide an early source of brief, 5 ϫ 105 cells in 50 ␮l RPMI 1640 medium (Life Technologies, immunoregulatory cytokines. Human NK cells comprise ϳ15% of Eggestein, Germany) supplemented with 10% FCS and 0.1% sodium azide ␮ all lymphocytes and are defined phenotypically by their expression (referred to as complete medium) were incubated with 100 l BsDb prep- aration for 45 min on ice. After washing with complete medium, the cells of CD56 and lack of expression of CD3 (26). The majority were incubated with 100 ␮lof10␮g/ml anti-c-Myc mAb 9E10 in the same (ϳ90%) of human NK cells express CD56 at low density buffer for 45 min on ice. After a second washing cycle, the cells were (CD56dim) and Fc␥RIII (CD16) at a high level (27). An effective incubated with 100 ␮l FITC-labeled goat anti-mouse IgG (Life Technol- CD16-mediated cytotoxicity induced by BsAb and BsDb has been ogies) under the same conditions as before. The cells were then washed again and resuspended in 100 ␮lofa1␮g/ml solution of propidium iodide documented for Hodgkin’s lymphoma (28, 29). To develop a sim- (Sigma-Aldrich, Taufkirchen, Germany) in complete medium to exclude ilar approach for NHL, we constructed a recombinant anti-human dead cells. The fluorescence of stained cells was measured using a FAC- CD19 ϫ CD16 BsAb in a diabody format and examined its Ag- Scan flow cytometer (BD Biosciences, Mountain View, CA). Mean fluo- binding and antitumor activities both in vitro and in vivo. rescence (F) was calculated using CellQuest software (BD Biosciences), and background fluorescence was subtracted. Equilibrium dissociation con- stants (Keq) were determined as previously described (30) by fitting the experimental values to the Lineweaver-Burk equation 1/F ϭ 1/F ϩ Materials and Methods max (Keq/Fmax)(1/[BsDb]) using the software program PRISM (GraphPad Soft- Materials and cell lines ware, San Diego, CA).