Leukemia (2000) 14, 474–475
`2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00
`www.nature.com/leu
`
`Monoclonal antibodies to the myeloid stem cells: therapeutic implications of CMA-
`676, a humanized anti-CD33 antibody calicheamicin conjugate
`ID Bernstein
`
`Fred Hutchinson Cancer Research Center and the University of Washington School of Medicine, Seattle, WA, USA
`
`There are several competing models of stem cell involvement
`in acute myeloid leukemia (AML). At issue is whether the dis-
`ease origin is in the pluripotent stem cell or whether it arises
`later in a more mature progenitor cell. The observation that the
`CD33 antigen is present on AML cells, and on normal and leu-
`kemic progenitors, suggested that one might be able to target
`these cells while sparing the normal stem cells. Response rates
`of acute myelogenous leukemia patients treated with the newly
`developed anti-CD33 antibody-calicheamicin conjugate sug-
`gest that at least for a proportion of patients early precursors
`responsible for re-establishing hematopoiesis are likely to be
`predominantly normal in origin. Leukemia (2000) 14, 474–475.
`Keywords: AML; CD33; calicheamicin immunoconjugate
`
`In the past few decades, there has been great interest in defin-
`ing antigens on the surface of normal and malignant myeloid
`stem cells and their progeny. In early hematopoiesis, a pluri-
`potent stem cell is thought to give rise to precursors committed
`to the various myeloid lineages, including precursors of gra-
`nulocytes, monocytes, erythrocytes, and platelets. These early
`precursors, including stem cells, are marked by the CD34 anti-
`gen. The CD33 antigen, which is on progenitors and commit-
`ted precursors, but not on stem cells, is also on virtually all
`acute myelogenous leukemia (AML) cells. This observation
`raised the possibility of whether CD33 leukemic cells could
`be targeted and then deleted along with the leukemic cell pre-
`cursors, as well as normal CD33-bearing cells, while sparing
`the normal stem cells.
`Research on which lineages are involved in the leukemic
`process in patients with AML was studied by evaluating
`females heterozygous for the X-linked gene, G6PD.1–3 In nor-
`mal cells, about half the cells expressed one G6PD type, and
`the other half the other type. A single G6PD type was found
`in the blast cells, demonstrating their clonal origin, and for
`most patients with AML, in the granulocyte/monocyte lineage,
`but not in other lineages. This could suggest the possibility
`that the precursor of this granulocyte and monocyte-restricted
`disease was a committed granulocyte/monocyte precursor. If
`not, it originated in early cells, but expansion of the precursors
`occurred only once they were committed to this lineage. If
`this were the case, then by ablating these cells, the remaining
`CD33- cells could be predominantly or even completely
`normal in origin.
`This process is in contrast to chronic myelogenous leuke-
`mia, in which similar studies demonstrated that the disease
`originates in a pluripotent stem cell.4,5 There is other evidence
`as well that for a small proportion of patients, particularly the
`elderly, a multipotent precursor may be involved in AML.3
`Nonetheless,
`in research focused on patients with disease
`restricted to the granulocyte/monocyte lineage,1 the CD33-
`
`ID Bernstein, Fred Hutchinson Cancer Research
`Correspondence:
`Center, 1100 Fairview Avenue North, C1-169, Seattle, WA 98109,
`USA
`Received 26 September 1999; accepted 3 October 1999
`
`cells or CD34+/CD33- cells of these patients were placed in
`long-term marrow culture and grown on an irradiated marrow
`stromal cell feeder layer. Selected cells were then grown in
`colony-forming assays and studied for their clonal or non-
`clonal origin. It was found that a proportion of patients grew
`CFU-GM that were polyclonal and thus predominantly or
`completely normal in origin, supporting the possibility that
`ablation of CD33- cells could eliminate leukemia and leu-
`kemic precursors, while allowing the remaining normal cells
`to establish normal hematopoiesis.
`A number of investigators have challenged this notion. For
`example, Bonnet and Dick6 isolated primitive CD34+/CD38-
`precursors and transplanted them to immunodeficient mice.
`For most patient samples, they found transplanted leukemia
`rather than normal hematopoiesis. This finding led to a com-
`peting model of stem cell involvement in which there is an
`expansion of leukemic stem cells with self-renewal in the
`primitive pool.7 This is in contrast to models at the other
`extreme in which the leukemia may arise in fairly mature pre-
`cursors, and while it may arise in an early precursor, it does
`not expand and dominate hematopoiesis until a later stage in
`precursor development.
`Based on these latter two models, a collaboration was
`established between investigators at Wyeth Ayerst (Radnor,
`PA, USA) and our laboratory to develop a humanized anti-
`CD33 antibody-calicheamicin conjugate (CMA-676) for test-
`ing whether ablation of these cells in vivo would allow primi-
`tive cells to establish normal hematopoiesis (Hamann et al,
`submitted). Calicheamicin is a small molecule, 1000 times
`more potent than doxorubicin, that should be poorly immuno-
`genic, and when attached to antibody should not materially
`affect
`its pharmacokinetics, as seen when attaching larger
`molecules such as ricin. It seems to form radical intermediates
`intracellularly, and is a DNA minor groove binder that causes
`double-strand breaks and apoptosis. The conjugate was able
`to kill HL60 cells in vitro, as well as in two xenografts, and
`was effective in inhibiting the growth of leukemic-forming
`cells in vitro. In preclinical studies, the conjugate was able to
`selectively inhibit HL60 tumor cells in vitro as well as in an
`in vivo xenograft model, but most importantly, it was effective
`in inhibiting the growth of leukemic colony-forming cells in
`vitro. Moreover, other studies have demonstrated that radio-
`labeled anti-CD33 antibody showed very rapid access to leu-
`kemia cells in the marrow and spleen and was rapidly
`internalized by the target cells, suggesting a usefulness for
`drug delivery.8
`A phase I trial was conducted in collaboration with Wyeth
`Ayerst.9 This dose escalation study was conducted to deter-
`mine whether normal hematopoiesis could be restored in
`patients with AML by selective ablation of cells expressing the
`CD33 antigen. Forty patients with refractory or advanced AML
`received up to three doses of CMA-676 at 2-week intervals.
`Elimination of morphologically detectable leukemia occurred
`in 20% of patients. Three patients also completely recovered
`their hematopoietic counts. No significant CNS, cardiac, or
`
`IMMUNOGEN 2148, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:211)
`

`
`frequent adverse
`renal toxicities were observed. The most
`events were fever and chills. In terms of nonhematopoietic
`toxicity, a small number of transient liver abnormalities were
`seen. Prolonged myelosuppression was observed in some of
`the patients, especially after three doses at the higher dose
`levels.
`In subsequent on-going studies, administration was
`limited to two doses. A dose-limiting toxicity was never
`reached; at the highest dose level of 9 mg/m2, the CD33 target
`sites were essentially saturated. This was accepted as the dose
`for further study. Humoral responses to the conjugate were
`limited to two patients. One of
`these patients initially
`responded to CMA-676, later relapsed, and upon retreatment
`with CMA-676 developed an antibody response.
`In an ongoing phase II trial, patients with CD33+ AML in
`their first untreated relapse following a remission of .6
`months were treated with a fixed dose.10 Patients received a
`total of two i.v. infusions of 9 mg/m2 of the conjugate. Prelimi-
`nary results obtained with the 23 evaluable patients showed
`that 43% achieved disappearance of leukemic blasts from
`blood and marrow;
`recovery of
`their granulocyte count
`(.1500/mm3); and were platelet transfusion independent with
`a platelet count .50 000/mm3. However, a delayed platelet
`recovery has been seen in many of these patients. In addition
`to beginning to define response rate, around 38% thus far,
`these data have also confirmed the relatively low toxicity of
`this drug.
`In summary, based on knowledge of stem cell development,
`it has been possible to develop an anti-CD33 antibody-
`calicheamicin conjugate, termed CMA-676. This conjugate
`has been shown to be well tolerated, to saturate target sites
`and, in some patients with AML, to eliminate leukemia in vivo
`without ablation of normal hematopoiesis. The observed
`response rates support the idea that early precursors respon-
`sible for re-establishing hematopoiesis are likely to be pre-
`dominantly normal in origin for many patients with AML.
`
`Monoclonal antibodies to the myeloid stem cells
`ID Bernstein
`
`References
`
`475
`
`1 Bernstein ID, Singer JW, Smith FO et al. Differences in the fre-
`quency of normal and clonal precursors of colony-forming cells in
`chronic myelogenous leukemia and acute myelogenous leukemia.
`Blood 1992; 79: 1811–1816.
`2 Singer JW, Bernstein ID, Davis PC, Walters TR, Raskind WH, Fial-
`kow PJ. The effect of long-term marrow culture on the origin of
`colony-forming cells in acute myeloblastic leukemia: studies of
`two patients heterozygous for glucose-6-phosphate dehydro-
`genase. Leukemia 1988; 2: 148–152.
`3 Fialkow PJ, Singer JW, Raskind WH, Adamson JW, Jacobson RJ,
`Bernstein ID, Dow LW, Najfeld V, Veith R. Clonal development,
`stem-cell differentiation, and clinical remissions in acute non-
`lymphocytic leukemia. New Engl J Med 1987; 317: 468–473.
`4 Fialkow PJ, Jacobson RJ, Singer JW, Sacher RA, McGuffin RW,
`Neefe JR. Philadelphia chromosome (Ph1)-negative chronic mye-
`logenous leukemia (CML): a clonal disease with origin in a multi-
`potent stem cell. Blood 1980; 56: 70–73.
`5 Anastasi J, Feng J, Dickstein JI et al. Lineage involvement by
`BCR/ABL in Ph+ lymphoblastic leukemias: chronic myelogenous
`leukemia presenting in lymphoid blast vs Ph+ acute lymphoblastic
`leukemia. Leukemia 1996; 10: 795–802.
`6 Bonnet D, Dick JE. Human acute myeloid leukemia is organized
`as a hierarchy that originates from a primitive hematopoietic cell.
`Nature Med 1997; 3: 730–737.
`7 Head DR. Revised classification of acute myeloid leukemia.
`Leukemia 1996; 10: 1826–1831.
`8 Appelbaum FR, Matthews DC, Eary JF et al. The use of radio-
`labeled anti-CD33 antibody to augment marrow irradiation prior
`to marrow transplantation for acute myelogenous leukemia. Trans-
`plantation 1992; 54: 829–833.
`9 Sievers EL, Appelbaum FR, Spielberger RT, Forman SJ, Flowers D,
`Smith FO, Shannon-Dorcy K, Berger MS, Bernstein ID. Selective
`ablation of acute myeloid leukemia using antibody-targeted
`chemotherapy: a phase I study of an anti-CD33 calicheamicin
`immunoconjugate. Blood 1999; 93: 3678–3684.
`10 Sievers E, Larson RA, Estey E, Stadtmauer EA, Roy D-CC, Spiel-
`berger R, Tarantolo S, Berger MS, Eten C, Manley L, Bernstein I,
`Appelbaum F. Preliminary results of the efficacy and safety of
`CMA-676 in patients with AML in first relapse. Proc Am Soc Clin
`Oncol 1999; 18: 7a (Abstr.).
`
`Leukemia
`
`IMMUNOGEN 2148, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676