Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-12-03
2004-02-17
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C436S064000, C424S009100, C530S387500, C435S007230
Reexamination Certificate
active
06693177
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel process for the preparation of a biomarker which is an antibody for O-acetylated sialic acid and useful for the diagnosis, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia. More particularly, the present invention relates to a novel process for the preparation of O-acetylated sialic protein immunoglobin useful as a biomarker for the diagnosis of acute lymphoblastic leukemia.
BACKGROUND OF THE INVENTION
Leukemia is a heterogeneous group of neoplastic cells arising from the malignant transformation of hematopoietic (i.e. blood forming) cells. Leukemia can be broadly classified according to the cell type involved myeloid or lymphoid and as acute and chronic depending on the natural history of the disease. Acute lymphoblastic leukemia (ALL) is the commonest type of leukemnia in children and adolescents. It occurs in all races with a peak incidence in children between 3 and 5 years of age. ALL is diagnosed in 2000-3000 new cases of children in the United States every year, whereas acute myelogenous leukemia is diagnosed in only 500 children and chronic myeloid leukemia in fewer than 100. About 40 million children under the age group of 15 years are affected and nearly about 75% of these have ALL. Pediatric hematopoietic malignancies rank first in cancer incidence and mortality in children and are responsible for roughly 40% of childhood related death.
The causes of leukemia are not known but environmental agents including irradiation, chemical carcinogens, cytogenetic abnormalities and retrovirus infections are known to play an important role in the etiology of leukemia. For instance, individual with occupational radiation exposure, patients receiving radiation therapy or Japanese survivors of the atomic bomb explosions have a predictable and dose related increased incidence of leukemia.
For oncologists, acute lymphoblastic leukemia (ALL) represents a major therapeutic success as this can be achieved in nearly 65% of patients (Pui C H, Crist W M Jr. Current Opinion in Oncology, Vol 7, p 36, 1995 and Pui C H, Crist W M Jr. Lancet, Vol 347, p1783, 1996). However, relapse and eventually treatment failure occurs in many cases receiving identical treatment and this area is a major challenge for leukemia specialists (Pui C H, Crist W M Jr. New England Journal of Medicine, Vol 332, p1618, 1995).
At the time of diagnosis, the leukemic cell mass is usually between 10
11
-10
12
cells and available chemotherapeutic agents produce a fractional cell kill capable of 3 to 5 log kill resulting in the elimination of 99.99 to 99.999% of leukemic cells. The persistence of the remaining 0.01 to 0.001% leukemic cells tantamounts to the persistence of 10
8
to 10
9
cells respectively (Champlin and Golde, Harrison Textbook of Internal Medicine pg 1552). What is important is these persisting leukemic cells are not detectable by standard morphology in bone marrow or peripheral blood. It is these cells that are responsible for relapse, if post induction chemotherapy fails to eradicate them. To eliminate this non-detectable yet existing leukemic cell mass, maintenance therapy is given for an extended period (2-2.5 years) for preventing relapse by eradicating this “ice berg” like leukemic cell and reducing and possibly eradicating blast cells. This “ice berg” is conventionally addressed as “Minimal Residual Disease” (MRD) (Knechtli et. Al. J. Clin. Path. 48,1995). MRD is defined as the presence of leukemic cells not detectable by morphology. Despite advances in the treatment of childhood ALL, the risk of relapse remains about 30 percent as patients in remission may harbor residual leukemic blasts, the cause of disease persistence and resurgence is referred to as minimal residual disease, MRD. If the detection of residual leukemia is to be used in clinical practice, the analysis should be performed as early as possible and the laboratory technique should be simple and rapid so that treatment can be tailored to the adjusted assessment of risk. Assays to detect residual blast cells is the need of the hour as these will help the clinician to assess the effect of treatment on tumor burden and allow anticipation of relapse with greater precision (Brisco M J, Condan J, Hughes E, et al. Lancet, Vol 343, p196, 1994).
Currently no specific marker is available to pinpoint the dose of chemotherapy and duration of maintenance therapy in acute lymphoblastic leukemia. Existing methods for the detection of leukemia blast cells are i) cytomorphology and karyotyping ii) immunological methods iii) molecular detection.
Cytomorphology and karyotyping: Acquired non-random chromosomal translocation occur in 30-70% of ALL patients and can serve as marker of disease. But the approach has limited sensitivity (1-5%) primarily due to the paucity of leukemic cells during clinical remission (Campana D, Pui C H, Blood, Vol 85, p1416, 1995). Fluorescent in situ hybridization using chromosome specific or locus specific probes allow identification of abnormalities in cells at metaphase (Le Beau M M, Blood, Vol 81, p1979, 1993). However, sensitivity remains at 1% level.
Immunological Methods: Immunological methods based on the recognition of leukemia associated phenotypes not usually found in normal bone marrow have had promising results (Cole et al. Baillieres din Haematol. 7, 183, 1994). Immunophenotyping has been complemented by flow cytometric analysis where a combination of markers have been able to quantify MRD with a sensitivity usually in the order of 10
2
to 10
3
which is inferior to available DNA based method (Hun & Andreef 8, 713, 1994).
Molecular approaches: In majority of the cases, relapse of acute lymphoblastic leukemia is thought to involve the same leukemic clone as the original disease (Bunin N J et al, Leukemia, Vol 4, p727, 1990). Around 80% of cases of childhood acute lymphoblastic leukemia are due to clonal expansion of precursor B cells and have rearrangement of lgH gene, from which specific DNA probes have been generated. Several PCR methods (Brisco M J. Condan J, Hughes E, Lancet, Vol 343, p196, 1994: Veelken H. Tyeko B. Sklar J., Blood. Vol 78, p1318, 1991; Wasserman R. Galili N. I to J. et al., Journal of Clinical Oncology, Vol 10, p1879, 1992) have been reported for detection of MRD in childhood acute lymphoblastic leukemia. This technique detects leukemia specific DNA sequences such as fusion regions of immunoglobulin (Ig) and TcR genes with a sensitivity of 10
5
for the detection of residual disease in childhood acute lymphoblastic leukemia. All these published methods are successful in only half of the patients since different individuals show different rearrangement of immuloglobulin genes or T cell receptor genes.
The principal drawbacks of the PCR methods for routine follow up of the patients are (i) occurrence of false positive results due to contamination of reaction mix with previously employed samples (ii) occurrence of false negative results owing to degraded RNA or DNA or clonal evolution in approximately 20% of cases (iii) not all leukemic specific gene rearrangements are amenable to initial amplification of PCR using universal primers (iv) a heterogeneous distribution of residual leukemic cells may result in sampling error since gene rearrangements may be different in different individuals (v) these methods are costly, lengthy and sophisticated requiring technical expertise. Therefore, the reliability of PCR assays depends on the use of stringent quantitation protocols and analysis of multiple genetic targets to prevent false negative results due to changes in the pattern of gene rearrangement during the disease course. These mandatory technical requirements further complicate an already laborious procedure and also make it expensive thereby limiting its suitability for routine clinical use. Under these circumstances, use of a novel biomarker effectively will serve as an index to reflect the clinical status of individual patients following therapy.
Since the determination of PCR undetectable r
Chatterjee Mitali
Mandal Chitra
Pal Santanu
Caputa Anthony C.
Jain, Esq. Mishrilal
Nickol Gary B.
Nirmel & Associates
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