Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-18
2002-09-17
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S387300, C530S388850
Reexamination Certificate
active
06451995
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to single chain antibody constructs which specifically bind to the disialoganglioside G
D2
, and to the use of such constructs for targeted delivery of imaging agents or therapeutic agents to human neuroectodermal derived cancers.
Gangliosides are acidic glycosphingolipids found on the outer surface of most cell membranes.
1
Many tumors have abnormal glycolipid composition and structure. Disialoganglioside G
D2
has been found in a wide spectrum of human tumors, including neuroblastoma, osteosarcomas and other soft tissue sarcomas, medulloblastomas, high grade astrocytomas, melanomas, and small cell lung cancer.
2-4
Among glioblastoma multiforme and anaplastic astrocytoma, anti-G
D2
demonstrated the most restrictive pattern when compared with anti-G
D3
and anti-GM2 antibodies.
5.6
Gangliosides are ideal targets for monoclonal antibodies (MAb) because of the high antigen density, lack of modulation, relative homogeneity in many tumors and the possibility of up-regulation by cytokines.
7
The only normal tissues with high ganglioside expression are neurons, and biodistribution studies have shown that MAb do not localize to the nontumorous brain or spinal cord because of the blood brain barrier. In contrast, in patients with primary or metastatic brain tumors, specific antibodies can localize preferentially to tumor tissues, but not to normal brain.
8
Murine monoclonal antibodies have been prepared to ganglioside G
D2
. Using somatic cell hybridization, murine MAbs were produced against the ganglioside G
D2
.
9
They were shown to react with disialoganglioside G
D2
, but not with GD3, GT1b, GD1b, GD1a, GM1, GM3 and GM4. When base-treatment step was omitted from the standard neuroblastoma ganglioside extraction procedure, immuno-thin-layer-chromatography (ITLC) using 3F8, 3G6 and other anti-G
D2
MAbs revealed a new ganglioside band with Rf of 0.342, besides G
D2
(Rf 0.183).
4
Immunochemical analysis showed that this new neuroblastoma ganglioside contained alkali-sensitive O-acetylated sialic acid residues recognized by MAb D1.1.
Of 15 anti-G
D2
MAbs studied, 13 reacted strongly with the novel ganglioside. 3F8 was chosen for our initial clinical studies because of its being an IgG3 and its strong binding in vitro to G
D2
. Based on the cDNA sequence and the anti-idiotype cross-reactivity, the antigen specificity and affinity of 3f8 and 3G6 were similar if not identical. We have chosen 3G6 for scFv development for ease of comparison with the other 14 MAbs which are IgM antibodies.
TABLE 1
SPECIFICITY OF ANTI-G
D2
MAb.
MAb
G
D2
GD3
“0-G
D2
”
GD1b
1A8
4+
−
±
1+
1F9
4+
±
3+
1+
1H12
4+
±
3+
1+
2F7
3+
−
1+
−
3A7
3+
−
−
−
3A10
4+
1+
2+
1+
3B4
4+
−
3+
1+
3F8
4+
−
4+
−
3G6
4+
−
2+
−
4C11
4+
−
3+
±
5E11
4+
−
4+
−
5F4
4+
1+
2+
1+
5F11
2+
±
3+
1+
6E8
4+
1+
3+
1+
6H4
4+
±
3+
1+
“O-G
D2
”, a novel neuroblastoma alkali-labile ganglioside band consistent with the O-acetylated form of disialoganglioside G
D2
.
4+ = deep, dark staining band;
3+ = dark staining;
2+ = clearly staining;
1+ = faint staining;
± = very faint staining;
− = negative.
All MAbs were tested negative to G
MI
, G
M3
, G
D1a
AND G
TIb
.
In order to determine the general applicability of the ganglioside G
D2
as a target for immunotherapy, its expression in human cancers has been studied by immunostaining tumor specimens using these monoclonal antibodies. These anti-G
D2
antibodies reacted with all the neuroblastoma surgical specimens tested to date in our laboratory. A recent update
10
analyzed a series of 39 neuroblastomas. Staining of both primitive neuroblastic and differentiating ganglioneuromatous elements were seen, although tumor cell heterogeneity was noted in some. 23/39 tumors showed a more intense reactivity with MAb 3A7 than with 3F8, and this was particularly evident in the primitive neuroblastoma group. In a separate study, the expression of G
D2
was analyzed in 67 solid tumors and normal tissues from children by using the antibody 3A7.
11
G
D2
expression was found in 28 of 28 neuroblastomas, and was most abundant in stroma-poor tumors. Differentiating stroma-rich neuroblastomas, neuroblastic clusters, neurofibrils, and most ganglion-like cells were found to be G
D2
positive, whereas Schwann's-cell stroma did not express G
D2
. In ganglioneuromas, only a few ganglion-like cells showed GD
2
, whereas all other structures were negative. Scattered foci of G
D2
were also found in some non-neuronal tumors, such as rhabdomyosarcomas and osteosarcomas, but not in lymphomas, Askin tumors, or most Wilm's tumors. 3A7 was also found to react with retinoblastomas.
12
Previous studies have shown that anti-G
D2
antibodies reacted with the majority of osteosarcomas.
13
Sixty freshly frozen human soft-tissue sarcomas were studied by avidinbiotin immunostaining using purified monoclonal antibodies 3F8 (anti-G
D2
) and R24 (anti-GD3).
14
Ninety-three percent of the tumors tested by the immunohistochemical staining expressed G
D2
and 88% expressed GD3. The intensity of expression varied among different histologic types. Liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma and spindle cell sarcoma reacted strongly with both antibodies. Embryonal rhabdomyosarcoma and synovial sarcoma demonstrated substantially weaker staining by either MAb. Ganglioside extraction and immuno-thin layer chromatography (ITLC) confirmed the identities of these gangliosides as G
D2
and GD3 respectively.
Among brain tumors, 3F8 and 3A7 have also shown excellent reactivities. Two separate studies were carried out the first study in collaboration with Dr. Paul Zeltzer of Texas and the second with Dr. Ira Bergman (now Associate Professor of Neurology at the University of Pittsburgh) in our laboratory. In the first study, 12/15 medulloblastoma and 16/18 astrocytoma were positive, the majority staining homogeneously. In the second study, similar results were obtained. Medulloblastoma and a number of brain tumors reacted strongly with 3F8 and 3A7. The pattern of reactivity was generally homogeneous. For small cell lung cancer, all have reacted homogeneously in vitro using immunoperoxidase techniques.
Despite in vitro evidence for exquisite specificity of these antibodies for the ganglioside G
D2
on neuroblastoma cells, a critical test of in vivo delivery is the actual amount of MAb uptake in the tumors. Biodistribution of 131I-anti-G
D2
antibody was tested in preclinical experiments using athymic mice xenografted with human neuroblastoma.
Between 8 to 50% injected dose of 131I-MAb/gm of tumor was found, with variability depending primarily on the size of the tumor.
15
There was no localization to G
D2
-negative tumors like Ewing's sarcoma. Pooled mouse IgG and an irrelevant MAb also did not localize to neuroblastoma xenografts. Both small tumors (50 mg) and large tumors (over 2 g) showed radiolocalization with this technique. Optimal tumor to normal tissue ratios were rapidly reached by 24 to 48 hours. There was no increased uptake in the reticuloendothelial system, and the MAb did not cross the intact blood-brain barrier. The efficacy of tumor targeting was then tested by imaging neuroblastoma patients with 131I-MAb. Radiolocalization was demonstrated in primary tumors of the mediastinum and abdomen, as well as metastatic disease in the lymph nodes, bone marrow and bone.
61.17
The specificity was validated by tumor and marrow biopsies, as well as by CT/MRI and bone scans. A comparison with 131I-meta-iodobenzylguanidine (MIBG) suggested that 131I-MAb was twice as sensitive in detecting metastatic sites of disease. The tumor uptake in patients was
Cheung Nai-Kong V.
Guo Hong-Fen
Larson Steven M.
Rivlin Ken
Sadelain Michel
Helms Larry R.
Huff Sheela
Oppedahl & Larson LLP
Sloan-Kettering Institute for Cancer Research
LandOfFree
Single chain FV polynucleotide or peptide constructs of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Single chain FV polynucleotide or peptide constructs of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Single chain FV polynucleotide or peptide constructs of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2832005