Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties

Although monoclonal antibody (MoAb) therapy of the humunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony man malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic maformation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent lignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman BT-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in 2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synvivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antiment of B-cell malignancies. q 1997 by The American Society of Hematology. genic specificity was confirmed by a competition radioim-


ITH THE EXCEPTION of a chimeric anti-CD20
monoclonal antibody (MoAb), which has produced tumor regressions in patients with relapsed B-cell non-Hodg-(SCID) mice by the induction of apoptosis (Funakoshi et al, manuscript in preparation).In antibody-dependent cellular kin's lymphoma (NHL), 1 unconjugated MoAbs have demonstrated limited therapeutic responses. 2Radioimmunotherapy, cytotoxicity (ADCC) assays, however, murine Lym-2 mediates low tumor lysis with human mononuclear effector cells.on the other hand, has shown considerable promise in clinical studies, particularly in the treatment of B-cell NHL. 3 The A human-mouse chimeric derivative designated chCLL-1 has therefore been constructed to increase its effector func-efficacy of radioimmunotherapy is restricted, however, either by dose-limiting thrombocytopenia or more severely by the tions.To further enhance the immunotherapeutic potential of this chimeric antibody for the treatment of B-cell malig-presence of bone marrow disease.In these settings, effective therapy with unconjugated MoAbs would be desirable for nancies, antibody fusion proteins containing human GM-CSF and IL-2 have been generated.In this study, we describe the induction of tumor remission.For this purpose, the combination of MoAbs and biologic response modifiers has been the effector functions mediated by these recombinant molecules and demonstrate their tumor targeting abilities in a investigated as a means of increasing tumor lysis.Cytokines including interleukin-2 (IL-2) and granulocyte-macrophage nude mouse xenograft model.colony-stimulating factor (GM-CSF) have been shown to

enhance both in vitro cytotoxicity mediated by MoAbs against tumor targets and in vivo killing of tumor xenografts
5][6][7][8][9] Because of the toxicity of sys- The plasmid pcD-hGM/Eo-CSF containing the human GM-CSF temically administered cytokines, however, methods are cDNA 22 was obtained from the American Type Culture Collection needed to target these biologically potent immunologic mediators to the tumor site.One approach, gene transfer, has

kines stimulate antitumor immunity and rejection in animal
Oncology, the Department of Medicine, UCLA School of Medicine, models, [10][11][12][13][14][15][16][17] illustrating the importance of localizing cyto-Los Angeles, CA; and the Section of Pediatric Hematology-Oncolkines to tumors.However, at the present time, this approach ogy, Mayo Clinic, Rochester, MN.   is impractical in the clinical setting.An alternative method Submitted December 16, 1996; accepted January 31, 1997.9][20] In this Techniclone Corp (Tustin, CA), Brilliance Pharmaceuticals (Shanghai, China), and a grant from Children's Cancer Research Fund, way high local concentrations of cytokines within tumors can the Hedberg Foundation (Minneapolis, MN).be achieved and systemic toxicity is minimized or avoided.

is a murine IgG 1 MoAb directed against a human major
The publication costs of this article were defrayed in part by page histocompatability complex (MHC) class II variant that is charge payment.This article must therefore be hereby marked strongly reactive with a high percentage of human B-cell shown to have a direct inhibitory effect on human lymphoma (clone 57594; Rockville, MD).The plasmid pBC12/HIV/IL-2 con-from the pcD-hGM/Eo-CSF plasmid template.To amplify the human IL-2 cDNA from the pBC12/HIV/IL-2 plasmid template, two taining the human IL-2 cDNA 23 was obtained from the American Type Culture Collection (clone 67618).The plasmids pEE6hCMV-primers, 5 -GGTAAAGCGGCCGCAGGAGGTGGTAGCGCA-CCTACTTCAAGTTCTACA -3 and 5 -TCATGCGGCCGCTCA-B and pEE12 were purchased with the Glutamine Synthetase Gene Amplification System from Celltech Biologics (Slough, UK).Re-AGTTAGTGTTGAGATGATGCT -3, were used.The PCR fragments were each inserted into the Not I site of 12/chCLL-1/HL, striction endonucleases, T4 DNA ligase, and other molecular biology reagents were purchased from New England Biolabs (Beverly, MA) resulting in the expression vectors 12/chCLL-1/HL/GM-CSF and 12/chCLL-1/HL/IL-2, encoding the chimeric light chain and a fusion or Boehringer Mannheim (Indianapolis, IN).RPMI-1640 medium, minimal essential medium (MEM) nonessential amino acids solu-protein consisting of the chimeric CLL-1 heavy chain with human GM-CSF or human IL-2 at its C-terminus.tion, penicillin-streptomycin solution, Dulbecco's phosphate-buffered saline (PBS), dialyzed fetal bovine serum, Sephadex, buffer salts, and other reagents such as chloramine T, sodium metabisulfite,

Expression and Purification of Fusion Proteins
hydrogen peroxide, and ABTS (2,2-azino-bis (3-ethylbenzthiazo-The fusion proteins were expressed from NS0 murine myeloma line-6-sulfonic acid) diammonium salt) were purchased from Sigma cells according to the protocol of the manufacturer (Celltech Biolog-Chemical Co (St Louis, MO).Hybridoma-SFM medium with and ics).Briefly, linearized plasmids were electroporated into NS0 cells, without glutamine was purchased from Life Technologies (Gaitherswhich were plated in nonselective Hybridoma-SFM medium.Selecburg, MD).Fetal bovine serum was obtained from HyClone Laborative glutamine-free medium was added 24 hours later.When tories, Inc (Logan, UT).Iodine-125 and iodine-131 were obtained transfectants appeared approximately 3 weeks later, supernatants as sodium iodide in 0.1 N sodium hydroxide from DuPont/New were tested for the presence of chimeric fusion protein by indirect England Nuclear (North Billerica, MA).Balb/C and athymic nude enzyme-linked immunosorbent assay (ELISA).The highest-producmice were purchased from Harlan Sprague Dawley (Indianapolis, ing clones were identified by 24-hour rate of production assays.To IN).
maximize the yield of chCLL-1/GM-CSF, amplification of vector copy number was achieved by expanding the clone and incubating

Antibodies and Cell Lines
the cells in increasing concentrations of methionine sulfoximine, a specific inhibitor of glutamine synthetase.Three to 4 weeks later, The murine MoAb Lym-2 (muLym-2, IgG 1 ), directed against a B-cell surface antigen, 21 was obtained from Techniclone Interna-viable clones were again assayed for rate of chimeric fusion protein production.After subcloning by limiting dilution, the highest-pro-tional, Inc (Tustin, CA).The human-mouse chimeric MoAb Lym-1 (chLym-1, IgG 1 k) was generated as previously described. 24The ducing clones were expanded, incubated in 10 L bioreactors, and chCLL-1/GM-CSF and chCLL-1/IL-2 were purified stepwise from chimeric MoAb CLL-1 (chCLL-1, IgG 1 k) was produced as previously described (Funakoshi et al, in preparation).The chimeric cell culture medium by protein A affinity chromatography and ionexchange chromatography, as described previously. 24The purity of MoAb TNT-1 (chTNT-1, IgG 1 k), the cDNAs for whose variable regions were cloned from the murine TNT-1 hybridoma, 25 was con-each fusion protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a reducing gel ac-structed and expressed in the same manner as chCLL-1.The murine Lym-2 antiidiotype MoAb (7E2) was generated as previously de-cording to the method of Laemmli 27 and by high performance liquid chromatography (HPLC).The samples were filtered through a 0.22 scribed for the antiidiotype to Lym-1 (1A7). 24Iodine-125 and iodine-131-labeled MoAbs were prepared using a modified chloramine T mm Nalgene disposable filter unit before injection.The fusion proteins were analyzed with a Beckman HPLC Gold System (Beckman method as previously described. 24The NS0 murine myeloma cell line, which was obtained from Celltech Biologics, was grown in Instruments, Fullerton, CA) equipped with two 110B solvent pumps, a 210A valve injector, a 166 programmable UV detector, and a nonselective medium consisting of Hybridoma-SFM supplemented with 10% fetal bovine serum, L-glutamine, MEM nonessential amino 406 analog interface module.Size exclusion chromatography was performed on a G4000SW column (TosoHaas; Montgomeryville, acids solution, penicillin G (100 U/mL), and streptomycin (100 mg/ mL).Selective medium consists of Hybridoma-SFM without gluta-PA) with 0.1 mol/L PBS, pH 7.2 as the solvent system, eluting at a flow rate of 1 mL/min.The UV absorbance of the HPLC eluate mine supplemented with 10% dialyzed fetal bovine serum, glutamic acid, asparagine, nucleosides, penicillin G, and streptomycin, ac-was detected at 280 nm.cording to the protocol provided with the Glutamine Synthetase Gene Amplification System (Celltech Biologics).The ARH-77 hu-Immunoassays man myeloma cell line, 26 obtained from the American Type Culture ELISA.Chimeric fusion protein-containing supernatants were Collection, was grown in RPMI-1640 medium supplemented with initially identified by indirect ELISA using murine Lym-2 antiidio-10% fetal bovine serum, L-glutamine, penicillin G, and streptomytype 7E2 MoAb, as described previously. 24For production rate cin.
assays, 10 6 cells were plated in 1 mL of selective medium and allowed to incubate for 24 hours.ELISA was then performed as

Construction of Expression Vectors
before.Supernatants were serially diluted and applied to wells of microtiter plates coated with goat antihuman IgG (H/L) (CalTag, The expression vectors were constructed using standard techniques.The expression vector for chCLL-1, 12/chCLL-1/HL, was South San Francisco, CA).Dilutions of a control chimeric antibody were used to generate a standard curve using 4-parameter fit by an used as the parent vector.This plasmid contains the cDNA sequences for the human-mouse chimeric CLL-1 heavy and light chains, each automated ELISA reader (Bio-Tek Instruments, Inc, Winooski, VT), from which concentrations of unknowns were estimated.Rates of under the control of the cytomegalovirus (CMV) major immediate early promoter, and the cDNA sequence for glutamine synthetase, production expressed as mg/mL/10 6 cells/24 hours were compared to identify the highest producing clones.under the control of the SV40 early promoter.Two oligonucleotide primers, 5-GGTAAAGCGGCCGCAGGAGGTGGTAGCGCA-ARH-77 cell competition radioimmunoassay.The antigen-binding activity of chCLL-1/GM-CSF and chCLL-1/IL-2 was determined CCCGCCCGCTCGCCCAGC -3 and 5 -TCAATGCGGCCG-CTCACTCCTGGACTGGCTCCCAGCA -3, were used to amplify by a competition radioimmunoassay for binding to fixed ARH-77 myeloma cells.For these studies, 2 1 10 6 ARH-77 cells previously by polymerase chain reaction (PCR) the human GM-CSF cDNA fixed in 2% paraformaldehyde 28 were incubated with 20 ng of 125 I-containing more than 30 cells were enumerated after 14 to 16 days in culture.labeled muLym-2 and serial dilutions of cold muLym-2, chCLL-1/ GM-CSF, chCLL-1/IL-2, or an irrelevant MoAb (chLym-1).The cells and MoAbs or fusion proteins were incubated for 1 hour at

IL-2 Bioassay
room temperature with constant mixing.The cells were then washed Biologic activity of chCLL-1/IL-2 was determined by a standard twice, and the cell pellet-associated radioactivity was measured in IL-2-dependent T-cell proliferation assay. 30Carrier-free recombia gamma counter.Maximal binding was determined from tubes connant IL-2 obtained from Hoffmann La Roche, Inc (Nutley, NJ) was taining no cold antibodies.used as a standard.Roche IL-2 stock (7.8mg/mL, specific activity É12 1 10 6 IU/mg) was diluted to yield a stock solution containing Determination of Avidity 2 1 10 6 IU/mL.Growth of the IL-2-dependent murine T-cell line, CTLL-2, was used to determine the amount of IL-2 bioactivity in a To determine the avidity constants of chCLL-1/GM-CSF and chCLL-1/IL-2, a fixed cell radioimmunoassay was performed using sample.Briefly, serially diluted samples and standard were incubated with 2 1 10 4 CTLL-2 cells in triplicate for 20 hours at 37ЊC in 96-the method of Frankel and Gerhard. 29Each experimental variable was run in duplicate.ARH-77 myeloma cell suspensions containing well flat bottom microtiter plates.The cells were then pulsed with 0.5 mCi of 3 H-thymidine for 6 hours, and the samples were harvested 10 6 cells/mL were incubated with 10 to 110 ng of 125 I-labeled chCLL-1/GM-CSF or chCLL-1/IL-2 in 200 mL PBS for 1 hour at room and counted.temperature with constant mixing.The cells were then washed three times with PBS containing 1% bovine serum albumin to remove Cytotoxicity Assays unbound antibody and counted in a gamma counter.The amount of ADCC was performed using the CytoTox 96 Non-Radioactive fusion protein bound was then determined by the remaining cell-Cytotoxicity Assay (Promega, Madison, WI), which is a colorimetric bound radioactivity (cpm) in each tube and the specific activity (cpm/ assay that quantitatively measures lactate dehydrogenase release. 31ng) of the radiolabeled fusion protein.Scatchard plot analysis was Effector cells were peripheral blood MNC or neutrophilic polymorused to obtain the slope.The equilibrium or avidity constant K a was phonuclear leukocytes (PMN).MNC were isolated from healthy calculated by the equation K Å 0(slope/n), where n is the valence human donors by Ficoll-Paque gradient centrifugation, and PMN of the antibody (2 for IgG).
were purified by centrifugation through a discontinuous percoll gradient (70% and 62%) followed by hypotonic lysis to remove residual

Isolation of Bone Marrow Cells
erythrocytes as described previously. 32ARH-77 myeloma cells were used as target cells.ARH-77 cells were suspended in Hybridoma-Bone marrow samples were obtained in preservative-free heparin from normal donors after receiving their informed consent (with the SFM medium supplemented with 2% fetal bovine serum and plated in 96-well V-bottom microtiter plates at 2 1 10 4 cells/well.Antibody approval of UCLA Institutional Review Board).Cells were diluted with an equal volume of PBS containing 0.6% ACD-A (anticoagu-or fusion protein preparations (chCLL-1/GM-CSF, chCLL-1/IL-2, chCLL-1, muLym-2, or chTNT-1 as an isotype-matched irrelevant lant citrate dextrose solution, Formula A; Baxter-Fenwal Corp, Deerfield, IL) and mononuclear cells (MNC) were isolated by gradient control) were added in triplicate to individual wells at 1 mg/mL, and effector cells were added at various effector:target cell ratios (12.5:1 centrifugation on Ficoll-Paque (Pharmacia LKB; Uppsala, Sweden) followed by two washes with PBS/ACD-A.CD34 / cells were puri-to 50:1).The plates were incubated for 4 hours at 37ЊC, after which the supernatants were harvested, lactate dehydrogenase release was fied from MNC using a CD34 / Progenitor Cell Isolation Kit (Miltenyi Biotec; Auburn, CA) without modification of the manufac-determined, and % specific lysis was calculated according to the protocol of the manufacturer.Data are reported as mean { standard turer's instructions.deviation (SD).Differences between groups were analyzed by unpaired Student's t-test.

Colony Assays
Bone marrow MNC (7.5 1 10 4 cells/well) or CD34 / cells (1 1 whole body activity at injection and at selected times thereafter was measured with a CRC-7 microdosimeter (Capintec, Inc, Pittsburgh, Cultures were maintained humidified at 37ЊC in 5% CO 2 .Colonies   The fusion proteins were expressed from NS0 murine myeach group (n Å 5), individual mice were injected intravenously eloma cells using the glutamine synthetase gene amplifica-(IV) with a 0.1 mL inoculum containing 100 mCi/10 mg of 125 Ilabeled fusion protein.Animals were killed by sodium pentobarbital overdose at 72 hours postinjection, and various organs, blood, and tumors were removed and weighed.The radioactivity in the samples was then measured in a gamma counter.For each mouse, data were expressed as percent injected dose/gram (% ID/g) and tumor:organ ratio (cpm per gram tumor/cpm per gram organ).From these data, the mean and SD were calculated for each group.

Imaging Studies
ARH-77 human myeloma tumors were grown in the left thighs of athymic nude mice as described above.When the tumors had reached approximately 1 cm in diameter, the mice were injected IV with a 0.1 mL inoculum containing 100 mCi/10 mg of 131 I-labeled chCLL-1, chCLL-1/GM-CSF, or chCLL-1/IL-2.At 1, 3, and 5 days postinjection, the mice were anesthetized with a SC injection of 0.8 mg sodium pentobarbital.The immobilized mice were then imaged in a prone position with a Spectrum 91 camera equipped with a pinhole collimator (Raytheon Medical Systems, Melrose Park, IL) set to record 5,000 to 10,000 counts using the Nuclear MAX Plus image analysis software package (MEDX Inc, Wood Dale, IL).tion system (Celltech Biologics).After subjection to vector amplification, the highest chCLL-1/GM-CSF-producing subclone secreted approximately 26 mg/mL/10 6 cells/24 hours in static culture.The highest chCLL-1/IL-2-producing subclone expressed approximately 16 mg/mL/10 6 cells/24 hours.Upon scale-up, greater than 100 mg/mL of chCLL-1/ GM-CSF were obtained after purification.When the chCLL-1/IL-2-producing cell line was grown in a 10-L bioreactor, approximately 70 mg/mL of fusion protein were obtained.Both chimeric antibody fusion proteins were properly assembled as demonstrated by reducing SDS-PAGE; two welldefined bands were resolved for chCLL-1/GM-CSF at approximately 25 and 66 kD and for chCLL-1/IL-2 at approximately 25 and 65 kD, corresponding to the molecular weights of the immunoglobulin light chain and heavy chain plus cytokine (Fig 2).Both fusion proteins appeared as a single peak by HPLC analysis (data not shown).

Immunobiochemical Analysis
The immunoreactivity of purified chCLL-1/GM-CSF and chCLL-1/IL-2 with the target antigen of muLym-2 was assessed by determining the binding to antigen-bearing ARH-77 myeloma cells.In a radioimmunoassay, increasing conr
tion of the IL-2-dependent cell line compared with the recombinant IL-2 standard.This corresponds to a specific activity of approximately 8 1 10 5 IU/mg of fusion protein.At higher concentrations (eg, ú1 nmol/L), maximum proliferation was achieved as evidenced by the plateau of the incorporation of 3 H-thymidine into DNA.As expected, chCLL-1 had no activity.
Avidity binding studies were then conducted in which 125 Ilabeled chCLL-1/GM-CSF or chCLL-1/IL-2 was incubated In Vivo Pharmacokinetic and Tumor Targeting Studies with ARH-77 cells and the bound radioactivity used to calcu-Whole body clearance studies were performed to establish late the avidity constant K a by Scatchard analysis as dedifferences in pharmacokinetics among chCLL-1/GM-CSF, scribed in the Materials and Methods.chCLL-1/GM-CSF chCLL-1/IL-2, and chCLL-1.Mice were injected with 125 Iand chCLL-1/IL-2 had similar binding constants of 3.3 1 10 8 labeled fusion proteins or chimeric antibody, and the whole mol/L 01 and 3.0 1 10 8 mol/L 01 , respectively.The binding body activity at injection and selected times thereafter was constant of muLym-2 was determined to be 2.9 1 10 8 mol/ measured with a microdosimeter.chCLL-1/IL-2 cleared rap-L 01 .These studies demonstrate that the presence of the cytoidly with a whole body half-life of 11 hours (Fig 7).chCLLkines on the C-terminus of the heavy chain does not affect 1/GM-CSF had a half-life of approximately 30 hours, while binding to the antigenic target.
chCLL-1 cleared slowly, with a half-life of 100 hours.
The difference among clearance rates was evident when Colony-Forming Activity of chCLL-1/GM-CSF tumor and normal organ biodistribution was examined in Biologic activity of the GM-CSF moiety was determined ARH-77 myeloma-bearing nude mice.As indicated in Fig by colony assays using both bone marrow MNC and CD34 / 8A, tumor uptake of chCLL-1 after 72 hours was 2.54% { cells.As indicated in Fig 4, chCLL-1/GM-CSF compares 0.14% injected dose/gram, while tumor uptake of chCLL-1/ favorably with recombinant human GM-CSF in its ability to IL-2 and chCLL-1/GM-CSF was significantly lower (1.14 stimulate colony formation from the MNC fraction of normal { 0.08 and 1.07 { 0.10, respectively; P õ .001).However, bone marrow.In addition, the fusion protein is capable of uptake of the fusion proteins in normal tissues was considerinducing the formation of colonies from isolated CD34 / ably lower than chCLL-1, which can be attributed to the progenitor cells (data not shown).No colonies formed in the rapid clearance of the fusion proteins.This low normal tissue presence of chCLL-1.uptake produces higher tumor/organ ratios, as can be seen in Fig 8B.

IL-2 Bioactivity of chCLL-1/IL-2
Imaging studies were also performed to examine tumor targeting with the fusion proteins.Tumor-bearing nude mice Biologic activity of the IL-2 moiety was determined by assaying the ability of chCLL-1/IL-2 to support IL-2-depen-were injected with 131 I-labeled chimeric antibody or fusion protein and imaged at 1, 3, and 5 days postinjection.In Nevertheless, by day 5, localization of chCLL-1 to the tumor been generated, which retain both tumor targeting and cytosite is clear.In mice that received chCLL-1/GM-CSF or kine functions.The GS gene amplification system was used chCLL-1/IL-2, by day 5 no signal remained except in the for high level expression of the fusion proteins from mytumor.These data demonstrate that chCLL-1/GM-CSF and eloma cells so that large-scale production can yield sufficient chCLL-1/IL-2 effectively localize to the ARH-77 human products to enable clinical studies to be undertaken.With myeloma xenografts.
this expression system, gram quantities of the fusion proteins can be produced in batch cultures.Biochemical analysis Cytotoxicity studies clearly demonstrate the improved ef-fusion protein compared with those containing other cytokines has previously been described for the antiganglioside fector functions of chCLL-1 over muLym-2 and of both fusion proteins over chCLL-1 (Fig 6).Human IgG 1 constant MoAb ch14.18. 42It has yet to be demonstrated whether similar differences in clearance between chimeric MoAbs and regions were selected for construction of the chimeric MoAb based on earlier observations of the enhanced antitumor cy-cytokine-containing antibody fusion proteins exist in patients.Biodistribution and imaging studies in human my-totoxic activity of chimeric IgG 1 over chimeric MoAbs of other isotypes. 33At each effector:target cell ratio, chCLL-1/ eloma-bearing nude mice illustrate the tumor targeting abilities of chCLL-1/IL-2 and chCLL-1/GM-CSF (Figs 8 and 9).IL-2 mediates higher specific tumor lysis by human MNC than the chimeric MoAb alone (Fig 6B).This is in agreement Despite their rapid clearance profiles, they retain the capacity to localize to tumor xenografts effectively.,35 chCLL-1/GM-CSF also mediates higher specific tumor lysis by human MNC than chCLL-1 (Fig 6C).
tissues would be minimized.][45][46] There is considerable evidence that high local concentra-against colorectal carcinoma and lymphoma cell lines.Other investigators have observed no effect of GM-CSF on MNC tions of cytokines within tumors can stimulate antitumor immunity and rejection in animal models.The majority of ADCC against malignant B-cell lines. 9There is evidence that GM-CSF and IL-2 can act synergistically in vitro.1][12][13][14][15][16][17] Although these stud-regard, GM-CSF has been shown to augment the induction of lymphokine-activated killer (LAK) activity by IL-2 against ies demonstrate the utility of delivering cytokines directly to tumors, they are presently impractical in the clinical set-a human Burkitt's lymphoma cell line through monocytes. 37n addition, GM-CSF and IL-2 enhance ADCC against a ting.A more feasible approach to generating high local concentrations of cytokines within tumors is targeting cytokines colorectal carcinoma cell line, 38 leading the investigators to suggest combination therapy consisting of low dose IL-2, via antibody fusion proteins. 18,19This approach combines the cytotoxicity that MoAbs can mediate against tumor targets GM-CSF, and MoAb.
No specific lysis of target cells by PMN in ADCC medi-with the host antitumor immune response, which is stimulated by high local concentrations of cytokines.Several ated by chCLL-1 or chCLL-1/GM-CSF was observed in our studies.8][49] Intact MoAbs may have greater effectiveness than to mediate such ADCC. 32In these studies, Lym-1 and 1D10, both of which recognize HLA class II related epitopes, did fragments, however, because they can mediate ADCC.An alternative approach that also employs antibody-cytokine fu-not mediate ADCC by PMN from healthy donors, although both MoAbs mediated ADCC with PMN from patients sion proteins is engineering a cancer vaccine using idiotypecytokine fusion proteins including IL-2 and GM-CSF. 50,51In treated with granulocyte colony-stimulating factor.Similar results have been observed with Lym-1 in combination with a murine B-cell lymphoma model, such fusion proteins have been shown to induce antitumor responses.The efficacy of GM-CSF. 9GM-CSF has also been shown to enhance PMN ADCC against solid tumor cell line targets, including neuro-antibody-targeted IL-2 has been elegantly demonstrated in both a SCID mouse human neuroblastoma model 20,52 and a blastoma, melanoma, and colorectal carcinoma. 36,39,40It is as yet unclear why MoAbs directed against particular antigens syngeneic murine melanoma model. 53,54In these studies, the effector cell population responsible for antitumor responses on malignant B cells possess the ability to mediate PMN ADCC, while those with specificity for other B-cell antigens was identified as CD8 / T cells.As the fusion protein retained a therapeutic effect in natural killer (NK) cell-deficient mice, do not.Based on in vitro ADCC data and clinical experience with a murine MoAb, a clinical trial using the combination the investigators concluded that tumor eradication was not dependent on NK cells. 55Whether such a mechanism of of the MoAb and GM-CSF for the treatment of metastatic colorectal carcinoma was initiated. 41In this study, complete antitumor cytotoxicity holds for other antibody-cytokine fusion proteins in the treatment of other malignancies remains remissions were achieved in some patients, providing clinical evidence for the benefit of combination therapy.
to be determined.The chimeric antibody fusion proteins described in the In the current study, pharmacokinetic analysis in Balb/C mice demonstrated the marked difference in whole body current study have the potential for producing tumor killing by a number of mechanisms.The parent muLym-2 is reactive clearance among chCLL-1 and the fusion proteins (Fig 7).We have recently shown that a fusion protein consisting of with a majority of human B-cell lymphomas, chronic lymphocytic leukemias, and multiple myeloma, 21 suggesting chLym-1 and IL-2 has a half-life of 11 hours, 35 which is identical to that observed for chCLL-1/IL-2.chCLL-1/GM-that this MoAb and derivatives may be of use in treating a variety of B-cell malignancies.Both muLym-2 and chCLL-CSF has a whole body half-life intermediate between the For personal use only.on October 3, 2017.by guest www.bloodjournal.orgFrom

From
the Department of Pathology, University of Southern Calidemonstrated that tumor cells engineered to secrete cytofornia School of Medicine, Los Angeles; the Division of Hematology- Address reprint requests to Alan L. Epstein, MD, PhD, Depart-In this report, we describe the development of such molement of Pathology, University of Southern California School of Medcules for the treatment of hematologic malignancies.Lymicine, 2011 Zonal Ave, HMR 210, Los Angeles, CA 90033.

Fig 1 .
Fig 1.Schematic diagram depicting the linker containing the Not I cloning site between the human g1 and human GM-CSF or human IL-2 cDNAs in the chimeric CLL-1 heavy chain/cytokine fusion genes.

3
weeks until they reached approximately 1 cm in diameter.Within

Fig 7 .
Fig 7. Whole body pharmacokinetic clearance of 125 I-labeled