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`'HWNormal and Malignant Herfidpoiesis
`
`HEMTH SEBENEES ”WAR?
`University of Wisconsin
`
`MAR 2 [l 1995
`
`1305 Linden Drive
`Madison, WI 53706 -
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`z-‘r
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`STOCKTON I
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`Leukemia [1996} 10, 321-326
`© 1996 Stockton Press All rights reserved 0887-6924/96 $12.00
`
`Antileukemic activity of recombinant humanized M195-gelonin immunotoxin in nude
`mice
`
`y )(ul, Q Xu‘, MG Rosenblum2 and DA Scheinberg1
`
`I Memorial Sloan-Kettering Cancer Center, NY; and 2lmmunopharmacol'ogy Section, Department of Clinical immunology and Biological
`Therapy, MD Anderson Cancer Center, Houston, TX USA
`
`.‘f‘
`4!
`
`A leukemia-selective immunotoxin was constructed by iinking
`recombinant gelonin (rGel), a single chain ribosome inhibitory
`protein, to recombinant humanized M195 antibody (HuM195),
`which recognizes the cell-surface protein designated CD33.
`0033 is an antigen found on myeloid leukemia blasts as well
`as myeloid progenitor cells but it is not expressed in detectable
`amounts on the ultimate hematopoietic progenitor stem cell.
`our previous
`studies
`indicated that a non-recombinant
`humanized immunotoxln displayed specific, potent
`toxicity
`towards CDaa-positive cells but not to CD33-negative cells in
`vitro. In the current study, a recombinant humanized immuno-
`toxin, HuM195-rGel, was evaluated in vivo in a nude m0use
`model of human myeloid Ieukemias. HuM195-rGel was ,found
`to target leukemia cells rapidly in vivo and was subsequently
`internalized into the cells. For trials in viva, nude mice were
`injected (ip) with 107 log-phase HL60 human leukemia cells 10
`days prior to the start of i.p. HuM195-rGel treatments. HuM195-
`rGel demonstrated significant tumor suppressive activity in
`this model. While all mice treated with either saline, rGel alone,
`or HuIIlI195 plus unconiugated rGel (at 10 or 14 days after
`transplantation) had rapid tumor growth or early deaths, 50%
`of mice treated with HuM195-rGei failed to develop leukemic
`tumors for 5 months and the other 50% had significantly
`retarded tumor growth after treatment with HuM195-rGel. Mice
`treated at later times (28 days after transplantation of leukemia
`cells) also showed delayed leukemia cell growth, but no cures.
`These data show that HuM195-rGel can target leukemia cells in
`vivo and can result in pronounced anti-leukemic effects.
`Keywords: myeloid
`leukemia; CD33;
`immunotoxin;
`gelonin;
`HuM195
`
`Introduction
`
`lmmunotoxins (IT) are a class of proteins that consist of a
`monoclonal antibody (mAb) covalently linked or genetically
`fused to a cytotoxic molecule and are thus able to direct
`potent cytotoxic protein to particular cells.“2 Ribosome
`inhibitory proteins (RlPS) can be specifically targeted to certain
`tissues through chemical conjugation or genetic fusion with
`' mAb and thereby acquire cell-specific cytotoxicityFl4
`Gelonin toxin originally isolated from the seeds of Colon-
`tum multiflorum is a single polypeptide chain in a class of
`proteins designated type | RIPS. Unlike the type II RIPS, for
`example ricin, which is composed of a ricin A chain and a
`lectin-Iike B chain, gelonin has a relatively low native cyto—
`toxic activity due to the lack of a lectin B chain, which can
`nonspecifically bind to cell membrane glycoproteins. Like all
`planbderived RiPs, gelonin damages 285 rRNA through a gly—
`cosidase that cleaves the glycosidic bond at a unique adenine
`base in the rRNA,
`thereby inhibiting protein synthesis?“6
`Recently, the native gelonin protein was sequenced, a syn-
`
`
`
`Correspondence: DA Scheinberg, Memorial Sloan-Kettering Cancer
`Center, 1275 York Avenue, New York, NY 10021, USA
`Received 9 August 1995; accepted 16 October 1995
`
`thetic gene encoding gelonin was sythesized, and biologicalw
`active recombinant gelonin (rGel) was synthesized in E. coil?
`Gelonin has several advantages for use in immunotoxin-
`therapy compared to other RIPS, including the lack of the B
`chain containing the galactosespecific lectin domain respon—
`sible for much non—specific binding and toxicity?”5 As a result,
`free gelonin is much less toxic to intact mammalian cells in
`vitro and in vivo than type II RIP. Despite this relative safety,
`in a cell—free rabbit reticulocyte translation assay, gelonin
`demonstrates nearly equal biological activity to heterodimeric
`toxinsarg In addition,
`immunotoxins containing an A chain
`separated from a B chain are, in general, less potent on intact
`cellslovl‘ and the larger heteroclimers may also be more immu—
`nogenic than the single chain toxins. Therefore, gelonin may
`have properties advantageous for the generation of potent and
`specific immunoconjugates.‘2
`CD33 is a useful target antigen for therapy of myelogenous
`leukemias, as it is expressed on the cell surface of greater than
`80% of leukemia isolates from patients with myeloid leukemia
`with an average antigen density of 10 000 sites per cell.13“"’
`However, CD33
`is not
`found on tissues outside the
`hematopoietic system.l3 Its expression within the hematopo-
`ietic system is limited to early myeloid progenitor cells, mono—
`cytes and dendritic cells. lmportantly, CD33 is not found on
`the ultimate hematopoietic progenitor stem cell, thus allowing
`in principle, selective elimination of leukemia cells and early
`progenitors while preserving capacity for long-term regener—
`ation of marrow cells.“"”'“3
`HuM195, a humanized version of M195 constructed by
`genetically grafting the murine complementarity—determining
`region (CDR)
`to a human lgGl
`framework and constant
`regions,
`is reactive with CD33.19 The humanized antibodies
`may be advantageous clue to reduced immunogenicity, higher
`avidity, and longer serum half lives?”22 In addition, rapid
`internalization occurs upon binding of mAb HuM195 to
`CD33 both in vitro and in VlV0.19‘22’23 This suggests that
`HuM195 can be a suitable candidate for immunotoxin studies
`in humans.
`
`We recently described a HuMiQS-gelonin immunotoxin.‘2
`HuMi95-gelonin did not completely prevent hematopoietic
`reconstitution in vitro after treatment as evidenced by bone
`marrow colony experiments, but bone marrow treated with
`HuMi 95-gelonin demonstrated a log reduction of colony for-
`mation. This is expected since colony forming unit-granulo—
`cyte monocytes (CFU—GM) express CD333“24
`Clinical trials using 131I
`labeled M195 have demonstrated
`rapid and specific localization of antibody to tumor sites, satu~
`ration of all available CD33 antigen, followed by intracellular
`internralizationfizr23 Dose escalation studies using 13ll—iV‘llgS
`resulted in greater than 99% killing of leukemic blasts with
`negligible toxicity outside of
`the hematopoietic compart-
`rnents.25 However, due to the long range cytotoxicity of .the
`conjugated nuclide (approximately 50 cell diameters) killing
`of normal bystander cells occurs as well, redtriring bone mar-
`,1"/
`
`IMMUNOGEN 2159, Pg. 3
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`lPR2014-00676
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`IMMUNOGEN 2159, pg. 3
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`IPR2014-00676
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`

`

`Flow cytometry assays
`
`Cells were washed and resuspended in 2% rabbit serum (Pei
`Freeze, Rogers, AK, USA)
`to reduce nonspecific binding.
`5 x105 cells in a final volume of 0.1 ml were incubated I h
`on ice in the presence of primary antibody HuFd79 control
`or HuM195. Cells were washed twice, incubated 30 min on
`ice with secondary fluorescein isothiocyanate (FlTC) labeled
`antibody (goat anti-human immunoglobulin) (Kirkegaard and
`Perry, Gaithersburg, MD, USA), washed twice, and fixed with
`0.5% paraformaldehyde. FlTC fluorescence intensity was
`measured on an EPICS Profile II
`flow cytometer (Coulter,
`Miami, FL, USA).15
`
`inhibition of tritiated thymidine or leucine
`incorporation
`
`An aliquot containing 100 pl of cells were washed and incu-
`bated at 37°C in 96-well plates in the presence of 50 pl of
`antibody, conjugate or toxin. After an incubation time of 3—7
`days, 50 ul of 10 uCi/ml of tritiated thymidine or leucine (Du
`Font—New England Nuclear) was added to each well and
`allowed to incorporate for 5 to 6 h. Trichloroacetic acid was
`added to a final concentration of 10% to precipitate protein
`for 3H—leucine incorporation experiments. Cells were har-
`vested using a semiautomatic harvester (Skatron) and read in
`a scintillation counter LS 6000IC (Beckman, Fullerton, CA,
`USA).
`
`3-(4,5—dimethylthiazol—Z—yll—Z,5~diphenyltetrazolium
`bromide : thiazolyl blue (M TT) assays
`
`One hundred microliters of cells were washed and incubated
`at 37°C in 96—well plates in the presence of 50 ,ul of antibody,
`conjugate or toxin. After an incubation time of 3—7 days, the
`plate was centrifuged 5 min at 1000 r.p.m. MTT diluted in
`phosphate-buffered saline, was added to each well and incu-
`bated. for 4 h. Plates were washed and the formazan product
`was solubilized with 0.04 M l-lCl
`in 2—propanol and quantit-
`ated spectrophotometrically at 570 nm.
`
`Transplantation HL60 cells into nude mice
`
`A 0.2-ml aliquot containing 107 HL60 cells from suspension
`culture was transplanted ip into nude mice. Tumors grew sub—
`cutaneously and the cutaneous tumor size was measured as
`a cross product to derive surface area. For studies ex vivo;
`pieces of tumor were minced and intact single cells were iso-
`lated on a Ficoll—Hypaque density, gradient or after passage
`through a 70 um nylon filter (Spectrum, Houston, TX, USA)-
`
`ln vivo measurement of antibody targeting to tumor
`
`The rate at which 125I—I-IuM195 was bound to and sub-
`sequently internalized into leukemia cells in vivo was meas—
`ured. Tumor bearing mice at 4 weeks (two mice per group)
`infused ip with 2 or 20 pg of 125I—l-IuMi 95 or 125I—HuFd7’9 (a
`negative control), were sacrificed at 4 or 24h after
`the
`infusion. Tumors were excised and weighed at 4°C to aVO'd
`internalization during the assay process. Cell surface boll”CF
`‘25I-HuM195 or ”5I-HuFd79 was then stripped using 50 mM
`
`IMMUNOGEN 2159, Pg. 4
`Phigenix v. Immunogen
`|PR2014-00676
`
`Recombinant humanized M195-gelunin lmmunotoxin
`Y Xu et al
`
`row transplantation at high doses. We propose that use of an
`anti-CD33 immunotoxin may avoid this problem by targeting
`cells bearing the CD33 antigen. In this paper, we describe the
`activity of
`immunotoxins constructed by chemical conju-
`gation of HuM195 with recombinant gelonin in a mouse
`model of HL60 leukemia.
`
`a 3
`
`22
`
`Materials and methods
`
`Animals
`
`Six-week-old female outbred Swiss nu/nu mice were obtained
`from the colony at Sloan Kettering Institute. All bedding
`material was sterilized before use; the cages were covered
`with an air filter and maintained in isolation cabinets. Animal
`handling and experiments were performed in aseptic atmos—
`phere using a laminar flow hood.
`
`Cell lines
`
`HL60 (acute myeloid leukemia, CD33 positive), RAJI and
`DAUDI (both B lineage Burkitt’s lymphomas, CD33 negative)
`were maintained in culture using RPMI 1640 supplemented
`with 10% Serum Plus (JRH Biosciences, Lenexa, KS, USA) and
`10% heat
`inactivated fetal calf serum (lntergen, Purchase,
`NY, USA).
`
`Antibodies and radiolabelecl antibodies
`
`HuM195, a humanized IgGi reactive with human CD33 anti—
`gen, and HuFd79, a genetically engineered human lgGi con—
`struct reactive with a herpes simplex virus antigen not found
`on HL60 cells, were prepared as described.19 Antibodies were
`trace labeled with 125| (New England Nuclear, Boston, MA,
`USA) using the chloramine T method to a specific activity of
`240 ”Ci/pg as described previously.‘5 lmmunoreactivity of
`the radiolabeled antibody was determined by incubating serial
`dilutions (107 to 106 cells) of l-IL60 cells with 24 ng of radio—
`labeled antibody for i h at 4°C. Cells were resuspended in
`phosphate buffered saline twice and the pellets were counted
`to determine total cell-bound 125l-.antibody.
`
`Toxins
`
`Recombinant gelonin (rGel) was derived and purified as
`described.7 Functional activity studies demonstrated that this
`protein behaved similarly to chemically purified natural
`gelonin.7
`
`Preparation of HuM195 Conjugates
`
`l-luM‘i95 was conjugated with rGel using N-succinimidyl 3~
`(2—pyridyldithio) propionate (SPDP);
`the immunoconjugates
`were purified by gel-permeation chromatography and separ—
`ation on cibachron blue sepharose as previously described.l2
`The molecular weight of HuMi95-rGel was about 180 kDa
`demonstrating a l
`: 1 molar ratio of HuM195 to rGel.
`
`IMMUNOGEN 2159, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Recombinant humanized M195-gelunin immunutoxin
`Y Xu et al
`
`323
`
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`leuflut)
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`
`Cytotoxicity and inhibition of protein synthesis in HL60
`Figure1
`or RAJI cells by recombinant geionln (rGel), HuMi95 and Huh/11957
`rGel. (a) Inhibition of protein synthesis in HL60 or RAJI cells by rGel,
`HuM195 and HLIMIQS-rGel. HL60 or RAJI cells at a final concen—
`nation of 105 cells/ml were incubated 3 days at 37°C in the presence
`of HUM1957rGel, HuM195 and rGel. Leveis of protein synthesis were
`determined by fish incorporation of tritiated leucine into trichloro-
`acetic acid precipitable protein. The treatment is shown in paren-
`thesis. (b) Cell viability determined by trypan blue exclusion. l-IL60 or
`RAJI cells at a final concentration of 105 cells/ml were incubated 3
`days at 37°C in the presence of HuM195—rGel. Trypan blue was added
`and live and dead cells were counted under the microscope.
`
`the expression of CD33 on the tumors was
`Therefore,
`assessed. The cells
`from the leukemic tumors
`retained
`expression of CD33 positive antigen after growth in vlvo, as
`determined by flow cytometry at saturating mAb concen-
`trations (Figure 2). The internalization of 125I-Huiv1195 into the
`target cells in vivo was rapid, and similar to the observations
`in vitro.19 At 4 h after infusion of 2 or 20 ,ug antibody, 23—26%
`of bound‘15|~H uM195 was internalized, whereas a higher rate
`of internalization (38—43%) was seen at 24 h (Figure 3).
`
`In vivo antltumor effects of HuM795—rGel
`
`The leukemic cell growth in the subcutaneous space and peri—
`toneum of nude mice was substantially reduced by HuM195-
`rGel. At 10 days after transplantation of HL60 cells into the
`peritoneum of nude mice, tumors of about 2 mm2 in size are
`present in the subcutaneous space (Figure 4). .fititer three injec—
`tions of HuMi 95—rGel at a dose of 36 ug penthouse beginning
`at 10 days, two out of four mice did not develop tumors for
`1‘
`{1
`
`IMMUNOGEN 2159. Pg. 5
`Phigenix v. Immunogen
`|PR2014-00676
`
`glycine/HCI, ”{50 mM NaCl, pH 2.8, and internalized c.p.m.
`(residual c.p.m. in the cell pellets) were calculated.19 Specific
`surface bound and internalized HuM195 were calculated by
`subtraction of nonspecific surface bound and internalized
`Hchl79.
`
`lmmunotherapy
`
`The test animals werg’ treated i.p. with HuM195-rGel
`(36 pig/dose), recombinant gelonin (6 tag/dose), or I-luM195
`and recombinant gelonin mixture (30 Mg Huh/i195 plus 6 ug
`gelonin/dose) twice a week in a final volume of 0.2 ml (the
`molar amounts of toxin and antibody were kept constant).
`Assuming a circulation volume of about 2 ml,
`the dose
`injected was equivalent to 100 nM initial concentration. Con-
`trol mice were treated with 0.2 ml saline twice a week.
`
`Results
`
`In vitro cytotoxicity
`
`HuM195~rGel was tested for its ability to kill CD33 positive
`and CD33 negative cells in comparison to free rGel. Activity
`and cytotoxicity were determined by inhibition of incorpor—
`ation of 3H-ieucine into protein and by trypan blue exclusion.
`Doseeresponse curves were generated by testing the inhibitory
`effects HuMi95-rGel on the protein synthesis of HL60 cells
`(CD33 positive) and RAJI (CD33 negative) in culture (Figure
`1a).
`In the in vitro assays, the concentration of HuM195-rGel
`required to inhibit protein synthesis in HLGO cells by 50% was
`0.6 nM, whereas the concentration of rGel alone required to
`nonspecifically inhibit protein synthesis in both Ht60 and
`RAJI cells by 50% was about 200 nM (Figure ia). In the con-
`centration range of 10—100 nM HuMi95-rGel, protein syn»
`thesis in HL60 cells was almost completely inhibited while no
`cytotoxicity was observed with the CD33 negative cell
`lines
`RAJI (Figure ta) and DAUDI (not shown). However, HuM195
`alone did not affect the protein synthesis in CD33 positive
`HL60 cells (Figure 1a). This shows that the inhibition of pro—
`tein synthesis was due to specific binding and activity of the
`immunotoxin, and not a nonspecific property of the antibody
`itself. The specific targeting of leukemic cells by HuM195-Gel
`appeared to occur via the CD33 antigen binding site and not
`through the Fc region or other non-specific binding sites on
`target cells as shown previously.i2
`The cytotoxicity of HuM195—rGel was directly determined
`by trypan blue analysis. The concentration of HuM195-rGel
`required to kill 50% of cells was 0.7 nM (Figure 1b), similar
`to the concentration of HuM195 required to inhibit protein
`synthesis by 50% (Figure 1a). However, HuM195-rGel did not
`kill CD33 negative RAJI cells at the highest concentration of
`100 nM, suggesting that it may be used safely for study in vivo.
`The cytotoxicity was also confirmed by 3H—thymidine incor—
`poration and MTT assays
`(not shown), confirming that
`HuM195-rGe| causes HL60 leukemia cell death in vitro.
`
`Targeting of radiolabeled HUMl95 into leukemic cells
`in Vii/O
`
`We have previously shown that nude mice retain limited
`ability to generate antibodies to the HL60 cells after transplant
`and that CD33 can be down-regulated by this response.26
`
`IMMUNOGEN 2159, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Recombinant humanized M195—geinnin immunotuxin
`Y )(u at ai
`
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`
`Humps-ma
`
`“Ha yea
`--O - Control
`
`"'+"' HuMlOSplu: rOei
`
`0
`
`f
`i1
`{I
`
`200
`
`500
`
`400
`
`300
`
`100
`
`800
`
`700
`600
`
`HuMlES-rGai (m2 weeks)
`----- HuMlFi-rflai (m4 week)
`
`,
`
`I
`
`500
`
`400
`
`300
`
`100
`
`200
`
`
`
`
`
`
`
`Tumorsurfacearea(1111112)
`
`.1
`
`‘l
`
`10
`
`100
`
`1000
`
`Log Fluorescence
`
`Tumor cell surface antigen expression. Expression of
`Figure2
`CD33 by HL60 cells grown at 4 weeks in a representative tumor mass
`in va0 determined by indirect
`immunofluorescence as described
`under Materials and methodse—u, HuFd79;—, HuMlQS.
`
`0.4
`
`
`I Susanna pg)
`El
`Intnmalizaflm (2 pg)
`
`El] Surfacempg]
`
`
`E Intemulizaliml (mug)
`
`
`
`0.3
`
`0.2
`
`0.1
`
`SpecificradiolabeledHuh/1195(ngper
`
`
`
`
`mflhontumorcells) 0.0
`
`llllll
`
`4
`
`24
`
`Time (hours)
`
`Specific binding and internalization of ‘25I~HuM195 in
`Figure3
`tumor in vivo. Tumor bearing mice at 4 weeks after transplantation
`received infusions of 2 or 20 pg of ”SI-lmluMIQS shown in parenthesis.
`Mice were sacrificed at 4 or 24h after the infusion. Tumors were
`excised, weighed, and counted. Specific surfacenbound and internal-
`ized HuMlEiS were calculated as described under Materials and
`methods. SD. was less than 10%.
`
`up to 5 months after transplantation, Tumors grew slowly in
`the other two immunotoxin—treated mice. Control groups of
`mice (treatment with saline alone, gelonin alone, or HuMi 95
`admixed with rGel at the same final concentrations) did not
`show significant tumor inhibition or any cures.
`To assess whether activity could be observed against larger
`tumors,
`in a second trial, we also tested six injections of
`HuM195—rGel (twice a week for 3 weeks at the same dose of
`36 pg per mouse at 14 clays and 28 days after transplantation
`with HL60 cells. Despite the increase in the number of doses
`from three to six, the delay in treatment to 28 days caused
`less inhibition'oftumor growth by HuMl 95-rGel (Table 1 and
`Figure 4b). After 3 weeks of treatment with HuMi 95—rGel, two
`out of four mice had no tumors in the group treated 14 days
`after transplantation; however, all four mice developed local
`
`Treatment of human leukemia cells in vivo model by
`Figure4
`HuMiQS—recombinant gelonin (rGel). Mice were ip transplanted 107
`HL60 human leukemia cells as described under Materials and
`methods. (a) At the tenth clay, mice were treated by three iniections
`of TOO nM: HuM195—rGel
`(four mice);
`rGel
`(four mice); HuMi95
`mixed with rGel (five mice); or control saline (five mice). At times
`indicated in x axis, tumor surface area was measured, One of five
`mice in control group and one of five mice in Huh/1195 admixed with
`I‘Gel died in the sixth week. (b) At the l4th or 28th days, mice were .
`treated by six injections of 100 nM: HuMl95—rGel (four mice at the
`14th day); HuMl 95—rGel (four mice at 28th day), Control saline (five
`mice). At times indicated in x axis, tumor surface area was measured.
`
`tumors when treated 28 days after transplantation. This may
`be due to difficulty in delivering HuM195 to larger solid
`tumors or to the development of resistant cells within the
`larger tumors.
`,
`
`Discussion
`
`Leukemia-specific immunotoxins (IT) have been shown tO
`exhibit potent cytotoxic activity in viti'rllz’l“mm”s In this
`study we have used HuM195rrGel directed to human CD33
`antigen to show anti—leukemic effects in vivo in a nude mouse
`model of human leukemia. Anti-tumor effects have been seen
`in models of lymphoid neoplasmsm‘32 and in humansfflias but
`this is the first report of in vivo activity of an lT in myeloid
`leukemia. CD33 is a useful target antigen for therapy of mye‘
`logenous leukemias, as it is expressed on the cell surface‘Of
`greater than 80% of leukemia isolates from patients With
`myeloid leukemia and on leukemia colony-forming cells with
`
`IMMUNOGEN 2159. Pg. 6
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2159, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Table1
`
`Tumor size at 7 weeks after HuMl95—rGel treatment
`
`
`325
`
`Recombinant humanized M195—gelonin immunotoxin
`Y Xu et at
`
`Groups
`
`Control
`
`rGel'
`
`Hull/l195 + rGelb
`
`HUM195~rGel
`
`Mouse 1
`Mouse 2
`Mouse 3
`Mouse 4
`Mouse 5
`Meanisd.
`
`420
`525
`900
`1225,
`Death at 6 weeks
`7653:1368
`
`648
`696
`760
`803
`N/Ac
`727i68
`
`342
`400
`550
`784
`Death at 6 weeks
`Sig: i9?
`
`No tumor
`No tumor
`49
`96
`NIA
`38i46
`
`No tumor
`No tumor
`81
`‘I17
`NIA
`50i59
`
`96
`160
`i80
`380
`NIA
`2041:123
`
`(at 10 days)
`
`(at 14 days?
`
`(at 28 days)a
`
`Ten million HLBO leukemia cells were transplanted ip into nude mice. Mice were treated with three injections of equimolar amounts of
`HuM1957rGei, rGel. or HuMi95 plus rGeI, beginning at 10 days after HLGO transplantation. Two other groups of mice were treated with
`six injections of HuM195~rGeL beginning at the 14 or 28 days after HL60 transplantation. Subcutaneous tumor size (cross product in mmz)
`at 7 weeks is shown
`aThese mice received six injections instead of three injections.
`bInjected together, but not conjugated to each other.
`”NIA, these groups had four mice only.
`
`an average antigen density of 10 000 sites per cell._16 In
`addition, rapid internalization occurs upon binding of mAb to
`CD33 both in vitro and in viva.”23 l-luMl95, a humanized
`version of M195, constructed by genetically grafting the
`murine complementarity—determining region (CDR)
`to a
`human IgGl framework and constant regions,
`is reactive with
`CD33.19 The humanized antibodies may be advantageous due
`to reduced immunogenicity, higher avidity, and longer serum
`half lives-.191“
`_
`Human CD33 is not expressed on normal mouse tissues.
`The distribution of CD33 positive cells on human normal tisu
`sues is likely to have an important impact on the clinical utility
`of this immunotoxin. The expression of CD33 antigen in
`humans is restricted to a small fraction of hematopoietic cells,
`including colony forming unit-granulocyte monocytes (CFU-
`GM), some burst forming units—erythroid (BFU-E), a fraction
`of more primitive progenitorsflllfir‘fi'“ monocytes, and blood
`dendritic cells. However, CD33 is not found on tissues outside
`the hematopoietic system, not on the normal pluripotent
`hematopoietic progenitor stem cells.
`Major obstacles to the effective therapeutic use of IT in
`humans include immunogenicity of the IT, toxicity to cells that
`non—specifically internalize the IT, and difficulty in delivering
`sufficient IT to tumor sites.‘i33'35 HuMlQS—rGel may bypass
`some of these difficulties by the use of a CDR-grafted non-
`immunogenic mAb that has already demonstrated efficient,
`specific, and saturable targeting to leukemia cells in humans
`in vivo. In this paper we now demonstrate that the HuMl 95-
`rGel will target and internalize into human leukemia cells in
`vivo in a murine model, and subsequently will eliminate or
`slow tumor growth.
`It
`is possible that conjugation of the mAb to this smaller,
`less toxic RIP, will provide an immunotoxin that is less immu—
`nogenic and more tolerable in humans. The IDS.) of HuM195-
`rGel and free rGel
`in vitro was 400 to i000-f'0ld different.
`Therefore, some dissociation of the HuMl95—rGel bond in
`vivo is not likely to affect the specificity of the treatment; nor
`should there be significant additional cytotoxic effects on
`CD33-negative normal cells.
`Other investigators have characterized immunotoxins com-
`posed of anti-CD33 mAb and ricin A chains in vitro. La Russa
`et all“ and Roy et at27 developed an anti CD33—IT, MY9-
`blocked ricin (MY9—bR), which also demonstrates potent and
`selective cytotoxicity towards CD33 positive cells.
`In the
`blocked ricin system, non-specific binding of the ricin B chain
`
`was blocked by chemically modifying the galactose binding
`domain. MYQ—bR has demonstrated selective inhibition of
`greater than 85% of the CD33—positive CFU~GM clonog'enic
`growth while sparing the CD34—positive/CD33—negative hem—
`atopoietic stem cell. The use of MYQ—bR is under investigation
`as
`a purging agent prior
`to autologous bone marrow
`reinfusion.37
`HuMl95-rCiel conjugates showed specific and potent cyto-
`toxicity for CD33 positive leukemia cells in viva. Under the
`conditions of this model, where I-IL60 cells grow in the peri—
`neum and subcutaneous space of nude mice as a solid tumor,
`the HuMl954rGel was still effective at
`inhibiting tumor
`growth.
`lmportantly, the early treatment of leukemia tumors
`‘ with HuM195~rGel was able to cure half of the mice that had
`already developed local tumors. Although the leukemic tumor
`size at 10 days is only about 2 mm}, this represents about 107
`cells in the subcutaneous tumor site. Although cell numbers
`are much larger
`in humans,
`leukemia cells are more dis—
`persed, allowing for even more rapid penetration and possibly
`greater efficacy. At later time—points after transplantation into
`