~ Ch.eek for updates]
`
`Downloaded from http://ashpublications.org/blood/article-pdf/116/20/4099/1489743/zh804610004099.pdf by guest on 12 January 2022
`
`GENE THERAPY
`
`Brief report
`Eradication of B-lineage cells and regression of lymphoma in a patient treated
`with autologous T cells genetically engineered to recognize CD19
`James N. Kochenderfer,1 Wyndham H. Wilson,2 John E. Janik,2 Mark E. Dudley,1 Maryalice Stetler-Stevenson,3
`Steven A. Feldman,1 Irina Maric,4 Mark Raffeld,3 Debbie-Ann N. Nathan,1 Brock J. Lanier,1 Richard A. Morgan,1 and
`Steven A. Rosenberg1
`
`1Surgery Branch, 2Metabolism Branch, and 3Laboratory of Pathology, National Cancer Institute, Bethesda, MD; and 4Department of Laboratory Medicine,
`Clinical Center, National Institutes of Health, Bethesda, MD
`
`Adoptive transfer of genetically modified
`T cells is an attractive approach for gener-
`ating antitumor immune responses. We
`treated a patient with advanced follicular
`lymphoma by administering a preparative
`chemotherapy regimen followed by au-
`tologous T cells genetically engineered
`to express a chimeric antigen receptor
`(CAR) that recognized the B-cell antigen
`CD19. The patient’s lymphoma under-
`went a dramatic regression, and B-cell
`Introduction
`
`precursors were selectively eliminated
`from the patient’s bone marrow after infu-
`sion
`of
`anti–CD19-CAR-transduced
`T cells. Blood B cells were absent for at
`least 39 weeks after anti–CD19-CAR-
`transduced T-cell infusion despite prompt
`recovery of other blood cell counts. Con-
`sistent with eradication of B-lineage cells,
`serum immunoglobulins decreased to
`very low levels after treatment. The pro-
`longed and selective elimination of B-
`
`lineage cells could not be attributed to
`the chemotherapy that the patient re-
`ceived and indicated antigen-specific
`eradication of B-lineage cells. Adoptive
`transfer of anti–CD19-CAR-expressing
`T cells is a promising new approach for
`treating B-cell malignancies. This study
`is registered at www.clinicaltrials.gov as
`#NCT00924326.
`(Blood. 2010;116(20):
`4099-4102)
`
`T cells can be genetically modified to express chimeric antigen
`receptors (CARs).1-5 CARs consist of an antigen-recognition
`moiety, such as antibody-derived, single-chain variable fragments,
`coupled to T-cell activation domains.1-4 T cells have been geneti-
`cally engineered to express CARs that can recognize a variety of
`tumor-associated antigens, including the B-lineage antigen CD19,
`in a non-human leukocyte antigen-restricted manner.4-15 Expres-
`sion of the cell-surface protein CD19 is restricted to normal mature
`B cells, malignant B cells, B-cell precursors, and plasma cells.16-19
`We have designed a CAR that targets CD19 and initiated a clinical
`trial of autologous T cells expressing this CAR (www.clinicaltrials.
`gov; #NCT00924326).
`
`Methods
`
`This clinical trial was approved by the National Cancer Institute Institu-
`tional Review Board. Design and construction of the mouse stem cell
`virus-based splice-gag retroviral vector MSGV-FMC63-28Z encoding the
`anti-CD19 CAR used in our clinical trial have been described (GenBank
`HM852952).7 The anti-CD19 CAR contains an antigen-recognition
`moiety consisting of the variable regions of the FMC63 monoclonal
`antibody joined to part of the CD28 molecule and the signaling domains
`of the CD3␨ molecule.
`Peripheral blood mononuclear cells were transduced with retroviruses
`encoding the anti-CD19 CAR and cultured in an almost identical manner as
`previously described.20 As measured by flow cytometry, the CAR was
`expressed on 64% of the infused cells, which were 98% CD3⫹ T cells
`(supplemental Figure 1, available on the Blood Web site; see the Supplemen-
`
`tal Materials link at the top of the online article). The T cells were 66%
`CD8⫹ and 34% CD4⫹. The anti–CD19-CAR-transduced T cells specifi-
`cally recognized CD19⫹ target cells (supplemental Table 1). Methods of
`T-cell preparation, flow cytometry, polymerase chain reaction, and immuno-
`histochemistry are in the supplemental data. For the immunohistochemistry
`images in Figures 1 and 2, images were obtained via digital microscopy
`using an Olympus BX51 microscope (Olympus America) equipped with a
`UPlanFL 10⫻/0.3 numeric aperture and UPlanFL 40⫻/0.75 numeric aperture
`objectives. Images were captured using an Olympus DP70 digital camera system.
`Imaging software was Adobe Photoshop CS3 (Adobe Systems).
`
`Results and discussion
`
`The patient was diagnosed with grade 1, stage IVB follicular
`lymphoma in 2002. Before enrollment on our protocol, he had
`received the following treatments for his lymphoma: PACE (pred-
`nisone, doxorubicin, cyclophosphamide, and etoposide), an idio-
`type vaccine, the anti–CTLA-4 monoclonal antibody ipilimumab,
`and EPOCH-R (etoposide, prednisone, vincristine, cyclophospha-
`mide, doxorubicin, and rituximab). The last cycle of EPOCH-R
`was administered in January 2008. The EPOCH-R caused a partial
`remission; however, progressive disease was noted in July 2008.
`The patient received no further treatment before he was evaluated
`for enrollment on our trial of anti–CD19-CAR-transduced T cells.
`When we evaluated the patient in May 2009, he had progressive
`lymphoma that involved all major lymph node areas (Figure 1A).
`He had bilateral pleural effusions, night sweats, and a recent weight
`loss of 10 pounds. Flow cytometry of a fine needle aspirate from an
`
`Submitted April 23, 2010; accepted July 20, 2010. Prepublished online as
`Blood First Edition paper, July 28, 2010; DOI 10.1182/blood-2010-04-281931.
`
`The online version of this article contains a data supplement.
`
`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 USC section 1734.
`
`BLOOD, 18 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 20
`
`4099
`
`Miltenyi Ex. 1045 Page 1
`
`

`

`4100
`
`KOCHENDERFER et al
`
`BLOOD, 18 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 20
`
`A
`
`18 weeks after treatment
`
`Downloaded from http://ashpublications.org/blood/article-pdf/116/20/4099/1489743/zh804610004099.pdf by guest on 12 January 2022
`
`Figure 1. B-lineage cells, including B-cell precur-
`sors, were eradicated from the bone marrow after
`treatment with anti–CD19-CAR-transduced T cells.
`(A) Representative pretreatment computed tomography
`scan images and images from 18 weeks after treatment
`demonstrate regression of
`lymphoma masses in the
`chest and abdomen after treatment with chemotherapy
`followed by anti–CD19-CAR-transduced T cells plus IL-2.
`(B) Flow cytometric evaluation of a pretreatment bone
`marrow aspirate was conducted with a forward versus
`side light scatter analysis gate of lymphoid cells. The left
`upper quadrant contains CD19⫹ B-lineage cells (35% of
`lymphoid cells), and the right lower quadrant contains
`CD3⫹ T cells. (C) Flow cytometric evaluation of a pretreat-
`ment bone marrow aspirate with a CD19⫹ analysis gate
`is shown. ␬- and ␭-negative, CD19⫹, mostly immature
`B-lineage cells that are not part of the malignant lym-
`phoma clone are in the rectangle. The cells outside the
`rectangle are mostly lymphoma cells. (D) Flow cytomet-
`ric evaluation of a pretreatment bone marrow aspirate
`with a forward versus side light scatter analysis gate of
`lymphoid cells. Immature B-cell precursors in the oval
`are CD22⫹ and CD20⫺. (E) Flow cytometric evaluation
`of a pretreatment bone marrow aspirate with a forward
`versus side light scatter analysis gate of lymphoid cells.
`Immature B-cell precursors in the polyhedral demon-
`strate decreasing CD10 correlating with increasing CD20
`expression. (F) Flow cytometric evaluation of a bone
`marrow aspirate from 36 weeks after treatment with a
`forward versus side light scatter analysis gate of lym-
`phoid cells. CD19⫹ B-lineage cells are absent.
`(G) Immunohistochemistry staining of a pretreatment
`bone marrow biopsy reveals a large population of CD19⫹
`cells that includes lymphoma cells as well as nonmalig-
`nant B-lineage cells. (H) Immunohistochemistry staining
`of a bone marrow biopsy from 36 weeks after infusion of
`anti–CD19-CAR-transduced T cells demonstrates a com-
`plete absence of CD19⫹ cells. (I) High-power view of the
`same anti-CD19 staining shown in panel H.
`
`B
`
`a,
`,-
`C
`
`(.) -
`
`..
`~!· ·:
`CD3
`
`,..,• • ·
`
`E
`
`C
`,-
`C
`(.)
`
`~·: :
`
`H CD19
`
`,..
`
`. . :,,:9f.;:
`•
`'l(:· ·-,i~~1
`Kappa
`
`CD20
`
`C
`
`I'll
`'tJ
`.0
`E
`I'll
`.J
`
`F
`
`O>
`,-
`C
`(.)
`
`CD3
`
`' -
`
`enlarged cervical lymph node demonstrated a monoclonal B-cell
`process consistent with follicular lymphoma that uniformly ex-
`pressed CD19, CD20, CD22, CD10, and IgM-kappa. Flow cytom-
`etry showed that 14.5% of the blood lymphoid cells had a
`phenotype that was consistent with the lymphoma and 0.7% of the
`blood lymphoid cells were normal polyclonal B cells (data not
`shown). Before treatment, 35% of bone marrow lymphoid cells
`expressed CD19 (Figure 1B). A total of 55% of these CD19⫹ cells
`were monoclonal ␬-positive and ␭-negative lymphoma cells; 45%
`of the bone marrow CD19⫹ cells were normal surface-immuno-
`globulin (Ig)–negative immature B-cell precursors (Figure 1C).
`The immature B-cell precursors demonstrated a pattern of antigen
`expression consistent with normal maturation, namely, CD22⫹
`B cells with decreasing CD10 expression correlating with increas-
`ing CD20 expression (Figure 1D-E).21,22 Large numbers of bone
`marrow CD19⫹ cells and CD79a⫹ cells were detected by immuno-
`histochemistry before treatment (Figures 1G, 2A).
`The patient underwent apheresis, and peripheral blood
`mononuclear cells were used to prepare anti–CD19-CAR-
`transduced T cells. The patient received a lymphocyte-depleting
`regimen consisting of 60 mg/kg cyclophosphamide daily for
`2 days followed by 5 daily doses of 25 mg/m2 fludarabine. The
`
`day after the last fludarabine dose, the patient received 1 ⫻ 108
`anti–CD19-CAR-transduced T cells intravenously. The next
`day, he received 3 ⫻ 108 anti–CD19-CAR-transduced T cells intra-
`venously. After the second anti–CD19-CAR-transduced T-cell
`infusion, the patient received 720 000 IU/kg interleukin-2 (IL-2)
`intravenously every 8 hours. Eight doses of IL-2 were adminis-
`tered. The only acute toxicities that the patient experienced were
`cytopenias that were attributable to chemotherapy and a fever that
`lasted 2 days (maximum temperature, 38.5°C). The patient was
`discharged 11 days after his second anti–CD19-CAR-transduced
`T-cell infusion, and he resumed full-time employment.
`After therapy, computed tomography scans revealed an impres-
`sive partial remission of the lymphoma that lasted 32 weeks (Figure
`1A); 32 weeks after treatment, progressive CD19⫹ lymphoma was
`noted in right cervical and retroperitoneal lymph nodes.
`Blood B cells were absent from 9 weeks after anti–CD19-CAR-
`transduced T-cell infusion until at least 39 weeks after anti–CD19-CAR-
`transduced T-cell infusion (Figure 2C; supplemental Figure 2). This
`prolonged B-cell depletion cannot be attributed to the chemotherapy that
`the patient received. Neither the New York esophageal squamous cell
`carcinoma antigen-1 (NY-ESO) nor the melanoma antigen gp100 is
`expressed by B cells.23,24 In prior clinical trials, patients treated with the
`
`Miltenyi Ex. 1045 Page 2
`
`

`

`BLOOD, 18 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 20
`
`ERADICATION OF B-LINEAGE CELLS
`
`4101
`
`A
`
`C1'79a
`
`B
`
`D
`
`Figure 2. Prolonged B-cell depletion after anti–CD19-
`CAR-transduced T-cell infusion. (A) Immunohistochem-
`istry staining of a pretreatment bone marrow biopsy
`shows a large population of CD79a⫹ cells. (B) Thirty-six
`weeks after anti–CD19-CAR-transduced T-cell infusion,
`rare CD79a⫹ cells were detected by immunohistochem-
`isty staining of a bone marrow biopsy. The cells did not
`appear to be plasma cells morphologically. The number
`of CD79a⫹ cells was substantially below normal limits.
`the rare CD79a⫹ cells.
`The arrow indicates one of
`(C) The blood B-cell count of the patient treated with
`anti–CD19-CAR-transduced T cells is shown before treat-
`ment and at multiple time points after treatment. B cells
`were measured by flow cytometry for CD19. The dashed
`line indicates the lower limit of normal. Day 0 is the day of
`the second anti–CD19-CAR-transduced T-cell infusion.
`(D) The mean ⫾ SEM blood B-cell count is shown for
`patients who received infusions of T cells targeted to
`either the NY-ESO antigen or the gp100 antigen. The
`patients all received the same chemotherapy and IL-2
`regimen as the patient who received anti–CD19-CAR-
`transduced T cells. NY-ESO and gp100 are not ex-
`pressed by B cells. Day 0 is the day of T-cell infusion. All
`available B-cell counts were included for each time point
`(pretreatment, n ⫽ 28; 4-5 weeks after T-cell
`infusion,
`n ⫽ 29; 8-11 weeks after T-cell
`infusion, n ⫽ 31;
`14-19 weeks after T-cell infusion, n ⫽ 20). All patients
`with available samples had a B-cell count in the normal
`range by 14 to 19 weeks after T-cell infusion. (E) The
`blood CD3⫹ T-cell count of
`the patient
`treated with
`anti–CD19-CAR-transduced T cells is shown before treat-
`ment and at multiple time points after treatment. (F) The
`blood NK cell count of the patient treated with anti–CD19-
`CAR-transduced T cells is shown before treatment and
`at multiple time points after treatment. NK cells were
`measured by flow cytometry as CD3⫺, CD16⫹, CD56⫹
`cells. (E-F) Day 0 is the day of the second anti–CD19-
`CAR-transduced T-cell
`infusion, and the dashed line
`indicates the lower limit of normal. (G) The serum IgG
`level of the patient treated with anti–CD19-CAR-trans-
`duced T cells is shown before treatment and at multiple
`time points after treatment. Day 0 is the day of the second
`anti–CD19-CAR-transduced T-cell
`infusion. (H) Real-
`time polymerase chain reaction was performed with a
`primer and probe set that was specific for the anti-CD19
`CAR. Anti–CD19-CAR-transduced T cells were undetect-
`able in pretreatment blood samples. The anti–CD19 CAR
`transgene was detected in the peripheral blood of the
`patient who received anti–CD19-CAR-transduced T cells
`from 1 to 27 weeks after anti–CD19-CAR-transduced
`T-cell infusion.
`
`C
`
`E
`
`G
`
`Downloaded from http://ashpublications.org/blood/article-pdf/116/20/4099/1489743/zh804610004099.pdf by guest on 12 January 2022
`
`40
`24 32
`16
`8
`0
`Weeks after T cell infusion
`
`3000
`...1 2500
`-¢2000
`~ 1500
`o 1000
`I- 500
`o......,---r--~---..-----r--,
`0
`16
`24
`32
`40
`8
`Weeks after T cell infusion
`
`F
`
`250
`
`~200
`~ 150
`ai
`O 100
`::.::
`Z 50
`01-'---i----r--,--,------,---,
`0
`16
`24
`32
`40
`8
`Weeks after T cell infusion
`
`H
`
`i~ 150
`
`00
`0 c,1
`Cl> C
`co
`~o
`
`1/) -C ,._
`11:1 Cl)
`... 0.
`I-
`
`27
`18
`9
`1 4
`Weeks after T cell Infusion
`
`Weeks after T cell infusion
`
`same chemotherapy and IL-2 regimen as the patient described in this
`report along with T cells retrovirally transduced with receptors that
`recognized either NY-ESO or gp100 did not experience prolonged
`B-cell depletion (Figure 2D).
`Except for B cells and a mild thrombocytopenia, all blood cell
`counts, including neutophils, erythrocytes, T cells, and NK cells, of
`the patient treated with anti–CD19-CAR-transduced T cells recov-
`ered to normal levels by 9 weeks after treatment (Figure 2E-F).
`Thirty-six weeks after anti–CD19-CAR-transduced T cells were
`infused, CD19⫹ cells were absent from the bone marrow as measured
`by flow cytometry (Figure 1F) and immunohistochemistry (Figure
`1H-I). CD79a⫹ cells were undetectable in the bone marrow by
`immunohistochemistry 14 weeks after treatment (data not shown).
`CD79a⫹ cells were detected at greatly below normal frequency 36
`weeks after anti–CD19-CAR-transduced T-cell infusion (Figure 2B).
`CD79a is expressed earlier in B-cell development than CD19,25 so the
`presence of a small number of CD79a⫹ cells while CD19⫹ cells were
`absent suggests early recovery of B-lineage cells.
`A decrease in serum IgG levels occurred after treatment (Figure
`2G). Serum IgM was undetectable from 9 to at least 39 weeks after
`treatment. Serum IgA was 66.8 mg/dL before treatment. Serum IgA
`decreased to below the detectable limit of 10 mg/dL after treatment
`(supplemental Figure 3). Five months after treatment, the patient
`
`developed pneumonia of unknown etiology that required hospital-
`ization. After a course of antibiotics, the patient recovered com-
`pletely. The patient has subsequently received intravenous Ig
`replacement, and he has not had further infections.
`The anti-CD19 CAR transgene was detected in peripheral blood
`mononuclear cells from one to 27 weeks after anti–CD19-CAR-
`transduced T-cell infusion with a quantitative real-time polymerase
`chain reaction assay (Figure 2H).
`This is the first patient treated on our trial and the only patient
`with long enough follow-up to evaluate B-cell depletion. The
`prolonged elimination of CD19⫹ cells in this patient indicates in
`vivo antigen-specific activity of anti–CD19-CAR-expressing T cells.
`Our findings should encourage continued study of anti–CD19-CAR-
`transduced T cells.
`
`Acknowledgments
`
`The authors thank Margaret Brown for flow cytometry, Manuel Van
`Deventer for Ig assays, Laura Devillier for T-cell preparation,
`and Hui Xu, Mary Black, and Zhili Zheng for
`technical
`assistance.
`
`Miltenyi Ex. 1045 Page 3
`
`

`

`Downloaded from http://ashpublications.org/blood/article-pdf/116/20/4099/1489743/zh804610004099.pdf by guest on 12 January 2022
`
`4102
`
`KOCHENDERFER et al
`
`BLOOD, 18 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 20
`
`This work was supported by the Center for Cancer Research,
`National Cancer Institute, National Institutes of Health (intramural
`funding).
`
`Authorship
`
`Contribution: J.N.K. designed the protocol, provided patient care,
`conducted experiments, analyzed data, and wrote the paper;
`W.H.W., J.E.J., D.-A.N.N., and B.J.L. provided patient care,
`
`assisted protocol design, and edited the paper; S.A.F. and R.A.M.
`provided reagents and interpreted data; M.E.D. conducted experi-
`ments and edited the paper; M.S.-S., I.M., and M.R. conducted
`experiments, interpreted data, and edited the paper; and S.A.R.
`designed the protocol, interpreted data, and edited the paper.
`Conflict-of-interest disclosure: The authors declare no compet-
`ing financial interests.
`Correspondence: James N. Kochenderfer, National Institutes of
`Health, 10 Center Dr, CRC Rm 3-3888, Bethesda, MD 20892;
`e-mail: kochendj@mail.nih.gov.
`
`References
`
`3.
`
`1. Eshhar Z, Waks T, Gross G, Schindler DG. Spe-
`cific activation and targeting of cytotoxic lympho-
`cytes through chimeric single chains consisting of
`antibody-binding domains and the gamma or zeta
`subunits of the immunoglobulin and T-cell recep-
`tors. Proc Natl Acad Sci U S A. 1993;90(2):
`720-724.
`2. Kershaw MH, Teng MWL, Smyth MJ, Darcy PK.
`Supernatural T cells: genetic modification of
`T cells for cancer therapy. Nat Rev. 2005;5(12):
`928-940.
`Irving BA, Weiss A. The cytoplasmic domain of
`the T cell receptor zeta chain is sufficient to
`couple to receptor-associated signal transduction
`pathways. Cell. 1991;64(5):891-901.
`4. Sadelain M, Brentjens R, Riviere I. The promise
`and potential pitfalls of chimeric antigen recep-
`tors. Curr Opin Immunol. 2009;21(2):215-223.
`5. Vera JF, Brenner MK, Dotti G. Immunotherapy of
`human cancers using gene modified T lympho-
`cytes. Curr Gene Ther. 2009;9(5):396-408.
`6. Kowolik CM, Topp MS, Gonzalez S, et al. CD28
`costimulation provided through a CD19-specific
`chimeric antigen receptor enhances in vivo per-
`sistence and antitumor efficacy of adoptively
`transferred T cells. Cancer Res. 2006;66(22):
`10995-11004.
`7. Kochenderfer JN, Feldman SA, Zhao Y, et al.
`Construction and preclinical evaluation of an anti-
`CD19 chimeric antigen receptor. J Immunother.
`2009;32(7):689-702.
`8. Lamers CH, Langeveld SC, Groot-van Ruijven CM,
`Debets R, Sleijfer S, Gratama JW. Gene-modified
`T cells for adoptive immunotherapy of renal cell can-
`cer maintain transgene-specific immune functions in
`vivo. Cancer Immunol Immunother. 2007;56(12):
`1875-1883.
`9. Hwu P, Yang JC, Cowherd R, et al. In vivo antitu-
`mor activity of T cells redirected with chimeric an-
`tibody/T-cell receptor genes. Cancer Res. 1995;
`55(15):3369-3373.
`
`10. Brentjens RJ, Latouche JB, Santos E, et al.
`Eradication of systemic B-cell tumors by geneti-
`cally targeted human T lymphocytes co-stimu-
`lated by CD80 and interleukin-15. Nat Med. 2003;
`9(3):279-286.
`11. Till BG, Jensen MC, Wang J, et al. Adoptive im-
`munotherapy for indolent non-Hodgkin lymphoma
`and mantle cell lymphoma using genetically
`modified autologous CD20-specific T cells.
`Blood. 2008;112(6):2261-2271.
`12. Pule MA, Savoldo B, Myers GD, et al.
`Virus-specific T cells engineered to coexpress
`tumor-specific receptors: persistence and antitu-
`mor activity in individuals with neuroblastoma.
`Nat Med. 2008;14(11):1264-1270.
`13. Milone MC, Fish JD, Carpenito C, et al. Chimeric
`receptors containing CD137 signal transduction
`domains mediate enhanced survival of T cells
`and increased antileukemic efficacy in vivo. Mol
`Ther. 2009;17(8):1453-1464.
`14. Brentjens RYR, Bernal Y, Riviere I, Sadelain M.
`Treatment of chronic lymphocytic leukemia with
`genetically targeted autologous T cells: a case
`report of an unforeseen adverse event in a phase
`I clinical trial. Mol Ther. 2010;18(4):666-668.
`15. Jensen MC, Popplewell L, Cooper LJ, et al. Anti-
`transgene rejection responses contribute to at-
`tenuated persistence of adoptively transferred
`CD20/CD19-specific chimeric antigen receptor
`redirected T cells in humans. Biol Blood Marrow
`Transplant. 2010;16(9):1245-1256.
`16. Nadler LM, Anderson KC, Marti G, et al. B4, a
`human B lymphocyte-associated antigen ex-
`pressed on normal, mitogen-activated, and malig-
`nant B lymphocytes. J Immunol. 1983;131(1):
`244-250.
`17. Pontvert-Delucq S, Breton-Gorius J, Schmitt C, et
`al. Characterization and functional analysis of
`adult human bone marrow cell subsets in relation
`to B-lymphoid development. Blood. 1993;82(2):
`417-429.
`
`18. Uckun FM, Jaszcz W, Ambrus JL, et al. Detailed
`studies on expression and function of CD19 sur-
`face determinant by using B43 monoclonal anti-
`body and the clinical potential of anti-CD19 immu-
`notoxins. Blood. 1988;71(1):13-29.
`
`19. Harada H, Kawano MM, Huang N, et al. Pheno-
`typic difference of normal plasma cells from ma-
`ture myeloma cells. Blood. 1993;81(10):2658-
`2663.
`
`20. Johnson LA, Morgan RA, Dudley ME, et al. Gene
`therapy with human and mouse T-cell receptors
`mediates cancer regression and targets normal
`tissues expressing cognate antigen. Blood. 2009;
`114(3):535-546.
`
`21. van Lochem EG, van der Velden VHJ, Wind HK,
`te Marvelde JG, Westerdaal NAC, van Dongen
`JJM. Immunophenotypic differentiation patterns
`of normal hematopoiesis in human bone marrow:
`reference patterns for age-related changes and
`disease-induced shifts. Cytometry B Clin Cytom.
`2004;60(1):1-13.
`
`22. Lucio P, Parreira A, van den Beemd MW, et al.
`Flow cytometric analysis of normal B cell differen-
`tiation: a frame of reference for the detection of
`minimal residual disease in precursor-B-ALL.
`Leukemia. 1999;13(3):419-427.
`
`23. Kawakami Y, Eliyahu S, Delgado CH, et al. Identi-
`fication of a human melanoma antigen recog-
`nized by tumor-infiltrating lymphocytes associ-
`ated with in vivo tumor rejection. Proc Natl Acad
`Sci U S A. 1994;91(14):6458-6462.
`
`24. Chen YT, Scanlan MJ, Sahin U, et al. A testicular
`antigen aberrantly expressed in human cancers
`detected by autologous antibody screening. Proc
`Natl Acad Sci U S A. 1997;94(5):1914-1918.
`
`25. Blom B, Spits H. Development of human lym-
`phoid cells. Annu Rev Immunol. 2006;24:
`287-320.
`
`Miltenyi Ex. 1045 Page 4
`
`