NEOPLASIA - - - -- - - - -- -- - - --
`
`- - - - - - - -- - - -- - - -- - - -- - -- --
`
`Endostatin, an antiangiogenic d1ug, induces tumor stabilization after
`chemotherapy or anti-CD20 therapy in a NOD/SCID mouse 1nodel
`of human high-grade non-Hodgkin lymph01na
`Francesco Bertolini, Lisa Fusetti, Patrizia Mancuso, Alberto Gobbi, Chiara Corsini, Pier Francesco Ferrucci,
`Giovanni Martinelli, and Giancarlo Pruneri
`
`Both chemotherapy and chimeric anti(cid:173)
`CD20 monoclonal antibodies are effective
`agents against B-cell non-Hodgkin lym(cid:173)
`phoma (NHL). However, patients achiev(cid:173)
`ing remission are at risk of relapse. To
`evaluate the effect of the antiangiogenic
`drug endostatin used alone and after the
`administration of cyclophosphamide
`(CTX) or the anti-CD20 antibody ritux(cid:173)
`imab, we generated a new model of
`human NHL by transplanting Namalwa
`cells intraperitoneally into nonobese dia(cid:173)
`betic/severe combined immunodeficient
`(NOD/SCIO) mice. First, we determined
`the most effective treatment schedule for
`the drugs assessed. When administered
`alone, CTX (3 courses of 75 mg/kg of
`
`body weight given intraperitoneally), ritux(cid:173)
`imab (3 courses of 25 mg/kg given
`(5
`intraperitoneally), and endostatin
`courses of 50 µg given subcutaneously)
`delayed tumor growth, and CTX was the
`most effective in controlling bulky dis(cid:173)
`ease. When given after chemotherapy or
`immunotherapy, endostatin effectively in(cid:173)
`duced tumor stabilization. When mice
`given CTX or rituximab on days 3, 5, and 7
`after transplantation were randomly as(cid:173)
`signed to receive endostatin or phos(cid:173)
`phate-buffered saline on days 15 to 19,
`in end-
`tumor growth was prevented
`ostatin-treated mice as long as the drug
`was administered. Furthermore, adminis(cid:173)
`tration of endostatin on days 25 to 29
`
`after tumor regrowth still induced signifi(cid:173)
`cant tumor regression, whereas CTX and
`rituximab were not effective. The specific
`antiangiogenic action of endostatin was
`confirmed by in vitro and in vivo studies
`indicating that the drug inhibited prolifera(cid:173)
`tion and induced apoptosis of endothelial
`(but not of NHL) cells. In conclusion,
`sequential administration of chemother(cid:173)
`apy and endostatin seems promising for
`treating bulky NHL, and the less toxic
`sequential administration of rituximab
`and endostatin is promising for treating
`limited disease. (Blood. 2000;96:282-287)
`
`'-'· 2000 by The American Society of Hematology
`
`Introduction
`
`Despite recent advances in the treatment of B-cell non-Hodgkin
`lymphoma (NHL) and the introduction into mainstream hematology(cid:173)
`oncology of the chimeric anti-CD20 antibody rituximab, approxi(cid:173)
`mately one half of patients with NHL who achieve partial or complete
`remission after chemotherapy or immunotherapy do relapse. 1
`Because the growth of most types of cancers,2 possibly including
`hematologic malignancics,3·5 depe nds on the generation of new
`blood vessels, it was proposed that antiangiogenic therapy may
`induce solid-tumor dormancy or stab ilization. 2 Preclinical studies
`of antiangiogenic therapy in nonhematologic malignancies con(cid:173)
`firmed this hypothesis 6 Moreover, the antiangiogcnic drug thalido(cid:173)
`mide was found to be effective in patients with refractory myeloma,
`another B-eell malignancy. 7 In addition, a new generation of
`antiangiogenic drngs was determined to be significantly more etlective
`than thalidomide in inhibiting the growth of endothelial cells. 2 We
`generated a novel murine model of human B-cell N 11 L to evaluate
`the effect of the new antiangiogcnic drug endostatin 8 used alone
`and sequentially after chemotherapy or immunotherapy.
`Mice bearing the nude or severe combined immunodefici ent
`(SCIO) mutation have been used extensively to evaluate human
`malignancies in vivo. However, these strains have residual immu(cid:173)
`nity that may limit posttransp lantation neoplastic cell growth, and
`
`evidence has indicated that the nonobese diabetic/SCIO (NOD/
`SCIO) strain is more convenient for human leukemia and lym(cid:173)
`phoma xenotransplantation. 9• 11 With the aim of generating a
`disease similar to human high-grade B-cell NHL, we used intraperi(cid:173)
`toneal instead of subcutaneous transplantations in NOD/SCID mice
`and found that Namalwa cells generated intraperitoneal tumors in
`the injection site. These tumors cou ld be precisely measured with
`ca lipers to evaluate the ctlicacy of different therapies.
`
`Materials and methods
`
`Cell lines and in vitro studies of endostatin
`
`line (phenotype CDJ - , CDI0 +, CDl3 - , CDl9 +,
`The Namalwa ce ll
`CD20 +, GlyA - ) derives from an Epstein-Barr virus- pos iti ve Burki II NI·IL.
`It was obtained from the American Type Culture Collect ion (Ma nassas, VA)
`and cu ltured in RPMI and 8% fetal bov ine scrum (FBS; HyClonc, L ogan ,
`UT). Human umbilical vein endothelial cells (HUVEC) were obtained from
`Cascade Biologics (Portland. OR) and cultured in medium 200 (Cascade
`Biologics) supplemented with 2% FBS and IO ng/mL each of vascular
`endothelia l growth factor (VEGF) and basic fibroblast growth factor
`(b-FGF; Pcprotech, Rocky Hill, NJ). lluman recombinant cndostntin
`
`From the Divisions of Hematology-Oncology, Experimental Oncology, and
`Pathology-Laboratory Medicine, IRCCS European Institute of Oncology,
`Milan. Italy.
`
`Submilled December 30, 1999; accepted February 24, 2000.
`
`Reprints: Francesco Bertolini, Hematology-Oncology Unit, European Institute of
`Oncology, via Ripamonti 435, 20141 Milan, Italy; e-mail: f.bertolini@agora.stm.it.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore , and solely to indicate this fact, this article is hereby
`marked "advertisement" in accordance with 18 U.S.C. section 1734.
`
`F.B. is a scholar of the US National Bl ood Foundation.
`
`([) 2000 by The American Society of Hematology
`
`282
`
`BLOOD, 1 JULY 2000 • VO LUME 96, NUMBER 1
`
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`BLOOD, 1 JULY 2000 • VOLUME 96, NUMBER 1
`
`ENDOSTATIN, CTX,AND RITUXIMAB IN MURINE NHL
`
`283
`
`produced in Pichia pastoris was obtained from Calbiochcm (San Diego,
`CA). In accordance with the findings of O' Reilly ct al,8 cndostatin activity
`was tested in vitro on HUVEC and Namalwa cells at concentrations ranging
`from 250 to I 000 ng/mL.
`
`Animal studies
`
`Our model of high-grade human NHL was generated by injecting 6- to
`8-,week-old NOD/SCIO mice intraperitoncally with 10 X 106 Namalwa
`cells. The mice were evaluated for tumor growth every other day. Tumor
`volume was measured with calipers, and the formula, width2 X length X
`0.52, was applied to approximate the volume of a spheroid, as described
`previously. 6 Injections of treatment agents were given in a site remote from
`the inoculated tumor. Cyclophosphamide (CTX; Sigma, St Louis, MO) and
`the chimeric anti-CD20 monoclonal antibody rituximab (Roche, Monza,
`Italy) were given intraperitoneally, whereas endostatin was given subcuta(cid:173)
`neously. Control mice received intraperitoneal or subcutaneous injections
`of phosphate-buffered saline (PBS). Tumor-bearing mice were killed by
`carbon dioxide asphyxiation, and tumor engraftment was confirmed by
`histologic, immunohistochemislly (IHC), and flow cytometry (FC) studies.
`All procedures involving animals were done in accordance with national
`and international laws and policies.
`For histologic and IHC evaluations, tumor samples were fixed in I 0%
`formalin and embedded in paraffin. Then, 4-µm-thick sections were stained
`with hematoxylin and eosin and with Giemsa stain for conventional
`histologic assessment. For IHC, sections were immunostained with anti(cid:173)
`CD l 0 and anti-CD20 monoclonal antibodies (DAKO, Glostmp, Denmark).
`Tumor expression of human CD 19 and CD20 antigens was evaluated by FC
`using monoclonal antibodies from Becton Dickinson (Mountain View; CA).
`Tumor angiogenesis was evaluated by FC using a new monoclonal antibody
`(Phanningen, San Diego, CA) against murine FLK, a VEGF-related
`receptor found on endothelial cells. Fresh tumors were dissolved at the
`single-cell level, and 500 to I 000 X l 03 cells were incubated at 22°C for 30
`minutes in PBS and I% bovine serum albumin with monoclonal antibodies.
`The percentage of stained cells was determined in comparison with an
`isotypic control by using a fluorescence-activated cell sorter (FACScalibur;
`Becton Dickinson). A portion of each sample was incubated with the
`appropriate isotype control antibodies to establish the background level of
`nonspecific staining, and positivity was defined as staining greater than
`nonspecific background staining. To identify apoptotic and dead cells,
`7-amino actinomycin D (7AAD) was used as described previously. 12
`
`Statistical analysis
`
`Statistical comparisons were done with r tests and analysis of variance when
`data were normally distributed. Nonparametric Spearman and Mann(cid:173)
`Whitney analyses were used when data were not normally distributed. P
`values lower than 0.05 were considered to represent significance.
`
`Resu lts
`
`Atier intraperitoneal injection of 10 X I 06 Namalwa cells, measur(cid:173)
`able tumors developed in the injection site in all mice given the
`transplants (in the middle of the right posterior quadrant of the
`abdomen; Figure I). Tumors grew as solid masses in the peritoneal
`cavity that infiltrated the peritoneum of the abdominal wall. In a
`few cases, infiltration of the small and large bowel also occurred.
`Tumors were composed of large cells growing in a diffuse pattern,
`with round or slightly convoluted nuclei, at least I eosinophilic
`nucleolus, and a thin rim of basophilic cytoplasm. A few larger cells
`with 2 or more nuclei were also present. Apoptotic bodies were
`observed frequently, and larger areas of necrosis were also
`detectable. Scattered among the neoplastic cells were a few
`histiocytes, although a "starry-sky" pattern was not evident.
`Visible and measurable tumors were observed beginning on day I 0
`after transplantation. When left untreated, tumors reached a volume
`of approximately 5000 mm3 by day 20 (Figures 2-9). This pattern
`
`Figure 1. Namalwa tumor grow1ti in nonobese diabetic-severe combined
`immunodeficient (NOD/SCIO) mice given transplants of 10 x 106 cells intraper(cid:173)
`itoneally. A control mouse is shown on the bottom. The tumor growing in the injection
`site (arrow) is visible and measurable in the mouse that had transplantation (top).
`
`of tumor growth was similar to that previously described in a
`subcutaneous model ofNamalwa cell xenotransplantation. 10 In the
`first 30 days after transplantation, tumor metastasis in sites other
`than the injection site was not observed. In some mice that were
`given transplants and not treated, metastasis to regional lymph
`nodes and multiple visceral organs was observed during the second
`month after transplantation.
`
`CTX, rituximab, and endostatin used as single agents
`
`We first determined the most appropriate treatment schedule for
`rituximab, CTX, and endostatin used as single agents (n = 4-10
`animals per group). In our NOD/SCID mouse model, the maximum
`
`100000
`
`E 10000
`
`.......
`
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`E -(l)
`
`E
`:J
`0
`>
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`E
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`
`I,,.
`
`1000
`
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`
`.1.
`
`100
`
`10
`
`iii
`,.,,-·;
`1 - ' 0 5 10 15 20 25 30 35
`
`Day
`
`Figure 2. Results of treatment with 2 different doses of cyclophosphamide
`(CTX). NOD/SCIO mice given transplants of 10 x 106 Namalwa cells intraperitone(cid:173)
`ally were treated with either 150 mg/kg of body weight of CTX or 75 mg/kg of CTX on
`days 3, 5, and 7. When 150 mg/kg of CTX was administered(• , n = 10), 60% of the
`mice died of drug-related toxicity. When 75 mg/kg of CTX was given ((cid:127)
`, n = 10),
`none of the mice died of drug-related toxicity, and the delay in tumor growth was not
`significantly different than at the higher dose. The dotted line indicates tumor growth
`in 6 untreated controls (A). Results are mean:±: SD values for tumor volume.
`
`NOVARTIS EXHIBIT 2076
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`
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`

`284
`
`BERTOLINI et al
`
`BLOOD, 1 JULY 2000 • VOLUME 96, NUMBER 1
`
`(Figure 4). For endostatin, our in vitro studies indicated that 250 to
`1000 ng/mL of this agent inhibited 34% to 66% ofHUVEC prolifera(cid:173)
`tion , whereas no effect was observed on Namalwa cell proliferation
`(P < .001; Figure SA). In vivo, 5 courses of 50 µg endostatin given
`on days 3 to 7 significantly delayed tumor growth (Figure SB).
`
`CTX, rituximab, and endostatin used sequentially
`
`Four to 6 animals per group were evaluated in sequential(cid:173)
`administration studies. When given after CTX or rituximab,
`
`__ ,,1
`J.· ..
`
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`
`100000
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`
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`
`10
`
`Rituximab
`
`! ! !
`
`I
`
`,•
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`A 100
`90
`80
`70
`60
`50
`40
`30
`20
`10 -
`0
`
`r:: -0 ... C:
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`0
`:;;
`.c
`.c:
`
`Q)
`~
`<1)
`a.
`
`5
`
`0
`
`10 15 20 25 30
`Day
`Figure 3. Results of treatment with 2 different doses of rituximab. NOD/SCIO
`mice given transplants of 10 x 106 Namalwa cells inlraperitoneally were treated with
`75 mg/kg (• )or 25 mg/kg((cid:127)) of riluxlmab. Delay in tumor growth was not significantly
`different in mice given 75 mg/kg of rituximab and those given 25 mg/kg on days 3, 5,
`and 7 (n = 4 per group). The dotted line indicates tumor growth in untreated controls
`(A). Results are mean :t SD values for tumor volume.
`
`tolerable dose of CTX was found to be 7 5 mg/kg per body weight
`given on days 3, 5, and 7. This treatment significantly delayed tumor
`growth, and dose escalation to I 50 mg/kg killed 60% of the treated
`mice while not producing a significantly greater response (Figure
`2). Three courses of25 mg/kg ofrituximab given on days 3, 5, and
`7 were found to delay Namalwa tumor growth, and dose escalation
`(up to 75 mg/kg) did not produce an additional benefit (Figure 3).
`Interestingly, when we evaluated the effect of CTX and rituximab
`on bulky disease by administering these drugs on days 15, 17, and
`19 after transplantation, we found that CTX induced a transient
`reduction in tumor burden, whereas rituximab was not effective
`
`100000
`
`E 10000
`
`1000
`
`100
`
`10
`
`-M
`E -<1)
`
`E
`::1
`0
`>
`'-
`0
`E
`::1
`I-
`
`Rituximab or CTX
`
`Ir
`
`lll ~ ~-1
`I
`".~
`I
`~
`
`I~ (cid:127)
`250 500 750 1000
`0
`Endostatin concentration
`(ng/ml)
`
`E
`
`B -M
`E -Q,)
`E
`::l
`0 > ,._
`0
`E
`::l
`.....
`
`100000
`
`10000
`
`1000
`
`100
`
`10
`
`1
`
`*
`
`*
`
`Endostatin
`
`lllll
`
`0
`
`5
`
`10 15 20 25
`Day.
`
`0
`
`5 10 15 20 25 30 35
`Day
`Figure 4. Treatment of bulky disease with CTX (75 mg/kg) or rituximab (25
`mg/kg). NOD/SCIO mice (n = 4 per treatment group) that received transplants of
`1 O x 106 Namalwa cells intraperiloneally were treated on days 15, 17, and 20 after
`transplantation. CTX ((cid:127) ) induced a significant but transient reduction in tumor
`burden, whereas rituximab ( • ) did not. The dotted line indicates tumor growth in
`untreated controls (.6. ). Results are mean :!: SD values for tumor volume.
`
`Figure 5. Results of in vitro and in vivo studies of endostatin. (A) Specific inhibition of
`human umbilical vein endothelial cells (HUVEC) ( •) by endoslatin. HUVEC were cullured
`in medium 200 supplemented with 10 ng/ml vascular endothelial growth factor and basic
`fibroblast growth factor. Namalwa ceUs ((cid:127)) were cultured in RPM!~% fetal bovine serum
`in a 72-hour proliferation assay.• Endostatin inhibited proliferation of HUVEC but not
`Namalwa cells in a dose-dependent fashion. Results are mean :t SD values (n = 3). (B)
`Endostatin treatment ( • , 50 µg per mouse on days 3 lo 7) significantly delayed tumor
`growth in NOD/SCIO mice given transplants of 10 x 106 Namalwa cells inlraperiloneally.
`The dotted line indicates tumor growth in untreated controls ((cid:127)). Results are mean :t SD
`values for tumor volume (n = 4 per group). ' P < 0.01.
`
`NOVARTIS EXHIBIT 2076
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`

`BLOOD, 1 JULY 2000 • VOLUME 96, NUMBER 1
`
`ENDOSTATIN, CTX, AND RITUXIMAB IN MURINE NHL
`
`285
`
`endostatin effectively induced tumor stabilization. As shown in
`Figure 6, mice given 3 courses ofrituximab on days 3, 5, and 7 after
`transplantation were randomly assigned to receive 50 µg of
`endostatin or PBS on days 15 to 19. In endostatin-treated mice, but
`not in PBS-treated mice, tumor growth was prevented as long as the
`endostatin was administered. Endostatin was similarly effective in
`mice given 3 courses ofCTX on days 3, 5, and 7 after transplanta(cid:173)
`tion and randomly assigned to receive 50 µg of endostatin or PBS
`on days 15 to 19 (Figure 7). It is noteworthy that administration of
`endostatin on days 25 to 29 after tumor regrowth still induced
`significant tumor regression (P = .02 by paired t test; Figure 7).
`Conversely, when rituximab was administered after CTX, it delayed
`tumor growth in comparison with results in PBS-treated controls but did
`not prevent tumor growth or induce a significant tumor regression after
`tumor regrowth (Figure 8). Similarly, when CTX was administered after
`rituximab, tumor growth was delayed in comparison with results in
`PBS-treated controls, but neither prevention of tumor growth nor
`regression after tumor regrowth was observed (Figure 9).
`
`Induction of in vivo endothelial cell apoptosis by endostatin
`
`Figure IO shows the frequency of apoptosis in endothelial cells
`from dissolved Namalwa tumors. As indicated by double staining
`for murine FLK and 7 AAD, 8.3% ± 1.9% of endothelial cells from
`endostatin-treated animals were apoptotic. This value was 4 times
`higher than that in control mice given PBS, CTX, or rituximab
`(1.7% ± 1.1%;P > .001).
`
`Discussion
`
`Studies using preclinical models of nonhematologic malignancies
`have indicated that antiangiogenic therapies may delay or even
`abrogate tumor growth.2•6•8 Moreover, several findings suggest that
`angiogenesis may be clinically relevant in hematopoietic malignan(cid:173)
`cies,3·5 and the antiangiogenic drug thalidomide has been found to
`
`100000
`
`Endostatin or PBS
`
`-
`E -Q)
`
`E
`:::i
`0
`>
`I.,
`0
`E
`::,
`1--
`
`100000
`
`M
`E 10000
`
`Endostatin or PBS
`
`Jilll
`
`• .. .1
`
`1000
`
`100
`
`10
`
`i
`
`CTX
`
`!!!
`
`... i
`
`;✓
`
`I
`
`Endostatin or PBS
`
`0 5 10 15 20 25 30 35
`Day
`Figure 7. Results with CTX followed by endostatin. NOD/SC ID mice that received
`transplants of 10 x 106 Namaiwa cells intraperitoneally were given 3 courses of CTX
`(75 mg/kg) on days 3, 5, and 7 after transplantation and randomly assigned to receive
`50 µg of endostatin (•) or PBS((cid:127)) on days 15 to 19. In endostatin-treated mice, but
`not in PBS-treated mice, tumor growth was prevented as long as endostatin was
`administered. Moreover, administration of endostatin on days 25 to 29 after tumor
`regrowth induced significant tumor regression (P = .02 by paired t test comparing
`tumor volume on day 25 with that on day 30). The dotted line indicates tumor growth
`in untreated controls (A). Results are mean :!: SD values for tumor volume (n = 6 per
`group).• P = .02 vs day 25.
`
`be effective in patients with multiple myeloma refractory to
`chemotherapy. 7 Furthermore, thalidomide also seems to be effec(cid:173)
`tive in myelodysplastic syndromes, myeloproliferative disorders,
`and myelofibrosis. 13,14 All these hematologic diseases, including
`myeloma, are associated with relevant bone marrow angiogen(cid:173)
`esis.4•5 We and others 15•18 have investigated the role of angiogenesis
`and angiogenic growth factors in NHL. Expression of VEGF and
`related receptors Flt-I and FLK-KDR has been observed in most
`B-cell hematopoietic malignancies, 17 and the autocrine and
`paracrine roles of this growth factor are being evaluated. In
`addition, remodeling and immature vessels were observed in biopsy
`
`I
`
`· ·-····"'
`
`100000
`
`M
`E 10000
`
`Rituximab or PBS
`
`!!!
`
`E
`:::i
`0
`
`1000
`
`100
`
`10 -
`
`CTX
`
`-
`E -(I)
`.. ~
`> ...
`0
`E
`::I
`1--
`
`-M
`E -Q)
`
`E
`:::J
`0
`>
`I..
`0
`E
`:::J
`I-
`
`E 10000
`
`1000
`
`100
`
`10 -
`
`Rituximab
`
`,,
`
`l.
`II!
`
`! ! !
`, .. ·
`1 •
`
`5
`
`0
`
`10 15 20 25 30
`Day
`Figure 6. Results of treatment with rituximab followed by endostatin. NOD/
`SC ID mice that received transplants of 10 x 106 Namalwa cells intraperitoneally
`were given 3 courses of rituximab (25 mg/kg) on days 3, 5, and 7 alter transplantation
`and randomly assigned to receive 50 µg of endoslatin ( •) or phosphate-buffered
`saline (PBS)((cid:127)) on days 15 to 19. In endostatin-treated mice, but not in PBS-treated
`mice, tumor growth was prevented as long as endostatin was administered. The
`dotted line indicates tumor growth in untreated controls (A). Results are mean :!: SD
`values for tumor volume (n = 4 per group).
`
`0 5 10 15 20 25 30 35
`Day
`Figure 8. Results with CTX followed by rituxlmab. NOD/SCIO mice that received
`transplants of 1 O x 106 Namalwa cells intraperitoneally were given 3 courses of CTX
`(75 mg/kg) on days 3, 5, and 7 after transplantation and randomly assigned to receive
`25 mg/kg olrituximab or PBS on days 15, 17, and 19 (first course) and on days 25, 27,
`and 29 (second course). Rituximab (•) delayed tumor growth in comparison with
`results in PBS-treated controls ((cid:127)) but did not prevent tumor growth or induce
`significant tumor regression after tumor regrowth. The dotted line indicates tumor
`growth in untreated controls (.t.). Results are mean :!: SD values for tumor volume
`(n = 4 per group).
`
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`
`BERTOLINI et al
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`BLOOD, 1 JULY 2000 • VOLUM E 96, NUMBER 1
`
`100000
`
`CTX or PBS
`
`E 10000
`
`--('I')
`E -Cl)
`
`E
`:J
`0
`>
`i..
`0
`E
`:J
`I-
`
`J.
`
`Rituximab
`
`!!!
`
`1000
`
`100
`
`10
`
`1
`
`CTX or PBS
`
`iii
`
`0 5 10 15 20 25 30
`Day
`Figure 9. Results with rituximab followed by CTX. NOD/SCIO mice that received
`transplants of 10 x 106 Namalwa cells intraperitoneally were given 3 courses of
`rituximab (25 mg/kg) on days 3, 5, and 7 after transplantation and randomly assigned
`to receive 75 of mg/kg CTX (• ) or PBS((cid:127) ) on days 15, 17, and 19 (first course) and
`on days 25, 27, and 29 (second course). CTX delayed tumor growth in comparison
`with results in PBS-treated controls but did not prevent tumor growth or induce
`significant tumor regression after tumor regrowth. The dotted line indicates tumor
`growth in untreated controls (J. ). Results are mean ± SD values for tumor volume
`(n = 4 per group). •p = .003; tP = .02.
`
`specimens of high-grade NHL (F. Pezzella, unpublished data), and
`high circulating levels ofVEGF and b-FGF (the other endothelial
`cell mitogenic factor) were found to correlate with a poor prognosis
`in patients with NHL. 15•16•18
`When generating animal models of human cancer, it is impor(cid:173)
`tant to consider that tumor angiogenic phenotype can be modulated
`by cytokines released by host cells in specific microenvironments.
`Human renal cancer cells, for instance, express high levels of the
`angiogenic growth factor b-FGF when growing in kidney, but not
`when growing subcutaneously. 19 Thus, we reasoned that an intra(cid:173)
`peritoneal tumor model was more appropriate than a subcutaneous
`model for evaluating the effect of endostatin in B-cell NHL. By using
`NOD/SCID mice given transplants ofNamalwa cells, we generated a
`reliable in vivo model of aggressive intraperitoneal human B-cell
`
`c·~.------c=,.,.--,
`~~ 1,----t,,ti,. -I
`
`,-.. ,....~
`
`~~~-...... 1---;
`
`0
`
`102' 10":I
`10
`Mc,t;se FLK
`
`1114
`
`Control
`
`3.0%
`
`8.8°/o
`
`Endostatin
`
`Figure 10. Representative dot plots indicating the frequency of endothelial cell
`apoptosis in Namalwa tumors generated in NOD/SCID mice. Tumors dissolved
`as single cells were evaluated by flow cytometry. Panel A shows forward and side
`scatters of the cell suspension and the analysis gate, panel B the negative control,
`and panel C the human CD19' phenotype of the tumor. As indicated by the
`percentage of murine cells positive for FLK and 7-amino actinomycin D (panels D and
`E), the frequency of endothelial apoptotic cells was significantly increased in mice
`given endostatin.
`
`NHL that allows measurement of tumor, closely resembles the
`human disease, and has 100% engraftment efficiency. In fact, the
`animal model described in this study was similar to human Burkitt
`NHL, a B-cell malignancy that, in most endemic and sporadic cases,
`affects abdominal viscera. The model had 100% engraftment effi(cid:173)
`ciency: measurable intraperitoneal tumors developed in the injec(cid:173)
`tion site in all mice given transplants. Remarkably, the Namalwa
`line was previously found to be the most aggressive in a panel of
`lymphoid lines tested in subcutaneous xenotransplant studies. JO
`To study the efficacy of endostatin used alone and in combina(cid:173)
`tion with established chemotherapy and immunotherapy protocols
`in our NHL model, we chose drug-administration schedules in
`accordance with previously published inforrnation. 1 When seeking
`the most effective dosages of CTX and rituximab, we found that
`toxicity limited CTX administration and that drug resistance was
`progressively acquired. Rituximab efficiency reached a plateau at a
`dose of 25 mg/kg, and in our model, as well as in NHL patients (F.
`Pezzella et al, unpublished data), this agent was less active than
`CTX in cases of bulky disease. To our knowledge, our study is the
`first to indicate that rituximab is effective in NOD/SCID mouse
`models of human B-cell NHL. The evidence that rituximab was
`active in vivo against human NHL, despite the fact that the host
`mice had a severe deficiency of B cells, T cells, natural killer cells,
`myeloid cells, and complement, 11 is of particular interest. In fact, it
`suggests that at least some ofrituximab-induced biologic responses
`may not be due to antibody-dependent cellular cytotoxicity or
`complement-dependent cell lysis.
`Endostatin is one of the more promising antiangiogenic drugs, and
`our in vitro data indicate that it has a direct effect on newly generated
`endothelial cells without producing a detectable effect on NHL
`cells. Our study indicates that sequential administration of end(cid:173)
`ostatin after rituximab or CTX effectively induces tumor stabiliza(cid:173)
`tion. It should be noted that in mice treated in the first week after
`tumor transplantation, sequential administration of the nontoxic drugs
`rituximab and endostatin was at least as effective as sequential
`administration of CTX (a highly toxic drug) and endostatin. Our
`data also confirm the results of a study6 in mice treated for 240 to
`320 days with multiple cycles of high-dose endostatin that indi(cid:173)
`cated that this antiangiogenic drug, in sharp contrast to chemother(cid:173)
`apy drugs such as CTX, does not induce acquired drug resistance.
`On the other hand, long-te1m administration of endostatin is needed
`because tumor relapse has been observed after discontinuation of the
`drug. The ultimate goal of antiangiogenic therapy is to induce
`long-term tumor stabilization. 20 Because data from studies in
`nonhuman primates have indicated that endostatin may be adminis(cid:173)
`tered for long periods without producing toxicity,21 we suggest that
`this agent might be evaluated in clinical trials as a consolidation
`drug for patients who have achieved remission. The data we obtained by
`using our murine model indicate that sequential administration of
`chemotherapy and endostatin seems appropriate in patients with
`bulky disease, whereas the less toxic sequential administration of
`rituximab and endostatin seems effective in patients with limited
`disease. In this context, B-cell malignancies are of particular
`interest because molecular probes for quantifying minimal residual
`disease in blood and bone marrow are available for most patients.
`
`Acknowledgments
`
`We thank Francesco Pezzella, Domenico Delia, Caimelo Carlo(cid:173)
`Stella, and Davide Soligo for critical reading of the manuscript.
`
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`

`BLOOD, 1 JULY 2000 • V OLUME 96, NUMBER 1
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`ENDOSTATIN, CTX, AND RITUXIMAB IN MURINE NHL
`
`287
`
`References
`
`1. Longo DL. Lymphoma treatment: you have to ad-
`mit it's getting better. Curr Opin On col. 1999; 11 :
`319.
`2. Folkman J. Angiogenesis in cancer, vascular, meu-
`matoid and other diseases. Nat Med. 1995; 1 :27.
`3, Perez-Atayde AR, Sallan SE, Tedrow U, Connors
`S, Allred E, Folkman J, Spectrum of tumor angio-
`genesis in the bone marrow of children with acute
`lymphoblastic leukemia.Am'J Pathol. 1997;150:
`815.
`4. Pruneri G, Bertolini F, Soligo D, et al. Angiogen-
`esis in myelodysplastic syndromes. Br J Cancer.
`1999;81:1398.
`5. Vacca A, Ribatti D, Presta M, et al. Bone marrow
`neovascularization, plasma cell angiogenic po-
`tential, and matrix metalloproteinase-2 secretion
`parallel progression of human multiple myeloma.
`Blood. 1999;93:3064.
`6. Bohem T, Folkman J, Browder T, O'Reilly MS.
`Antiangiogenic therapy of experimental cancer
`does not induce acquired drug resistance. Na-
`ture. 1997;390:404.
`7. Singha! S, Mehta J, Desikan R, et al. Antitumor
`activity of thalidomide in refractory multiple my-
`eloma. N Engl J Med. 1999;341 :1565.
`8. O'Reilly MS, Boham T, Shing Y, et al. Endostatin:
`an endogenous inhibitor of angiogenesis and tu-
`mour growth. Cell. 1977;88:277.
`
`9. Bonnet D, Dick JE. Human acute myeloid leuke-
`mia is organized as a hierarchy that originates
`from a primitive hematopoietic cell. Nat Med.
`1997;3:730.
`10. Hudson WA, Li Q, Le C, Kersey JH. Xenotrans-
`plantation of human lymphoid malignancies is
`optimized in mice with multiple immunologic de-
`fects. Leukemia. 1998;12:2029.
`11, Greiner DL, Hesselton RA, Shultz LD. SCIO
`mouse models of human cell engraftment. Stem
`Cells. 1998;16:166.
`12. Philpott NJ, Turner AJC, Scopes J, et al. The use
`of 7-amino actinomycin D in identifying apoptosis:
`simplicity of use and broad spectrum of applica-
`lion compared with other techniques. Blood.
`1996;87:2244.
`13. Thomas DA, Aguayo A, Giles FJ, et al. Thalido-
`mide anti-angiogenesis therapy in Philadelphia-
`negative myeloproliferative disorders and myelo-
`fibrosis [abstract]. Blood. 1999;94:702a.
`14, Raza A, Lisak L, Andrews C, et al. Thalidomide
`produces transfusion independence in patients
`with long-standing refractory anemias and myelo-
`dysplastic syndromes [abstract]. Blood. 1999;94:
`661a.
`15, Bertolini F, Paolucci M, Peccatori F, et al. Angio-
`genie growth factors and endostatin in non-
`
`Hodgkin's lymphoma. BrJ Haematol. 1999;106:
`504.
`16. Salven P, Teerenhovi L, Joensuu H. A high pre-
`treatment serum vascular endothelial growth fac-
`tor concentration is associated with poor outcome
`in non-Hodgkin's lymphoma. Blood.
`1997;90:3167,
`17. Bellamy WT, Richter L, Frutiger Y, Grogan TM.
`Expression of vascular endothelial growth factor
`and its receptors in hematopoietic malignancies.
`Cancer Res. 1999;59:728.
`18, Salven P, Teerenhovi L, Joensuu H. A high pre-
`treatment serum basic-fibroblast growth factor
`concentration is an independent predictor of poor
`prognosis in non-Hodgkin's lymphoma. Blood.
`1999;94:3334.
`19, Singh RK, Bucana CD, Gutman M, Fan D, Wilson
`MR, Fidler IJ, Organ-site dependent expression
`of b-FGF in human renal cell carcinoma cells. Am
`J Pathol. 1994;145:365.
`20. Harris AL. Antiangiogenesis for cancer therapy.
`Lancet 1997;349(suppl 11):13.
`21. Fortier AH , Fogler WE, Tomszewski JE, et al. Re-
`combinant human endostatin protein in cynomol-
`gus monkey produces no toxicological effects fol-
`lowing iv administration for 28 consecutive days
`[abstract]. Clin Cancer Res. 1999;5:3813s.
`
`NOVARTIS EXHIBIT 2076
`Breckenridge v. Novartis, IPR 2017-01592
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