JP2001509369A - Antigen binding fragments designated 4B5 that specifically detect cancer cells, nucleotides encoding the fragments, and their use for cancer prevention and detection - Google Patents

Abstract

  (57) [Summary] The present invention relates to monoclonal antibody 4B5 and an antigen-binding fragment that specifically bind to an antibody specific for GD2. Also disclosed are polynucleotide and polypeptide derivatives based on 4B5, including single chain V region molecules and fusion proteins, and various pharmaceutical compositions. When administered to an individual, the 4B5 antibody is effective in diagnosing and / or treating neoplasia. The invention further provides methods of treating neoplastic diseases, particularly melanoma, neuroblastoma, glioma, soft tissue sarcoma, and lung carcinoma. Patients who are in remission as a result of the traditional mode of cancer treatment can be treated with the compositions of the present invention in hopes of reducing the risk of relapse. Patients can also be treated simultaneously with antibodies and traditional anti-tumor agents.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a polynucleotide encoding an anti-idiotope antibody that mimics the GD2 antigen. Recombinant human monoclonal antibody (Mab) and its antigenic fragments are designated "4B5." The invention encompasses a wide variety of antibodies, and functional derivatives thereof that retain the immunological specificity of 4B5 and are referred to herein as "4B5." Polynucleotides encoding 4B5, and the polypeptides encoded thereby, and recombinant molecules containing these polynucleotides are also encompassed by the present invention. Methods of use, including therapeutic and diagnostic uses, of the 4B5 antibody are also encompassed by the present invention.

BACKGROUND OF THE INVENTION Despite many advances in medical research, cancer remains the second leading cause of death in the United States. In developed countries, nearly one in five people die from cancer. Traditional forms of clinical care such as surgical resection, radiation therapy, and chemotherapy have a significant failure rate, especially for solid tumors. Failure occurs either because the original tumor is unresponsive or because of relapse due to regrowth at the original site and / or metastases. Even for cancers such as breast cancer with reduced mortality, successful interventions rely on early detection of cancerous cells. The etiology, diagnosis and resection of cancer
It remains a central focus of medical research and development.

[0002] Neoplasias that give rise to benign tumors can usually be completely treated by surgically removing the mass. When a tumor becomes malignant, as represented by invasion of surrounding tissues, it becomes very difficult to eradicate. Once a malignant tumor has metastasized, it does not appear to be eradicated.

[0003] From a morbidity and mortality standpoint, the three major cancers are colon, breast and lung. New surgical procedures provide increased survival for colon cancer. Improved screening methods increase the detection of breast cancer and allow for earlier and less aggressive treatment. Many studies have shown that early detection increases survival and treatment options. Lung cancer remains largely refractory to treatment.

[0004] Except for basal cell carcinoma, there are more than 1 million new cases of cancer per year in the United States alone, and cancer is responsible for more than 500,000 deaths per year in this country. Worldwide, the five most common cancers are lung, stomach, breast, colon / rectum, and cervix, and the total number of new cases per year exceeds 6 million. Only about half of people who develop cancer die from cancer.

[0005] Melanoma is one of the human diseases that has an urgent need for new treatment modalities. This is a particularly aggressive form of skin cancer and results in increased frequency in individuals exposed to normal, unprotected sun exposure. In early stages of disease, melanoma is characterized by growth at the dermis-epithelial junction, which quickly invades adjacent tissues and spreads widely. Once this has metastasized, it is often impossible to eradicate and is consequently fatal. Worldwide, 70,000 patients are diagnosed with melanoma and are responsible for 25,000 reported deaths each year. The American Cancer Society estimates that by 2000, 1 in 75 Americans will be diagnosed with melanoma.

[0006] Neuroblastoma is a very malignant tumor that occurs in infancy and early childhood.
Except for Wilms tumor, it is the most common retroperitoneal tumor in children.
The tumor metastasizes early, with extensive involvement of lymph nodes, liver, bone, lungs, and bone marrow. Primary tumors can be resolved by resection, but the recurrence rate is high.

[0007] An estimated 178,100 new cases of lung cancer were diagnosed in 1997, accounting for 13% of cancer diagnoses. Estimated 160,400 deaths from lung cancer in 1997
It occurs yearly and accounts for 29% of all cancer deaths. The one-year survival rate for lung cancer increased from 32% in 1973 to 41% in 1993, mainly due to improved surgical techniques. The 5-year survival rate for all combined stages is only 14%. The survival rate for cases detected when the disease is still localized is 48%, but only 15% of lung cancers are found earlier.

[0008] Small cell lung cancer is the most malignant and fastest growing form of lung cancer, and accounts for 20-25% of new cases of lung cancer. 60,000 cases in 1996
Diagnosed in the United States in the year. Primary tumors are generally responsive to chemotherapy,
A widespread metastasis follows. The median survival time at diagnosis is about 1 year, and the 5-year survival rate is 5-10%.

[0009] Breast cancer is one of the most common cancers and is the third leading cause of cancer deaths in the United States, with about 180,200 annually among women in the United States during 1997 With the incidence of new cases. About 1,400 new cases of breast cancer are diagnosed in men in 1997. In developed countries, about one in eight women can be expected to develop breast cancer. The overall mortality rate for breast cancer has remained unchanged since 1930. This has increased by an average of 0.2% per year, but has decreased by an average of 0.3% per year for women under the age of 65. Preliminary data suggests that breast cancer mortality may begin to decrease, possibly as a result of increased diagnosis of cancer and carcinoma localized in situ. For example, Marchant (1994) Contemporary Management
nt of Breast Disease II: Breast Cance
r, Obstetics and Gynecology Clinics
of North America 21: 555-560; and Coldi
tz (1993) Cancer Suppl. 71: 1480-1489. An estimated 44,190 deaths in 1997 (43,900 women, 29
0 men) are caused by breast cancer. Women die of cancer after lung cancer
The second major cause. The five-year survival rate of localized breast cancer was 7 in the 1940s.
It has increased from 2% to 97% today. However, if the cancer has spread locally, the survival rate is 76%, and for women with distal metastases, the survival rate is 2%.
0%. Survival after diagnosis of breast cancer continues to decline for more than five years. 65% of women diagnosed with breast cancer survive for 10 years and 56% survive for 15 years.

[0010] Non-Hodgkin's B-cell lymphoma is a cancer of the immune system that is expected to afflict about 225,000 patients in the United States in 1996. These cancers differ in prognosis and treatment, and generally fall into one of three grades. The lowest grade median survival is 6.6 years, with higher grade cancers having much lower life expectancy. Virtually all non-Hodgkin B-cell lymphomas are incurable. New diagnoses of non-Hodgkin's lymphoma have increased by about 7% annually over the past decade,
In 1996, 52,700 new diagnoses are estimated. This increase is due, in part, to an increasing epidemic of lymphoma in the AIDS patient population.

[0011] Colon and rectal cancer accounted for an estimated 131,200 cases in 1997,
Includes 94,100 colon cancers and 37,100 rectal cancers. Colorectal cancer accounts for about 9% of new cancer diagnoses. An estimated 54,900 deaths from colorectal cancer occurred in 1997, accounting for about 10% of cancer deaths. Mortality for colorectal cancer has dropped by 32% in women and 14% in men over the past 20 years,
This reflects a reduced incidence and increased survival. However, mortality among African American men continues to rise. One year and 5 year relative survival rates for colon and rectal cancer patients were 82% and 6%, respectively.
1%. If colorectal cancer is detected at an early localized stage, the 5-year survival rate is 91%; however, only 37% of colorectal cancers are found at that stage. After the cancer has spread locally to include adjacent organs or lymph nodes,
Survival drops to 63%. The survival rate for people with distant metastases is 7%. Survival is
Continue to decline for more than 5 years, and 50% survive 10 years.

[0012] Despite the difficulties, effective therapies using anticancer drugs (alone or in combination with other treatments) have been conceived for some previously highly lethal cancers. I have. The most prominent of these are Hodgkin lymphoma, testicular cancer, choriocarcinoma, and some leukemias and other childhood cancers. For some of the more common cancers, early diagnosis, appropriate surgery, or local radiation therapy allows most patients to recover.

[0013] Current methods of cancer treatment are relatively non-selective. Surgery removes the affected tissue, radiation treatment shrinks the solid tumor, and chemotherapy kills rapidly dividing cells. Chemotherapy, in particular, produces a number of side effects, and in some cases, are so severe that the use of potentially effective drugs is not possible. In addition, cancer often develops resistance to chemotherapeutic drugs.

[0014] A number of efforts have been made to enhance the specificity of cancer treatment. For a review, see Kohn and Liotta (1995) Cancer Res. 55:
See 1856-1862. In particular, the identification of cell surface antigens that are exclusively or preferentially expressed on particular tumors allows for the formulation of more selective treatment strategies. Antibodies to these antigens have been used in immunotherapy of several types of cancer.

[0015] The basic immunoglobulin (Ig) structural unit in a vertebrate system is two identical light ("L") polypeptide chains (about 23 kDa) and two identical heavy ("H") chains.
") Chain (approximately 53-70 kDa). The four chains are linked by a disulfide bond in a “Y” configuration. At the base of Y, the two heavy chains are linked by a covalent disulfide bond.

FIG. 1 shows a schematic diagram of the antibody structure. The L chain and the H chain each have an N-terminal variable (
V) region and a C-terminal constant (C) region. In the light chain, the V region (
"V _L J _L ") is connected to the C region (C _L ) via the connection (J _L ) region.
(V _L ) region. The H chain, V region (V _H D _H J _H) is the diversity (D _H) region and the coupling (J _H) combined through a C region of a region (C _H) connected to a variable to (V _H) It consists of an area. V _L J _L region and V _H D _H J _H regions of the L chain and H chain, respectively, in association with the tip of the Y to form the antigen binding portion and determine antigen binding specificity.

The (C _H ) region defines the isotype, ie, the class or subclass of the antibody. Antibodies of different isotypes activate complement, bind to specific receptors (eg, Fc receptors) present on a wide variety of cell types, traverse mucosal and placental barriers, and form basic four-chain IgG molecules. Its effector functions, such as its ability to form polymers, are significantly different.

[0018] Antibodies include immunoglobulin molecules (IgM, IgG, IgD, IgE, or Ig).
Classification is made according to the _CH type used in A). At least five types of the C _H genes (Cμ, Cγ, Cδ, Cε , and C alpha) have, and some species, a plurality of the C _H subtypes (e.g., C gamma ₁ in humans, C gamma _2, C gamma ₃ , And Cγ ₄ ). There are a total of nine _CH genes in the human haploid genome, eight in mice and rats, and some fewer in many other species. In contrast, normally there are only two types of light chain C regions ( _CL ) (kappa (κ) and lambda (λ)), and only one of these C regions is a single L not present in the chain protein (i.e., every V _L J _L possible for L chain C produced
There is only one area). Each heavy chain class can be associated with any of the light chain classes (
For example, the _CH gamma region may be present in the same antibody as either the kappa or lambda light chain), but the C regions of the heavy and light chains within a particular class are not altered by antigen specificity (e.g., IgG antibodies always have a Cγ heavy chain C region, regardless of antigenic properties).

Each of the V, D, J, and C regions of the heavy and light chains is a separate gene.
Coded by nome sequence. Antibody diversity depends on the different Vs in the heavy chain._H, D_H,
And J_HA segment of the gene and the V_LAnd J_LIt is produced by recombination between the gene segments. Different V_H, D_H, And J_HGenetic recombination is achieved by DNA recombination during B cell differentiation. Briefly, H chain
The array is first shuffled to D_HJ_HComplex, and then the second recombination event is
V_HD_HJ_HProduce the complex. Functional heavy chains are produced during transcription, followed by splicing of the RNA transcript. Production of a functional heavy chain depends on the set in the light chain sequence.
Triggering the rearrangement and rearranged V_LJ_LRegion, which in turn produces a functional V_LJ_LC _L To form a region, ie, a functional light chain.

The value and potential of antibodies as diagnostic and therapeutic agents has long been recognized in the art. Unfortunately, this field has been hampered by the slow and tedious processes required to produce large quantities of antibodies of the desired specificity. Classical cell fusion techniques involve the fusion of B cells producing antibodies with immortalized cell lines to produce Ma
b enabled efficient production. The resulting cell line is a hybridoma cell line.

Antibodies and their functional derivatives have been used in various clinical settings. For example,
Digoxin-specific Fab antibody fragments were used to treat digital threatening digitalis intoxication. Antibodies have become more routinely useful in diagnostic techniques such as radioimmunodiagnosis of colon cancer. Koda et al. (1995) Am. J. Gastroen
terol. 90: 1644. Many uses of Mab, previously considered unacceptable, have been implemented recently. For example, Hall (1995) Scie
279: 915-916.

Many autoantibodies (antibodies that recognize and bind to self antigens) are found in humans. Many of these include rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, primary biliary cirrhosis, polymyositis, systemic vasculitis, idiopathic necrotizing and crescentic glomerulonephritis, and the amyotrophic side It is associated with certain diseases such as chordosis.
For a review, see Shattner (1986/1987) Immunol. L
ett. 14: 143-153. Other autoantibodies occur naturally.
Lutz and Wipp (1982) J. Mol. Immunol. 128: 1965;
And Guilbert et al. (1982) J. Am. Immunol. 128: 2779
-2787. Recently, human autoantibodies to certain cancer antigens have been detected, and in some cases, are produced by hybridoma technology. These antibodies have also been produced by active immunization. U.S. Patent No. 5,474,755. Initially, human B cells were immortalized using Epstein-Barr virus or mouse myeloma. For a review, see Buck et al. (1984) Monoclon.
al Antibodies, "NY, Plenum Press.
More recent technology has allowed immortalization without the use of this potentially harmful virus. See, for example, U.S. Patent No. 4,618,477; and Glassy (198).
7) Cancer Res. 47: 5181-5188. In most cases, the antibodies will be specific for one, or in some cases, a small number of cancer types. For example, it has been described that Mab specifically recognizes glioma cells but does not recognize other tumor or normal cells. These antibodies were used to image gliomas in the patient's brain. Fischer et al. (199
1) Immunobiol. Prot. Pep. VI (M. Atassi) P
lenum Press, NY, pp. 263-270. Antibodies that are capable of recognizing a wide range of tumors but not, or only slightly, normal non-cancerous cells are not described.

[0023] Recombinant gene technology has allowed the cloning and expression of antibodies, functional fragments thereof, and recognized antigens. These engineered antibodies offer new production methods and treatment modalities. For example, functional immunoglobulin fragments have been expressed in bacteria and transgenic tobacco seeds and plants.
Skerra (1993) Curr. Opin. Immunol. 5: 256-
262; Fiedler and Conrad (1995) Bio / Techno.
logic 13: 1090-1093; Zhang et al. (1993) Cancer.
Res. 55: 3384-3591; Ma et al. (1995) Science 2
68: 916; and for a review of synthetic antibodies, see Barbas (1995) N.
attune Med. 1: 834-839.

Several human Mabs against tumor-associated antigens have been produced and characterized. Tumor-associated antigens recognized by human Mab include cell surface, cytoplasm,
And nuclear antigens. Yoshikawa et al. (1989) Jpn. J. C
cancel Res. (Gann) 80: 546-553; Yamaguchi
(1987) Proc. Natl. Acad. Sci. USA 84: 241
6-2420; Haspel et al. (1985) Cancer Res. 45:39
51-3961; Cote et al. (1986) Proc. Natl. Acad. Sc
i. USA 83: 2959-2963; Glassy (1987) Cance.
r Res. 47: 5181-5188; Borup-Christensen
(1987) Cancer Detect. Present. Suppl. 1
: 207-215; Haspel et al. (1985) Cancer Res. 45:
3951-3961; Kan-Mitchell et al. (1989) Cancer.
Res. 49: 4536-4541; Yoshikawa et al. (1986) Jpn.
. J. Cancer Res. 77: 1122-1133; and McKnig.
ht et al. (1990) Human Antibod. Hybridomas 1:
125-129. Dan et al., (1992) J. Mol. Neurosurg. 76:66
0-669.

Human Mab has been used for cancer imaging, diagnosis, and therapy. Olsso
n (1985) J. Am. Nat. Cancer Inst. 75: 397-404;
Larrick and Bourla (1986) J. Am. Biol. Resp. Mo
d. 5: 379-393; McCabe et al. (1988) Cancer Res.
48: 4348-4353; Research News (1993) Scie
nce 262: 841; Ditzel et al. (1994) Cancer 73: 8.
58-863; and Alonso (1991) Am. J. Clin. Onco
l. 4: 463-471. Recombinant single-chain bispecific antibodies with high tumor cytotoxicity have been reported. This molecule recognizes both the CD3 antigen on human T cells and EpCAM, which is associated with sporadic tumor cells in patients with minimal residual colorectal cancer. Mack et al. (1995) Proc. Natl. Acad. Sci
. ScL USA 92: 7021-7025.

Immunologists have learned that poor antigens (in terms of eliciting an immune response) can be converted to strong antigens by altering the molecular environment. Changes in the hapten carrier activate T-cell helper cells, making the overall immune response more potent. Thus, changing carriers may also change an antigen that produces immunological tolerance into an effective antigen. McBridge et al. (1986) Br. J. Cance
r 53: 707. The immunological status of cancer patients is often suppressed, so that patients can only respond to certain T-dependent antigens, but not other forms of the antigen. From these considerations, it makes sense to introduce molecular changes to tumor-associated antigens before using them as vaccines. Unfortunately, this is not possible with most tumor antigens. Because they are poorly defined and very difficult to purify.

Lindemann ((1973) Ann. Immunol. 124: 171).
-184) and Jerne ((1974) Ann. Immunol. 125:
373-389) proposed an elaborate approach to convert the epitope structure into idiotypic determinants expressed on the surface of the antibody. According to the network concept, immunization with a given tumor-associated antigen results in the production of antibodies against this tumor-associated antigen (designated Ab1);
It is used to generate a series of anti-idiotypic antibodies to b1 (designated Ab2). Some of these Ab2 molecules can effectively mimic the three-dimensional structure of the tumor-associated antigen identified by Ab1. These particular anti-idiotypes, called Ab2β, are compatible with the paratope of Ab1 and represent the internal image of tumor-associated antigens. Ab2β can elicit a specific immune response similar to that elicited by the native tumor-associated antigen, and thus can be used as a surrogate for tumor-associated antigens. Immunization with Ab2β may result in the generation of anti-anti-idiotype antibodies (Ab3) that recognize the corresponding native tumor-associated antigen identified by Ab1. Due to this Ab1-like reactivity, Ab3 also
It is called Ab1 'to indicate that it can be different from b1.

[0028] A potential and promising approach for the treatment of cancer is immunotherapy using anti-idiotype antibodies. In this form of therapy, an antibody that mimics an epitope of a tumor-associated protein is administered in an attempt to stimulate the patient's immune system against the tumor via the tumor-associated protein. WO 91/11465 describes a method of stimulating an immune response in humans against malignant cells or infectious agents using primate anti-idiotypic antibodies. However, not all anti-idiotype antibodies can be used in a treatment regimen for tumors. First, only the fraction of antibodies raised against Ab1 is limited in their reactivity to the Ab1 paratope (i.e., due to the characteristics shared with other potential antibodies in the host, Is reactive). Second, anti-idiotype antibodies are not necessarily immunogenic. Third, even if the anti-idiotype elicited an immune response, only those fractions of these immunogenic anti-idiotypes elicited an immune response against tumor antigens, but against other antigens with less specificity. Does not trigger. Furthermore, because different cancers have a wide variety of molecular and clinical characteristics, it is suggested that anti-idiotype therapy should be evaluated on a case-by-case basis for tumor origin and antigen expression. ing.

Anti-Id monoclonal antibodies that are structurally similar to tumor-associated antigens have been used as antigen replacements in cancer patients. Herlyn et al. (1987) PNAS.
84: 8055-8059; Mittleman et al. (1992) PNAS 89.
: 466-470; Chatterjee et al. (1993) Ann. N. Y. Ac
ad. Sci. 690: 376-377. It has been proposed that anti-Ids provide partial analogs of tumor-associated antigens in immunogenic settings.

Some mouse monoclonal anti-GD2 antibodies may suppress the growth of tumors of neuroectodermal origin in athymic (nu / nu) mice or produce remission in patients with metastatic melanoma Was reported. Several antibodies specific for GD2 have been described and have been deposited with the ATCC. These are HB-
9326 (described in U.S. Pat. No. 4,849,509); HB-9325 (described in U.S. Pat. No. 4,849,509); and HB-8568 (described in U.S. Pat.
75, 287). Anti-idiotypic monoclonal antibodies that provide internal anti-idiotypic determinants on antibodies to GD2 have also been described. U.S. Patent No. 5,612,030; and Saleh et al. Im
munol. 151: 3390-3398. Anti-idiotype antibodies are used for melanoma,
It is described for use in treating small cell carcinoma. Human-mouse chimeric anti-GD
Antibody 2 produced remissions in patients with metastatic neuroblastoma. The mechanism of action of the antibody is thought to include antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CMC). A clinical response has been obtained by treating melanoma with a Mab directed to GM2, GD2, and GD3. Cheresh et al. (1985) P
rc. Natl. Acad. Sci. ScL USA 82: 5155-5159. G
Active immunization with ganglioside vaccines containing M2 showed anti-GM in 50/58 patients.
2 produced these antibodies, and these patients survived on average longer than patients without detectable anti-GM2 antibodies.

A Mab directed to GD2 has been found to specifically react with small cell lung carcinoma. Cheresh et al. (1986) Cancer Res. 46: 511
2-5118. Human Mab specific for other cancers, including lung, melanoma, gastric, squamous, cervical, and breast, have also been produced. Murakami
(1985) in Vitro Cell. Dev. Biol. 21: 593;
Schadendorff (1989) J. Am. Immunol. 142: 1621-
1625; Yoshikawa et al. (1986) Jpn. J. Cancer Re
s. 77: 1122-1133; Pickering and Misra (198
4) Clin. Immunol. Immunopathol. 32: 253-2
60; Hagiwara and Sato (1983) Mol. Biol. Med
. 1: 245-252; and Schlom et al. (1980) Proc. Natl
. Acad. Sci. ScL USA 77: 6841-6845. Human anti-cancer Mabs and the antigens they recognize have also been suggested for use in vaccines.
See, for example, Finn et al. (1995) Immunol. Rev .. 145: 61-89
checking ... Human Mab against Malignant Brain Tumors
Used in phase clinical trials. Matsumoto et al. (1994) The Clini.
cal Report 28: 118-126. The results of a Phase II clinical trial of combined treatment with mouse Mab and colony stimulating factor in metastatic gastrointestinal cancer have been reported. Saleh et al. (1995) Cancer Re.
s. 55: 4339-4346. Single chain immunotoxins have also been found to treat cancerous meningitis in a rat model. Pastan et al. (1995) P
rc. Natl. Acad. Sci. ScL USA 92: 2765-2769. A human Mab that specifically recognizes ovarian cancer cells has been shown to effectively image this cancer. Chaudhuri et al. (1994) Cancer 73: 1.
098-1104.

[0032] The clinical perspective of cancers is generally improved if there is a simple and reliable strategy to provide immunoreactivity to a common antigen in these cancers rather than cancer-specific immunity. All references cited herein are hereby incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION The present invention encompasses a composition comprising an antigen-binding fragment of an antibody, wherein the antibody has an H chain having the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: 4. The antigen recognized by the antibody containing the L chain is specifically recognized. Preferably, the antibody is recombinant 4B5. The invention further encompasses antibodies comprising the 4B5 heavy and light chains (SEQ ID NOs: 2 and 4, respectively). 4B5 specifically recognizes cancer cells from a wide variety of cancers but does not recognize normal non-cancerous cells. "Not recognizing" means that the non-cancerous cells are either not specifically bound by 4B5 or are only slightly recognized by the antibody. These antigen-binding fragments include whole native antibodies, exemplified by the recombinant 4B5 described herein; bispecific antibodies; chimeric antibodies; Fab, Fab ', single chain V region fragments (scFv), And fusion polypeptides.

The present invention further relates to a 4B5 antibody fusion molecule (H chain C region, single chain V region)._H-V_LOr V _L -V_HContains antigenic, therapeutic, toxic, or labeled molecules bound to the V region
And polypeptides encoding such polypeptides
Leotide.

The present invention also encompasses a polynucleotide comprising a sequence encoding a polypeptide having the immunological specificity of 4B5, wherein the encoded polypeptide comprises at least five polypeptides from the 4B5 V region. Contains consecutive amino acids. V region is 4
It can be from either the light or heavy chain of B5. Five consecutive amino acids preferably play a role in the immunological reactivity of 4B5 and may be from a CDR.

[0036] The present invention also provides at least a 20-fold that can form a stable duplex with the 4B5 L or H chain coding sequence, but cannot form a sequence with other previously described immunoglobulin molecules. Of isolated nucleotides of contiguous nucleotides. Any of these polynucleotides may be in the form of a cloning vector, an expression vector, or transfected into a host cell.

Another embodiment of the present invention is a polypeptide having an immunological specificity of 4B5. Here, the polypeptide comprises at least five consecutive amino acids from the V region of the 4B5 antibody. The V region can be from the light chain or from the heavy chain.
5 consecutive amino acids preferably play a role in immunological specificity,
And it may be derived from CDR (complementarity determining region of antibody). Intact recombination 4
Functionally active fragments of B5, 4B5, fusion proteins, chimeric antibodies, multiple antigenic proteins, and other polypeptide derivatives of the 4B5 antibody are included. Of particular interest are single-chain V regions and fusion proteins.

The compounds and compositions of the present invention may be used in particular for the detection and treatment of cancer (
Treatment of such cancers, including prophylactic management, especially to reduce the risk of recurrence).

The invention further encompasses cells and cell lines that produce 4B5.

[0040] A further embodiment of the present invention encompasses prophylactic treatment of cancer patients with at least one 4B5 antigen binding fragment. Preferably, 4B5 is fused to a therapeutic molecule to effect delivery of the therapeutic molecule to cancer cells. The individual may have a clinically detectable tumor, or the tumor may have been previously treated and made undetectable. The method may be to alleviate the disease or to reduce the risk of relapse.

A further embodiment of the present invention is a kit for the detection or quantification of an antibody specific for GD2 in a sample, comprising 4B5 in a suitable packaging.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention encompasses antigen-binding fragments exemplified by the recombinant antibodies described herein. An exemplary antibody is designated 4B5 and has the sequence
And SEQ ID NOs: 2 and 5 (SEQ ID NOs: 2 and 5 are the complements of 1 and 4, respectively) and recognizes an antibody specific for the GD2 antibody. Antibodies specific for GD2 include glioblastoma, neuroblastoma, malignant and / or metastatic melanoma,
It recognizes a variety of cancers, including but not limited to breast, lung, small cell lung, colon and prostate.

The invention further encompasses 4B5 derivatives that have immunological specificity for specific antibodies to GD2. These derivatives include a region of the polypeptide sequence that includes a portion of the 4B5 VDJ linkage. Also encompassed are regions spanning at least one, preferably two, and more preferably three or more 4B5 CDR amino acid sequences.

The specific compounds, compositions, and methods described in this application generally relate to 4B5 and its derivatives conventionally produced by classical techniques of immunochemistry.
This includes 4B5 linked to another compound by chemical bonding or by mixing with excipients or adjuvants. The term antigen binding fragment includes any peptide specifically recognized by an antibody specific for GD2. Typically, these derivatives are immunoglobulin fragments, such as Fab, F (ab ') ₂ , Fab', scFv (both monomeric and polymeric forms), and isolated heavy and light chains. including. The antigen binding fragment retains the antigenic specificity of 4B5, but may have altered avidity and / or affinity.

[0045] Antigen-binding fragments (also referred to herein as "derivatives") are typically produced by genetic engineering, or they may be obtained by other methods and combinations of methods. This classification includes, but is not limited to, engineered peptide fragments and fusion proteins. Preferred compounds include polypeptide fragments of the 4B5 CDR, antibody fusion proteins containing a cytokine effector component, antibody fusion proteins containing an adjuvant or drug, and single chain V region proteins.

The present invention further includes polynucleotides encoding the 4B5 antibody V region and derivatives thereof. These include isolated polynucleotide fragments (both coding and complementary), recombinant polynucleotides, and therapeutic plasmids and vectors containing the polynucleotides (eg, vaccinia vectors).
Is mentioned. These polynucleotides are exemplified by SEQ ID NOs: 1, 2, 4, and 5, and plasmid pNB2-4B5 (FIG. 5).

As 4B5 has been shown to mimic GD2, 4B5 is particularly useful for generating a host immune response against cancer. The pharmaceutical compositions and modes of treatment of the present invention are suitable for eliciting an immune response to a neoplasia. Suitable carcinomas include any cancer known in the field of oncology, including astrocytomas,
Oligodendroglioma, ependymoma, medulloblastoma, early neuroectodermal tumor (PNET),
Pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma, and their liver metastases, liver cancer, bile duct carcinoma, ductal adenocarcinoma and lobule adenocarcinoma Breast cancer, squamous and adenocarcinoma of the cervix, uterine and ovarian epithelial carcinoma, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, such as
B-cell and T-cell lymphomas, (nodular and diffuse) plasmacytomas,
Includes but is not limited to acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

The subject may have an advanced form of the disease, in which case the treatment objective may include alleviating or reversing the progression of the disease and ameliorating side effects. The subject may have a history of the condition that is already being treated, in which case the objectives typically include a reduced or delayed risk of relapse.

The “immunological activity” of 4B5 refers to the ability to specifically bind to an antibody specific for GD2. Such binding may or may not elicit an immune response. A specific immune response can include antibodies, B cells, T cells, and any combination thereof, and effector functions resulting therefrom. Antibody-mediated functions ADCC and complement-mediated cell lysis (CDC) are included. T cell responses include T helper cell function, cytotoxic T cell function, inflammation / inducer T cell function, and T cell mediated suppression. Compounds that can elicit a specific immune response according to any of these criteria are termed "immunogenic".

4B5 “activity” or 4B5 “function” refers to any immunological activity of 4B5 or any other biological activity attributed to 4B5 in the present disclosure;
Includes the role of 4B5 in detecting antibodies specific for GD2, ameliorating or reducing cancer.

The “V region” of 4B5 refers to either the V region of the 4B5 L chain or the V region of the 4B5 H chain, alone or in combination. These V regions are shown in SEQ ID NOs: 3 and 6; the DNAs encoding these regions are shown in SEQ ID NOs: 1 and 4, respectively.

[0052] GM-CSF, IL-2 and other biologically active molecules referred to herein are based on the respective parent molecule having the same biological or physiological function. It is meant to include fragments and derivatives.

“Polynucleotide” is a polymeric form of nucleotides of any length.
This includes analogs in deoxyribonucleotides, ribonucleotides, and any combination analogs. A polynucleotide can have any three-dimensional structure, and can perform any function, known or unknown. The term "polynucleotide" includes double-stranded, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide described herein is known to constitute a double-stranded form of either a DNA, RNA or hybrid molecule. Or both the predicted double stranded form and the two complementary single stranded forms.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, ribozyme,
cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. A polynucleotide is a modified nucleotide (
For example, it can include methylated nucleotides and nucleotide analogs), uracil, other sugars and linking groups (eg, fluororibose and thioate), and nucleotide branches. Purine and pyrimidine analogs are known in the art and include aziridinyl cytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylamino Methyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-
Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentynyluracil (p
entynylacyl) and 2,6-diaminopurine,
It is not limited to these. The use of uracil as a replacement for thymine in deoxyribonucleic acids is also considered to be an analog form of pyrimidine.

The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component.
Other types of modifications included in this definition include caps, replacement of one or more naturally occurring nucleotides with analogs, and polynucleotide proteins, metal ions, labeling components, other polynucleotides, or solid supports. The introduction of means for binding to the body.

If present, modifications to the nucleotide structure may be made before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. For example, other types of qualification included in this definition include "
Caps ", substitution of one or more naturally occurring nucleotides with analogs, for example, modifications and charges using uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) Internucleotide modifications, such as modifications using linkages (eg, phosphorothioates, phosphorodithioates, etc.), eg, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)
, Modifications involving intercalators (eg, acridine, psoralen, etc.), modifications involving chelators (eg, metals, radiometals, boron, oxidative metals, etc.), alkylating agents , Modifications using modified linkages (eg, alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide.

In addition, any hydroxyl groups commonly present on sugars may include phosphonate groups,
It can be substituted by a phosphate group, protected by a standard protecting group, or activated to prepare an additional linkage to an additional nucleotide, or attached to a solid support. The 5 'and 3' terminal OH groups can be phosphorylated or replaced with an amine or an organic capping group moiety of 1-20 carbon atoms. Other hydroxyls can also be derived to standard protecting groups.

The polynucleotide may also include analog forms of ribose or deoxyribose sugars commonly known in the art. These include 2'-O-methyl-,
2′-O-allyl-, 2′-fluoro-, or 2′-azido-ribose, carbocyclic sugar analog, α-anomeric sugar, epimeric sugar (eg, arabinose, xylose or lyxose), pyranose sugar, furanose sugar , Sedoheptulose, acyclic analogs and abasic nucleotide analogs such as methyl riboside.

As described above, one or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups are those in which the phosphate is P (O) S ("thioate").
), P (S) S ( "dithioate"), be a "(O) NR ₂ (" amidate "), P (O) R, P (O) OR ', CO or CH ₂ (" formacetal ") Wherein R or R ′ is independently H or substituted or unsubstituted alkyl (1 to
20C), if necessary, ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl
nyl) or araldyl, including but not limited to. Not all linkages in a polynucleotide need be the same.

While conventional sugars and bases are used in the application of the methods of the invention, substitution of analog forms of sugars, purines, and pyrimidines may be alternative backbone structures such as polyamide backbones. , May be advantageous in the design of the final product.

The term “recombinant” polynucleotide refers to a genomic, cDNA, either non-naturally occurring or linked to another polynucleotide in a non-naturally occurring arrangement.
, Semi-synthetic, or polynucleotide of synthetic origin.

An “isolated” polynucleotide or polypeptide is one that is substantially free of associated substances in its native environment. Substantially free means at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of these substances.

A “stable duplex” of a polynucleotide, or “stable complex” formed between any two or more components in a biochemical reaction is defined as the formation of a duplex or complex followed by (Including any optional washing steps or other operations that may take place in between) refers to a duplex or complex that has long enough to persist.

“Vector” refers to a recombinant DNA or RNA plasmid or virus containing a heterologous polynucleotide that is delivered to a target cell, either in vitro or in vivo. A heterologous polynucleotide can include a sequence of interest for therapeutic purposes, and can optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replicating in the ultimate target cell or subject. The term includes cloning vectors for replication of the polynucleotide and expression vectors for translation of the sequence encoded by the polynucleotide. Also included are viral vectors that contain the encapsidated or enveloped polynucleotide in a viral particle.

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues of any length.
Polymers can be linear or branched, can contain modified amino acids or amino acid analogs, and can be interrupted by chemical moieties other than amino acids. These terms also include amino acid polymers that have been modified, either naturally or by intervention (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or labeling or conjugation with a biologically active ingredient). Any other operation or modification). Unless otherwise stated or implied, the term 4B5 includes any polypeptide monomer or polymer having the immunological specificity of 4B5, including intact recombinant antibodies, and smaller and larger functionally equivalent Polypeptides.

A “fusion polypeptide” is a polypeptide that includes a region at a different position in the sequence than that found in nature. This region can normally be on a separate protein,
And are brought together in the fusion polypeptide; they can normally be present in the same protein, but are placed in a new configuration in the fusion polypeptide; or they can be placed synthetically. For example, as described below, the invention
Includes recombinant proteins containing the functional portion of B5 and toxins (and polynucleotides encoding the proteins). Methods for making these fusion proteins are known in the art and are described, for example, in WO 93/07286.

A “functionally equivalent fragment” of a 4B5 polypeptide differs from a native sequence by any combination of additions, deletions, or substitutions, but with respect to the context in which it is used, at least one of the fragments. Maintains functional characteristics. A functionally equivalent fragment of a 4B5 polynucleotide encodes a polypeptide that is functionally equivalent to 4B5 when produced by an expression system, or
Or, when used in a hybridization assay, has similar hybridization specificity to a 4B5 polynucleotide. 4B
Functionally equivalent fragments of the 5 polypeptides typically have one or more of the following properties: the ability to bind to an antibody specific for GD2; the ability to bind to at least one type of cancer cell in a specific manner. The ability to generate specific antibodies; and the ability to elicit an immune response with antigen specificity similar to that elicited by 4B5 or GD2.

“Cell line” or “cell culture” refers to a bacterial, plant, insect, or higher eukaryotic cell grown or maintained in vitro. The progeny of cells are (morphologically,
It may not be completely identical to the parent cell (either genotypically or phenotypically). Ma
b can be produced by hybridomas or other cells. Methods for making both mouse and human hybridomas are known in the art. Particular methods of producing human hybridomas are described and referenced throughout the specification.

A “host cell” is a prokaryotic or eukaryotic cell that has been genetically altered or can be genetically altered by the administration of an exogenous polynucleotide such as a recombinant plasmid or recombinant vector. Is shown. When referring to a genetically modified cell, the term refers to both the original modified cell and its progeny.

“Heterologous” means derived from an entity that differs in genotype from the rest of the entity being compared. For example, the polynucleotide can be placed by genetic engineering techniques on a plasmid or vector from a different source, and is a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence other than the native sequence is a heterologous promoter.

“Signal peptide” or “leader sequence” refers to the newly synthesized protein, either the membrane of cells, usually the endoplasmic reticulum of eukaryotic cells, and the bacterial inner membrane or both the inner and outer membranes. Or a short amino acid sequence that tells it to pass through. The signal peptide is typically at the N-terminal portion of the polypeptide, and is typically enzymatically removed between the biosynthesis and secretion of the polypeptide from the cell. Signal peptides are not present in secreted proteins, but only during protein production.

A “biological sample” can be any of a variety of fluids, including fluid samples of blood and other biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells and progeny derived therefrom. Includes sample type. The definition also includes a sample that has been manipulated in any manner after its acquisition, such as by treatment with a reagent, solubilization, or enrichment of a component (eg, a protein or polynucleotide). The term encompasses a variety of clinical samples obtained from any species, and also encompasses cells in culture, cell supernatants, and cell lysates. In particular, for the purposes described herein, a biological sample includes tumor tissue or tissue that is considered to be neoplastic and can be used, for example, in surgical resection, biopsy, aspiration, or in the art. It can be obtained by any known method.

“Immunogen” refers to a composition for human or animal use, which is administered at an intensity that provides a recipient with some specific immunological reactivity to a particular antigen. Immunological reactivity is defined as antibodies or cells that are immunologically reactive to the target (especially B cells, plasma cells, T helper cells, and cytotoxic T lymphocytes, and their precursors). Or any combination thereof. For purposes of the present invention, the target is primarily a tumor-associated C antigen or a tumor-specific portion thereof. Immunological reactivity may be desired for experimental purposes, for the treatment of certain conditions, for the elimination of certain substances, or for prophylaxis. An active immunogen is intended to elicit a sustained immune response in the absence of a vaccine component.

As used herein, “adjuvant” has several meanings, all of which are apparent depending on the context in which the term is used. In the case of a pharmaceutical preparation, the adjuvant is a chemical or biological agent provided in combination with the antigen to enhance the immunogenicity of the antigen, or recombinantly fused to the antigen. . In the case of cancer diagnosis or treatment, adjuvant refers to a class of cancer patients for whom a tumor mass cannot be detected clinically, but is at risk of recurrence.

When referring to a type of cancer that usually appears as a solid tumor, a “clinically detectable” tumor is detected based on the tumor mass, ie, by a procedure such as CAT scan, X-ray, or palpation. A possible tumor. Biochemical, histological, or immunological findings alone may not be sufficient to meet this definition.

As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the treated individual or cell, and to prevent or prevent clinical pathology. During the course of the process. The desired effect of the treatment includes preventing the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease Amelioration, amelioration or alleviation of disease symptoms, and remission or improved prognosis.

A “condition” associated with a disease state is any condition that impairs the health, normal physiology, or quality of life of the affected individual. This includes the destructive invasion of previously unaffected areas of diseased tissue, growth at the expense of normal tissue function, irregular or suppressed biological activity, inflammatory response or immunological Undesirable clinical signs such as worsening or suppressing response, increased susceptibility to other pathogenic organisms or pathogens, and pain, fever, nausea, fatigue, mood changes, and other characteristics that can be determined by the attending physician But are not limited to these.

An “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more doses. For treatment, an effective amount is
An amount sufficient to palliate, ameliorate, stabilize, reverse, or delay the progression of the disease, or otherwise reduce the pathological consequences of the disease. For an adjuvant, an effective amount is an amount sufficient to enhance an immune response to the immunogen. The effective amount will generally be determined by the physician on a case-by-case basis and is within the skill of those in the art. Typically, several factors are considered when determining the appropriate dose. These factors include the age, sex, and weight of the patient, the condition being treated, the severity of the condition, and the form of antibody administered. For example, the concentration of the scFv need not be as high as the concentration of the native antibody to be therapeutically effective.

An “individual,” “patient,” or “subject” is a vertebrate, preferably a mammal,
More preferably, it is a human. Mammals include, but are not limited to, humans, livestock, sport animals, and pets.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology, which Within the field. Such a technique is described in Molecular Cloni.
ng: A Laboratory Manual "2nd edition (Sambrook et al., 1989);" Oligoncleotide Synthesis "(M
. J. Gait, 1984); "Animal Cell Culture"
(Edited by RI Freshney, 1987); "Methods in Enz.
ymology "(Academic Press, Inc.);" Handb
OK of Expertial Immunology "(DM.
Wei and C.I. C. Blackwell); “Gene Transfer
Vectors for Mammarian Cells "(JM Mi
ller and M.E. P. Calos, 1987); "Current Pro.
tocols in Molecular Biology "(FM Aus
Ubel et al., 1987); "PCR: The Polymerase Cha.
in Reaction "(Mullis et al., eds., 1994);" Current
Protocols in Immunology "(JE Colliga)
n et al., 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention, and as such may be considered in making and practicing the present invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections.

The present invention also includes 4B5 linked to a chemical functional moiety. Typically, this moiety is a label that can produce a detectable signal. These combined 4B
5 is useful, for example, in detection systems such as quantification of tumor burden, and in imaging metastatic foci and tumor imaging. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors and inhibitors. Examples of patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,853.
No. 0,752; 3,939,350; 3,996,345; 4,27
Nos. 7,437; 4,275,149; and 4,366,241. This moiety can be covalently linked to 4B5, recombinantly linked, or linked to 4B5 via a second reagent, such as a second antibody, protein A, or biotin-avidin complex.

Other functional moieties include signal peptides, factors that enhance immunological reactivity, factors that facilitate coupling to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels, and drugs. Is mentioned. Signal peptides have been described above and include prokaryotic and eukaryotic forms. Factors that enhance immunological reactivity include, but are not limited to, bacterial superantigens. Factors that facilitate coupling to a solid support include, but are not limited to, biotin or avidin. Immunogenic carriers include, but are not limited to, any physiologically acceptable buffer. Biological response regulators include cytokines, especially tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor, and gamma interferon.

[0083] Suitable drug moieties include antitumor agents. These include radioisotopes, vinca alkaloids (eg, vinblastine, vincristine, and vindesine sulfate), adriamycin, bleomycin sulfate, carboplatin, cisplatin,
Cyclophosphamide, cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechlororeamine hydrochloride
thamine), melphalan, mercaptopurine, methotrexate, mitomycin, mitotan, pentostatin, pipobroman, procarbazine hydrochloride (
procarbaze), streptozotocin, taxol, thioguanine, and uracil mustard.

[0084] Immunotoxins, including single-chain molecules, can be produced by recombinant means. The production of various immunotoxins is well known in the art, and methods are described, for example, in "Monoclon".
al Antibody-toxin Conjugates: Aiming
the Magic Bullet ", Thorpe et al. (1982) Monoc.
lon Antibodies in Clinical Medicine
e, Academic Press, pp. 168-190; Vitatta (19
87) Science 238: 1098-1104; and Winter and Milstein (1991) Nature 349: 293-299. Suitable toxins include ricin, radionuclide, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin, and phospholipase enzymes, It is not limited to these. Generally, "Chimer
ic Toxins "Olsnes and Pihl, Pharmac. Ther
. 15: 355-381 (1981); and "Monoclonal Ant"
ibodies for Cancer Detection and The
rapy ", edited by Baldwin and Byers, pages 159-179, 224-2.
See page 66, Academic Press (1985).

A chemically functional moiety can be made recombinantly, for example, by producing a fusion gene encoding an antigen-binding fragment and a functional region from another gene (eg, an enzyme). In the case of a gene fusion, the two components are present in the same polypeptide gene. Alternatively, the 4B5 antigen binding fragment can be chemically conjugated to the moiety by any of a variety of well-known chemical procedures. For example, if the moiety is a protein, the linkage may be via a heterobifunctional crosslinker (eg, SPDP, carbodiimide, glutaraldehyde, etc.). The moiety can be covalently linked or bound via a second reagent, such as a second antibody, protein A, or a biotin-avidin complex. Paramagnetic moieties and their binding to antibodies are well known in the art. For example, Miltenyi
(1990) Cytometry 11: 231-238.

[0086] The 4B5 antibodies of the present invention can be prepared in several ways. This is SEQ ID NO: 1.
It is most conveniently obtained from cells that have been engineered to express other polynucleotides encoding GD2 mimetic fragments or 4B5 binding fragments, including and 4. For example, cells can be cultured in a suitable medium, and the medium used is
It can be used as a source of antibodies. If desired, matrix-coated channels or beads and cell co-cultures can be included to enhance the growth of antibody producing cells. For the production of large amounts of antibodies, it is generally more convenient to obtain ascites. Method of raising ascitic fluid (method of raising asci)
tes) generally involves injecting hybridoma cells into an immunologically untreated histocompatible or immunoresistant mammal, especially a mouse. A mammal can be primed for ascites production by pre-administration of a suitable composition (eg, pristane).

Alternatively, 4B5 can be synthesized chemically using the sequence data and other information provided in this disclosure, along with standard methods of protein synthesis. A suitable method is the solid phase Merrifield technique. Automated peptide synthesizers are available, for example, from App
lied Biosystems, Inc. (Foster City, CA)
It is commercially available, such as a synthesizer manufactured by the Company.

4B5 can also be obtained using conventional recombinant methods as described in Sambrook et al. (1989). For example, using the amino acid and polynucleotide sequences provided herein and the information provided herein, a polynucleotide encoding either the 4B5 heavy or light chain can be prepared using a suitable expression vector ( It contains control sequences for transcription, such as a promoter). Next, the expression vector is introduced into a host cell. Host cells are grown under appropriate conditions such that the polynucleotide is transcribed and translated into protein. The heavy and light chains of 4B5 can be produced separately and then combined by disulfide bond rearrangement. Alternatively, a vector having separate polynucleotides encoding each strand of 4B5, or a single polynucleotide encoding both strands as separate transcripts, can be transfected into a single host cell. The whole molecule can then be produced and assembled.
Preferably, the host cell is from a higher eukaryote, which can provide a normal carbohydrate complement of the molecule. Thus, the 4B5 produced can be purified using standard techniques in the art. Polynucleotides encoding 4B5 for use in producing 4B5 can be produced synthetically or recombinantly from the DNA sequences provided herein.

A “humanized” antibody is an antibody in which at least a portion of the sequence has been altered from its original form to be more similar to a human immunoglobulin. In one type, H
The chains and the C region of the light chain are replaced with human sequences. This is a fusion polypeptide that includes an H11 V region and a heterologous immunoglobulin C region. For other types, CD
While the R region contains the H11 amino acid sequence, the V framework region is also the converted human sequence. See, for example, EP 0329400. In a third type, the V regions are humanized by designing consensus sequences for the human and mouse V regions and by changing residues outside the CDRs that differ between the consensus sequences. The present invention includes a humanized Mab.

In making humanized antibodies, the choice of framework residues can be important in retaining high binding affinity. In principle, framework sequences from any HuAb can serve as a template for CDR grafting; however, direct CDR substitution into such a framework results in a significant loss of binding affinity for the antigen. It has been demonstrated that can be derived. Glaser et al. (1992) J. Mol. Imm
unol. 149: 2606; Tempest et al. (1992) Biotechn.
9: 266; and Shalaby et al. (1992) J. Mol. Exp. M
ed. 17: 217. The more similar a HuAb is to a native muAb, the less the human framework will introduce distortions into the murine CDRs that may reduce affinity. Based on sequence homology searches against the antibody sequence database, HuAb IC
4 is also muM4TS. 22 provides good framework homology to
Other highly homologous HuAbs, particularly κ light chains from human subgroup I or human subgroup II
Still suitable for the heavy chain from I. Kabat et al. (1987). ENCAD (L
evitt et al. (1983) J. Am. Mol. Biol. 168: 595) are available to predict the ideal sequence for the V region. Thus, the invention encompasses HuAbs with different V regions. Determining the appropriate V region sequences and optimizing these sequences are within the skill of the art. Methods for obtaining antibodies with reduced immunogenicity are also described in US Pat. No. 5,270,202 and EP 699,755.

[0091] Antibody production and isolation methods are well known in the art. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory.
Manual, Cold Spring Harbor Laboratory
, New York. The 4B5 antibody is a human immunoglobulin of the IgG subclass and can be isolated by any technique appropriate for this isotype of immunoglobulin. Purification methods include salt precipitation (eg, using ammonium sulfate), ion exchange chromatography (eg, using a cation or anion exchange column, run at neutral pH, and eluted with a step gradient of increasing ionic strength). ), Gel filtration chromatography (including gel filtration HPLC),
And chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin. 4
B5 can also be purified on an affinity column containing an anti-GD2 antibody. Preferably, 4B5 is purified using protein A-CL-Sepharose ^™ 4B chromatography followed by chromatography on a DEAE-Sepharose ^™ 4B ion exchange column.

[0092] The invention also encompasses hybrid antibodies, wherein one pair of heavy and light chains is derived from a first antibody, while the other pair of heavy and light chains is a different second antibody. Obtained from antibodies. For the purposes of the present invention, one pair of light and heavy chains is from 4B5. In one example, each LH chain pair binds a different epitope of the 4B5 antigen. Such hybrids can also be formed using humanized heavy or light chains.

[0093] Another 4B5 contemplated by the present invention is an antibody in which the heavy or light chain has been modified to provide additional properties. For example, changes in the amino acid sequence can result in reduced immunogenicity of the resulting polypeptide. This change can range from one or more amino acid changes to a complete redesign of a region such as a C region domain. Representative changes include, but are not limited to, changes associated with complement fixation reactions, interactions with membrane receptors, and other effector functions. Peptides in which the various immunoglobulin domains are arranged in an order other than that which occurs naturally are also encompassed by the present invention.

When 4B5 is administered to an individual, it is preferably at least 80% pure, more preferably at least 90% pure, and even more preferably at least 9% pure.
5% pure and free of pyrogens and other contaminants. In this situation, percent purity is calculated as weight percent of the total protein content of the preparation,
And it does not contain constituents that are intentionally added to the composition after 4B5 is purified.

Polynucleotides of the Invention The present invention provides various polynucleotides encoding antibodies 4B5 or fragments of 4B5, which comprises the polynucleotide sequences provided herein (SEQ ID NOS: 1 and 4). And complementary polynucleotides (SEQ ID NOs: 2 and 6). Various embodiments include a number of different combinations of the 4B5 heavy or light chain V region sequences described in this section. In general, a 4B5 polynucleotide of the invention encodes at least one characteristic unique (in comparison to other immunoglobulins) to the 4B5 molecule. Preferably, this feature is related in some manner to the immunological reactivity of 4B5.

The invention encompasses a polynucleotide encoding a portion of the 4B5 light chain V region, wherein the polynucleotide comprises at least about 70 contiguous nucleotides of SEQ ID NO: 4, preferably at least about 80 contiguous nucleotides Contain more preferably at least about 100 contiguous nucleotides, even more preferably at least about 150 nucleotides. The invention also encompasses a polynucleotide encoding a portion of the 4B5 light chain V region, wherein the polynucleotide comprises the CD
At least about 25 contiguous nucleotides of the R1 coding sequence, preferably, at least about 30 contiguous nucleotides, even more preferably, at least about 35 contiguous nucleotides.
Of consecutive nucleotides. The invention also encompasses a polynucleotide encoding a portion of the 4B5 light chain V region, wherein the polynucleotide comprises the CDRs thereof.
It contains at least about 20 contiguous nucleotides of two or CDR3 coding sequences, preferably at least about 25 contiguous nucleotides, and even more preferably at least about 35 contiguous nucleotides.

The invention also encompasses a polynucleotide encoding a portion of the 4B5 heavy chain V region, wherein the polynucleotide comprises at least about 70 contiguous nucleotides of SEQ ID NO: 1, preferably at least about 80 contiguous nucleotides. It contains contiguous nucleotides, more preferably, at least about 100 contiguous nucleotides, even more preferably, at least about 150 nucleotides. The invention also includes a polynucleotide encoding a portion of the 4B5 light chain V region, wherein the polynucleotide contains about 15 contiguous nucleotides of its CDR1 coding sequence. The present invention also encompasses a polynucleotide encoding a portion of the 4B5 light chain V region, wherein the polynucleotide comprises at least about 20 contiguous nucleotides, preferably at least about 25, of its CDR2 or CDR3 coding sequences. It contains contiguous nucleotides, even more preferably at least about 35 contiguous nucleotides.

The present invention includes an isolated 4B5 polynucleotide that encodes a polypeptide having 4B5 immunological activity, wherein the polypeptide comprises at least one of the 4B5 VL chains set forth in SEQ ID NO: 5. Encodes five amino acids. The invention also includes an isolated 4B5 polynucleotide encoding a polypeptide having 4B5 immunological activity, wherein the polypeptide comprises at least five of the 4B5 VH chains set forth in SEQ ID NO: 3. Encodes amino acids. The polynucleotide sequence is set forth in SEQ ID NO: 1 or SEQ ID NO: 4 with changes designed to optimize codon usage, stability, facilitate cloning, or any other purpose. It can be similar to a polynucleotide sequence. Given the amino acid sequence in SEQ ID NO: 3 or SEQ ID NO: 6, designing such polynucleotides is within the skill of the art. Preferred polynucleotides encode at least five amino acids of the 4B5 CDR.

The present invention also includes variants and derivatives functionally equivalent to 4B5, as well as polynucleotides that encode functionally equivalent fragments thereof. These polynucleotides may enhance, decrease, or not significantly affect the properties of the polypeptides encoded thereby. These functionally equivalent variants, derivatives, and fragments exhibit the ability to specifically recognize the C antigen. For example, changes in the DNA sequence that do not alter the encoded amino acid sequence, and changes in the DNA sequence that result in conservative substitutions of amino acid residues, deletions or additions of one or several amino acids, and amino acid residues by amino acid analogs Is a change that does not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions include glycine / alanine; valine / isoleucine /
Leucine: asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan.

A polynucleotide of the present invention may comprise additional coding sequences within the same transcription unit, regulatory elements (eg, a promoter, a ribosome binding site, and a polyadenylation site), an additional transcription unit under the control of the same or a different promoter,
It may include sequences that allow cloning, expression, and transformation of the host cell, as well as additional sequences, such as any such constructs that may be desirable to provide embodiments of the present invention.

The present invention forms a stable hybrid with the polynucleotide encoding the V region of the 4B5 L or H chain, but does not form with other immunoglobulin-encoding regions known at the time of filing the present application. At least about 15 contiguous nucleotides;
Preferably at least about 20 nucleotides, more preferably at least about 25 contiguous nucleotides, more preferably at least about 35 contiguous nucleotides, more preferably at least about 50 contiguous nucleotides, even more preferably at least about 75 nucleotides, More preferably at least about 100
Nucleotides, even more preferably at least about 200 nucleotides, and even more preferably at least about 300 nucleotides. Any set of conditions can be used for this test, as long as there is at least one set of conditions that demonstrate the specificity required for the test polynucleotide. Preferably, the 4B5 coding sequence to which the test polynucleotide binds is the sequence shown in SEQ ID NOs: 2 and 5. Since known immunoglobulin sequences divide into a hierarchy of similarities to the sequence of 4B5, the test is performed by comparing the hybridization of the test polynucleotide to the 4B5 sequence with the hybridization to the most closely related sequence. Can be done. A panel of about 10 sequences most closely related to SEQ ID NO: 1 or 4 is preferred.

[0102] Hybridization reactions can be performed under conditions of different "stringency". Conditions that increase the stringency of a hybridization reaction are well known. See, for example, Sambrook and Maniatis. Examples of relevant conditions include 25 ° C, 37 ° C (in order of increasing stringency).
C, 50 C, and 68 C incubation temperatures; 10 x SSC, 6 x SS
C, buffer concentration of 1 × SSC, 0.1 × SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and its equivalent using other buffer systems; 0%, 25%, Formamide concentrations of 50% and 75%; incubation times from 5 minutes to 24 hours; one, two or more washing steps;
Wash incubation time of 2 or 15 minutes; and 6 × SSC, 1 × S
SC, 0.1 × SSC, or a washing solution of deionized water.

Useful 4B5 polynucleotides encoding fragments of 4B5 can be obtained by generating a polynucleotide fragment (eg, based on SEQ ID NO: 1 or SEQ ID NO: 4), and converting the polypeptide encoded thereby to a desired function. It can be identified by the step of testing. Alternatively, polypeptide fragments encoded by a particular polynucleotide can be prepared and tested for a function of interest. Alternatively, given a 4B5 polypeptide having the desired properties, a polynucleotide encoding the polypeptide can be designed.

[0104] Polynucleotides having 4B5 polymers, fusion proteins, humanized immunoglobulins, single-chain V regions, and coding regions for other particular polypeptides of interest are encompassed by all of these embodiments. These polypeptides are described above.

The present invention also provides a polynucleotide covalently linked to a detectable label.
Such polynucleotides are useful, for example, as probes for the detection of related nucleotide sequences.

The polynucleotide of the present invention can be produced by chemical synthesis, recombinant cloning, PCR,
Or it can be obtained using any combination of these. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain the desired polynucleotide by using a DNA synthesizer or by ordering from a commercial service.

A polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification. A polynucleotide can be introduced into a host cell by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, f-conjugation, or electroporation. Once introduced, the exogenous polynucleotide may be maintained intracellularly as a non-integrated vector (eg, a plasmid) or may be integrated into the host cell genome. Amplified DNA can be isolated from host cells by standard methods. See, for example, Sambrook et al. (1989). RN
A can also be obtained from transformed host cells, or
It can be obtained directly from DNA by using A polymerase.

[0108] The invention further encompasses various vectors containing the 4B5 polynucleotide.
These vectors can be used for expression of the recombinant polypeptide, and these vectors are also a source of 4B5 polynucleotides. Cloning vectors are
They can be used to obtain duplicate copies of the 4B5 polynucleotides they contain, or as a means of storing the polynucleotides in a repository for future recovery. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. They can also be used where it is desired to have intact cells that express 4B5 in an individual and thus synthesize the polypeptide as in gene therapy. Suitable cloning and expression vectors include those known in the art, for example, vectors for use in bacterial, mammalian, yeast, and insect expression systems. Specific vectors and suitable host cells are known in the art and are not described in detail herein. For example, Gacesa and Ramji, Vec
See tors, John Wiley & Sons (1994).

[0109] Cloning and expression vectors are typically selected for with a marker (eg,
Other polynucleotide sequences co-introduced into the host cell, including those encoding proteins necessary for the survival or growth of the host cell transformed with the vector, may have such a marker gene. Only host cells into which the selectable gene has been introduced grow under selective conditions. Representative selection genes include (a) conferring resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate; (b) supplementing auxotrophic deletion; or (c) utilizing from complex media Either it supplies important nutrition that is not possible. Selection of the appropriate marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Vectors also typically contain a replication system that is recognized by the host.

[0110] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from the majority of cloning vectors available in the art. Although the cloning vector of choice will vary according to the host cell for which it is intended to be used, useful cloning vectors generally have the ability to replicate autonomously, and a single cloning vector for a particular restriction endonuclease. It may have a target or may have a marker gene. Suitable examples include plasmids and bacterial viruses such as pUC18, mp18, mp19, pBR322, pMB9, ColE1.
, PCR1, RP4, phage DNA, and shuttle vectors (eg, pS
A3 and pAT28). These and other cloning vectors are available from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

[0111] Expression vectors are generally replicable polynucleotide constructs that include a polynucleotide that encodes a 4B5 polypeptide of interest. A polynucleotide encoding a 4B5 polypeptide is operably linked to appropriate transcription control elements, such as a promoter, enhancer, and terminator. Expression (
That is, for translation), one or more translation control elements such as a ribosome binding site, a translation initiation site, and a stop codon are also usually required. These regulatory elements (transcription and translation) may be in the 4B5 gene, or in a heterologous gene (
That is, from other genes or other organisms). A polynucleotide sequence encoding a signal peptide may also be included to allow the 4B5 polypeptide to cross or be embedded in a cell membrane, or be secreted from a cell. Many expression vectors suitable for expression in eukaryotic cells, including yeast cells, bird cells, and mammalian cells, are known in the art. One example of an expression vector is pcDNA3 (Invitrogen, San Diego).
o, CA), where transcription is driven by the cytomegalovirus (CMV) early promoter / enhancer. The vector also contains recognition sites for multiple restriction enzymes for insertion of the 4B5 polynucleotide of interest. Another example of an expression vector (system) is the baculovirus / insect system.

[0112] Expression systems suitable for use in antibody-targeted gene therapy involving the 4B5 polynucleotide are also encompassed by the present invention. Suitable systems are described, for example, in Brown et al. (1994) V
irol. 198: 477-488; and Miyamura et al. (1994) P
rc. Natl. Acad. Sci. USA 91: 8507-8511.

[0113] Vectors containing the polynucleotides of interest can be prepared by electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where The vector may be introduced into the host cell by any of a number of suitable means, including an infectious agent such as vaccinia virus, described below. Vector or 4B to be introduced
The choice of 5 polynucleotides often depends on the characteristics of the host cell.

[0114] Once introduced into a suitable host cell, expression of the 4B5 polypeptide can be determined using any assay known in the art. For example, the presence of 4B5 polypeptide can be detected by RIA or ELISA of the culture supernatant (if 4B5 polypeptide is secreted) or cell lysate.

A particularly useful expression vector for 4B5 polynucleotides is vaccinia virus, which is composed of 4B5 polynucleotide sequences, which can also be used in vaccine preparations. Moss (1991) Science 252: 1662-1
667. In order to introduce a polynucleotide sequence encoding a 4B5 polypeptide, including a 4B5 polypeptide fragment, into vaccinia, the polynucleotide sequence of interest is first vaccinia virus with flanking sequences homologous to vaccinia DNA not required for replication. Inserted into plasmid containing promoter. The plasmid-containing cells are then infected with vaccinia, which leads to low levels of homologous recombination between the plasmid and the virus, and the vaccinia promoter and the polynucleotide sequence encoding the 4B5 polypeptide into the vaccinia virus genome. Population results. Typically, a 4B5 polynucleotide is inserted into the viral TK (thymidine kinase) gene. Insertion into the TK site attenuates the virus more than 10,000-fold compared to the wild type. Flexner et al. (19
80) Vaccine 88 (Cold Spring Harbor Lab)
org.), pages 179-184. Recombinant viruses are identified by TK ^- phenotype. Preferably, the expression of the 4B5 polynucleotide is under the control of the vaccinia early / late promoter (7.5K), whereby the resulting 4B5
5 polypeptides can be expressed in infected cells throughout the life cycle of the virus. However, other promoters known in the art, such as pH 6, or synthetic promoters can be used. 4B5 polypeptide expression occurs in cells infected with recombinant vaccinia or in individuals immunized with live recombinant vaccinia virus. WR, A
Any one of several strains of vaccinia can be used, including, but not limited to, LVAC, and NYVAC.

[0116] The vectors of the present invention may include one or more polynucleotides encoding a 4B5 polypeptide. This also means that lymphokines (IL-2, IL-4, GM-C
Polynucleotide sequences that encode other polypeptides that enhance, enhance, or regulate the desired result, such as, but not limited to, SF, TNF-α, and IFN-γ. A preferred lymphokine is GM-CSF.
Preferred GM-CSF constructs are those in which the AU-rich element from the 3 'untranslated region and the sequence of the 5' untranslated region capable of forming a hairpin loop have been deleted. Vaccinia vectors encoding recombinant 4B5 variants such as scFvs, chimeras, and polymers are also encompassed by the present invention.

Another embodiment of the present invention is directed to a 4B5 polynucleotide, as described above,
A host cell transformed with a vector containing the B5 polynucleotide sequence. Both prokaryotic and eukaryotic host cells can be used. Prokaryotic hosts include bacterial cells, for example, E. coli. coli and Mycobacteria. Among eukaryotic hosts are yeast cells, insect cells, bird cells, plant cells, and mammalian cells. Host systems are known in the art and need not be described in detail herein. Examples of mammalian host cells include CHO and NS0;
european Collection of Cell Cultures (
(England). For example, transfection of NS0 cells with a plasmid driven by a CMV promoter followed by amplification of this plasmid using glutamine synthetase provides a useful system for protein production. Cockett et al. (1990) Bio / Technology 8:
662-667.

The host cells of the present invention can be used, inter alia, as a repository for 4B5 polynucleotides or as a vehicle for production of 4B5 polynucleotides and polypeptides. They can also be used as vehicles for in vivo expression of 4B5 polypeptides. The 4B5 polynucleotides of the invention can be used in expression systems to produce 4B5 polypeptides, intact 4B5, or recombinant forms of 4B5, as described below.

The polynucleotides of the present invention have several uses. These are, for example,
Useful in expression systems for the production of 4B5. They are also useful as hybridization probes to assay for the presence of 4B5 polynucleotides or related sequences in a sample using methods well known to those skilled in the art. In addition, polynucleotides are also useful as primers to effect amplification of the desired polynucleotide. The polynucleotides of the present invention are also useful in pharmaceutical compositions, including vaccines, and for gene therapy.

[0120] Polynucleotides can also be used as hybridization probes for detection of the 4B5 coding sequence. Suitable samples include cells transformed ex vivo for use in gene therapy. In one example, DNA or RNA is extracted from a sample and optionally run on a gel and / or digested with restriction endonucleases. The processed sample polynucleotide is typically transferred to a medium suitable for washing. The sample polynucleotide is then contacted with a 4B5 polynucleotide probe under conditions that allow a stable duplex to be formed if the sample contains a compatible 4B5 sequence. Any stable duplexes formed are detected by any suitable means. For example, 4
B5 polynucleotide probes can be supplied in labeled form, and the label remaining with the sample after washing directly reflects the amount of stable duplex formed. In a second example, the hybridization is performed in situ. A properly prepared tissue sample is overlaid with a labeled probe to indicate the location of the 4B5 coding sequence.

[0121] Short 4B5 polynucleotides can also be used as primers for PCR reactions, particularly to amplify longer sequences, including regions that hybridize to the primers. This can be done preparatively to produce a polynucleotide for further genetic manipulation. This can also be done analytically, for example, to determine if a 4B5 encoding polynucleotide is present in a sample for diagnostic purposes.

[0122] Another use for polynucleotides is in vaccines and gene therapy.
The general principle is to administer the polynucleotide so as to enhance or attenuate the expression of the polypeptide encoded therein. Thus, the invention encompasses methods of inducing an immune response and methods of treatment that include administering to an individual an effective amount of a 4B5 polynucleotide. In these methods, the 4B5 polypeptide-encoding 4
A B5 polynucleotide is administered to an individual, either directly or via cells transfected with a 4B5 polynucleotide. Preferably, 4B5
The polynucleotide is in the form of a circular plasmid, preferably in a supercoiled configuration. Preferably, the 4B5 polynucleotide is replicated inside the cell. Thus, the 4B5 polynucleotide is operably linked to a suitable promoter, such as a heterologous promoter that is naturally active in cells of the target tissue type. Preferably, once in the cell nucleus, the plasmid persists as a circular, non-replicating episomal molecule. In vitro mutations can be made, for example, in a plasmid construct to encode a molecule with greater immunogenicity.

To determine whether a plasmid containing a 4B5 polynucleotide can express 4B5 in eukaryotic cells, cells such as COS-7, CHO, or HeLa can be transfected with the plasmid. The expression of 4B5 is then determined by immunoassay, eg, by Western blot. Smaller 4B5 polypeptides can be detected, for example, by constructing a plasmid such that the resulting 4B5 polypeptide is fused to a tag, such as a target epitope or enzyme label. Further characterization of the expressed 4B5 polypeptide can be achieved by purifying the peptide and then performing one of the functional assays described herein.

In one mode of gene therapy, the polynucleotides of the invention are used to genetically alter cells ex vivo. In this strategy, cells removed from a donor or cells obtained from a cell line are transfected or transduced with a vector encoding a 4B5 polypeptide and then administered to a recipient. Suitable cells for transfection include peripheral blood mononuclear cells.

In another mode of gene therapy, the polynucleotides of the invention are used to genetically alter cells in vivo. This purpose includes, but is not limited to, treating various types of cancer.

(Polypeptides of the Invention) The present invention also encompasses polypeptide fragments of 4B5 that include at least a portion of the V region of 4B5. Preferred fragments are those having an immunological activity of 4B5. Also preferred are fragments containing amino acid sequences that differ substantially from other immunoglobulins, and fragments containing CDRs. In one embodiment, the invention encompasses a polypeptide fragment of the 4B5 H chain V region, comprising at least 25 contiguous amino acids of SEQ ID NO: 3, more preferably 30 contiguous amino acids, or 5 contiguous of its CDR1. Amino acids or CDR2s thereof
Or at least 7 contiguous amino acids of CDR3, preferably at least 9
Contains two consecutive amino acids. The invention also encompasses a polypeptide fragment of the light chain V region of 4B5, wherein at least 25 contiguous amino acids of SEQ ID NO: 6, more preferably 30 contiguous amino acids, or seven contiguous amino acids of its CDR2, or It comprises at least 8 contiguous amino acids of the CDR1 or CDR3, preferably 10 contiguous amino acids.

[0127] The size of the 4B5 polypeptide may be only the minimum size necessary to provide the desired function. Polypeptides can, if desired, include any additional sequences specific to 4B5 or from heterologous sources. The 4B5 peptide may include only five contiguous amino acids from the 4B5 V region sequence. 7 amino acids from the L chain V region or 4B5 H chain V region of 4B5, more preferably about 10 amino acids, more preferably about 15 amino acids, more preferably about 25 amino acids, more preferably about 50 amino acids, more preferably about 75 amino acids. Polypeptides containing amino acids are also included. Polypeptides comprising the entire 4B5 L chain V region or 4B5 H chain V region are even more preferred. Preferably, the polypeptide is 4
It is derived from B5.

The present invention encompasses modified 4B5 polypeptides that are functionally equivalent to 4B5 or that have been altered but have measurable 4B5 immunological activity. Modified polypeptides having improved 4B5 immunological activity are preferred. Examples of modified polypeptides include peptides that have conservative substitutions of amino acid residues and one or more deletions or additions of amino acids that do not significantly deleteriously alter immunological activity.

One example of this is a 4B5 polypeptide that contains one or more amino acid substitutions as compared to a prototype 4B5 sequence. Substitutions can range from changing or altering one or more amino acid residues to complete redesign of a region such as a V region. Amino acid substitutions, if present, are preferably conservative substitutions that do not adversely affect the folding or functional properties of the peptide. The group of functionally related amino acids for which conservative substitutions can be made are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine /
Tyrosine / tryptophan. Polypeptides of the invention can be in glycosylated or non-glycosylated form, can be post-translationally modified (eg, acetylated, and phosphorylated), or can be synthetically modified (eg, addition of a labeling group). .

A 4B5 polypeptide derivative containing both 4B5 light and 4B5 heavy chains is
It can be formed as separate light and heavy chains and then constructed, or constructed in situ by an expression system for both chains. Such an expression system comprises L
It can be produced by transfecting appropriate cells with a plasmid containing separate transcribable regions for the chains and heavy chain, or by co-transfecting the same cells with the plasmid for each chain. In the third method, H
A suitable plasmid having a chain coding region is transfected into the heavy chain loss mutant.

The heavy chain loss mutant was a rabbit anti-mouse IgG fluorescently labeled according to the supplier's instructions.
(H chain specific, DAKO Corporation, Carpinteria
, CA) by treating about 2 x 10 ⁷ 4B5-producing cells. The stained and unstained cells are analyzed on a fluorescence activated cell sorter. Unstained cells are collected in sterile tubes and plated at 1 cell / well in a 96-well plate by limiting dilution. The culture supernatants were then purified using goat anti-mouse IgG (H chain specific) and goat anti-mouse κ
Assay by LISA. Clones with a kappa positive and IgG negative phenotype are subcloned at least three times to obtain stable 4B5 ^(-H) mutants. Estimated H
MRNA from the chain loss mutant 4B5 ^(-H) clone can be isolated and the sequence of the light chain V region cDNA determined. The inverted PCR of the mRNA for 4B5 V _H was combined with two sets of 5 ′ used for cloning of 4B5 ^(-H) cDNA.
-With primers and 3'-primers (Example 7). H chain loss mutants
Generates no detectable DNA band. Transfection of cells is performed using the appropriate H
Perform with strand plasmid.

Another 4B5 derivative encompassed by the present invention is an antibody in which the 4B5 heavy or light chain has been modified to provide additional properties. For example, changes in the amino acid sequence can result in greater immunogenicity of the resulting polypeptide. This change
It ranges from one or more amino acid changes to a complete redesign of a region such as the 4B5 region domain. The intended change affects complement binding, interaction with membrane receptors, and other effector functions. Proteins in which the various immunoglobulin domains are arranged in a non-naturally occurring order are also encompassed by the present invention.

The invention also encompasses single chain V region fragments of 4B5 ("scFv"). Single chain V region fragments are made by linking light and / or heavy chain V regions by using a short connecting peptide. Bird et al. (1
988) Science 242: 423-426. Any peptide having sufficient flexibility and length can be used as a linker in a scFv. Usually, the linker is selected to have little to no immunogenicity. An example of a connecting peptide is (GGGGS) ₃ , which bridges about 3.5 nm between the carboxy terminus of one V region and the amino terminus of the other V region. Other linker sequences may also be used and may provide additional functions such as a means for attaching a drug or solid support.

All or any part of the heavy or light chain can be used in any combination.
Typically, the entire V region is included in the scFv. For example, an L chain V region can be linked to an H chain V region. Alternatively, a portion of the L chain V region can be linked to the H chain V region or a portion thereof. ScFvs in which the heavy chain V region is from 4B5 and the light chain V region is from another immunoglobulin are also contemplated. It is also possible to construct biphasic scFvs, one component being a 4B5 polypeptide and the other being a different polypeptide, such as a T cell epitope.

[0135] scFv can be any order (for example, V _H - (linker) -V _H - (linker) -V _L or V _L) may be associated with. A tandem scFv can also be made as (X)-(linker)-(X)-(linker)-(X), where X is a 4B5 polypeptide, or a 4B5 polypeptide and another polypeptide. It is a combination of
In other embodiments, the single chain antibody polypeptide has no linker polypeptide or has only a short, inflexible linker. Exemplary configurations include:
_VL - _VH and _VH - _VL are included. This coupling is too short to permit interaction between V _L and V _H within the chain, and this chain forms a homodimer with V _L / V _H antigen binding site at each end. Such molecules are known in the art as "diabodies (
diabody) ".

[0136] scFvs can be produced either recombinantly or synthetically. scFv
For synthetic production of, an automatic synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be a prokaryotic organism, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell; E. coli can be introduced into any suitable host cell, such as E. coli, and the protein expressed by the polynucleotide can be isolated using standard protein purification techniques.

A particularly useful system for producing scFv is described in E. coli. plasmid pET-2 in E. coli
2b (+) (Novagen, Madison, WI). pET-2
2b (+) contains a nickel ion binding domain consisting of six consecutive histidine residues, which allows for purification of the expressed protein on a suitable affinity resin. Other examples of suitable vectors are pcDNA3 (Invitro) described above.
gen, San Diego, CA).

[0138] Expression conditions should ensure that the scFv assumes a functional, and preferably optimal, tertiary structure. Depending on the plasmid used (especially the activity of the promoter) and the host cell, it may be necessary to regulate the rate of production. For example, the use of a weaker promoter, or expression at lower temperatures, may be necessary to optimize the production of a properly folded scFv in a prokaryotic system, or it is preferably a eukaryotic system. It may be to express the scFv in the cell.

Preferred scFvs comprise at least 10 contiguous amino acids of SEQ ID NO: 3 and at least 10 contiguous amino acids of SEQ ID NO: 6, particularly where
And the amino acid of SEQ ID NO: 6 are linked by a 5-20 amino acid linker polypeptide or comprise the LB V region and H chain V region of 4B5.

The present invention also encompasses polymeric forms of the 4B5 polypeptide, including a plurality of 4B5 polypeptides. One embodiment is a 4B optionally coupled to a carrier.
It is a linear polymer of 5 polypeptides. These linear polymers consist of a single 4
It may include multiple copies of a B5 polypeptide, or a combination of different 4B5 polypeptides, and may have tandem 4B5 polypeptides, or 4B5 polypeptides separated by other amino acid sequences. Another embodiment is a 4B5 multiple antigen peptide (MAP). MAPs have a small immunologically inactive core with radially branched lysine dendrites, where many 4B5 polypeptides are covalently linked. See, for example, Posnett et al. Biol.
Chem. 263: 1719-1725; and Tam (1989) Me.
th. Enz. 168: 7-15. The result is a large macromolecule with a high molar ratio of 4B5 polypeptide to core. MAP is an effective immunogen and a useful antigen for immunoassays. 4B5 MAP
Can be made by standard peptide synthesis techniques, or
Or, for example, Quality Controlled Biochemical
als, Inc. , Hopkinton, MA. A representative core matrix is made from three levels of lysine and 8 amino acids.

When using a 4B5 polypeptide as an immunogen, preferably the polypeptide is delivered in combination with a carrier. Any carrier that is not harmful to the host can be used. Suitable carriers are typically proteins; polysaccharides (eg, latex-functionalized Sepharose, agarose, cellulose, cellulose beads, etc.); polymeric amino acids (eg, polyglutamic acid, polylysine, etc.); amino acid copolymers; Large, slowly metabolized macromolecules, such as particles or attenuated bacteria (eg, Salmonella). Particularly useful carrier proteins are serum albumin, keyhole limpet hemocyanin (KLH), certain Ig molecules, thyroglobulin, ovalbumin, and tetanus toxoid. KLH is particularly preferred.

A 4B5 polypeptide of the present invention can be identified in a number of ways. For example, the light and heavy chain V regions prepare a series of short polypeptides that span the entire V region amino acid sequence and determine whether they are specifically recognized by antibodies specific for GD2. Can be screened. Using a series of 20 or 50 amino acid long polypeptides, each 4B5 V region can be examined for useful functional properties. It is also possible to perform a computer analysis of the protein sequence to identify potentially interesting polypeptides, such as those having the shape of GD2 or participating in idiotype-anti-idiotype contacts.

The present invention further encompasses the various adaptations of 4B5 described in this section, combined in various ways to obtain other 4B5 polypeptides having the desired properties. For example, a 4B5 polypeptide having an altered amino acid residue can be included in a MAP. In another embodiment, the 4B5 scFv is fused to a cytokine such as IL-2. All such combinations are contemplated by the present invention.

The polypeptides of the present invention can be made by any suitable procedure, including proteolysis of the 4B5 antibody, by recombinant methods, or by chemical synthesis. These methods are known in the art and need not be described in detail herein. Examples of proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin. Intact 4B5 can be incubated with one or more proteinases simultaneously or sequentially. Alternatively, or in addition, intact antibodies can be treated with a disulfide reducing agent. The peptides can then be separated from one another by techniques known in the art, including, but not limited to, gel filtration chromatography, gel electrophoresis, and reverse phase HPLC.

4B5 polypeptides can also be made in a suitable expression system by expression from a polynucleotide encoding a peptide according to the information provided elsewhere in this application. Typically, a polynucleotide encoding a 4B5 polypeptide will be ligated into an expression vector under the control of a suitable promoter and used to genetically alter a given host cell. Both eukaryotic and prokaryotic host systems can be used. The polypeptide is then isolated from the lysed cells or from the culture medium and purified to the extent necessary for the intended use. Examples of suitable prokaryotic host cells for use in the present invention include E. coli. coli. Examples of eukaryotic host cells include avian cells, insect cells, plant cells, and animal cells, including, but not limited to, COS7, HeLa, and CHO cells.

For certain applications, such as when the 4B5 polypeptide is expressed in a suitable storage medium, such as plant seeds, the 4B5 polypeptide can be used without purification. Fie
dler et al. (1995) Biotechnology 13: 1090-1.
093. For most applications, it is generally preferred that the polypeptide be at least partially purified from other cellular components. Preferably, the polypeptide is at least about 50% pure as a weight percent of total protein.
More preferably, the protein is at least about 50-75% pure. For clinical use, the polypeptide is preferably at least about 80% pure.

[0147] 4B5 polypeptides can be characterized in several ways. For example, 4B5
The polypeptide may have an ability to specifically bind to an antibody specific for GD2.
Alternatively, it can be tested for its ability to specifically inhibit the binding between cancer cells and an antibody specific for GD2. 4B5 polypeptides can also react with anti-CDR3 polypeptides. 4B5 polypeptides can also be tested for their ability to alleviate or ameliorate neoplastic diseases such as carcinomas. It is understood that only one of these properties need be present for a polypeptide to fall within the scope of the invention, but preferably more than one of these properties is present.

[0148] The ability of a 4B5 polypeptide to bind to a specific antibody can be tested by immunoassay. Any form of direct binding assay is suitable. In one such assay, the antibody is labeled. Suitable labels include radioisotopes (eg, ¹²⁵ I), enzymes (eg, peroxidase), fluorescent labels (eg, fluorescein), and chemiluminescent labels. Typically, other binding partners are insolubilized to facilitate washing (eg, by coating on a microtiter plate). After combining the labeling component with the insolubilizing component, the solid phase is washed and the amount of bound label is determined. Another such assay is a sandwich assay, in which a putative 4B5 polypeptide is captured by a first anti-immunoglobulin on a solid phase and developed with a 4B5 antibody. In any of these examples, the degree of 4B5 binding is directly related to the amount of label bound to the solid phase.

To perform an inhibition assay, a putative 4B5 polypeptide is titrated for its ability to reduce the binding of an antibody specific for GD2 to GD2. Typically, anti-GD2 antibodies are labeled, while GD2 is typically insolubilized to facilitate washing. The putative 4B5 polypeptide is typically mixed with an anti-GD2 antibody, and the mixture is then combined with the solid phase. A polypeptide having the properties of 4B5 will proportionally reduce the amount of label attached to the solid phase as compared to a control polypeptide. This test may be more sensitive than measuring direct binding. Because, when present in sufficient concentration, the lower affinity interaction between 4B5 and the anti-GD2 antibody is too weak to form a stable bond, but may prevent the binding of another ligand-receptor pair. May be appropriate.

The invention also includes a method of detecting an antibody specific for GD2 in a biological sample. The method comprises the steps of obtaining a biological sample, contacting the sample with 4B5 under conditions that allow the antibody to bind antigen, and detecting the binding of 4B5, if any, to the anti-GD2 antibody. Is included.

When using intact mouse 4B5, it is generally preferred to deplete any possible sample of anti-mouse immunoglobulin activity. Anti-mouse immunoglobulin antibodies can be isolated, for example, by precipitation with normal mouse IgG or
It can be removed from the sample by adsorption with the adsorbent. Binding of anti-mouse immunoglobulin antibodies, particularly antibodies specific for the Fc region, can be minimized by judicious choice of assay reagents. Other reagents with fewer mouse determinants, such as the F (ab ') ₂ or Fab fragment of mouse 4B5 and humanized 4B5 or 4B5 are suitable.

After the sample is properly prepared, the sample is mixed with an excess of 4B5 under conditions that allow for the formation of a complex between 4B5 and any target antibodies that may be present. Then
The amount of the complex is determined and compared to the complex formed with a standard sample containing the expected range of known amounts of the target antibody. Complex formation can be observed by any method known in the art, such as immunoprecipitation or nephelometry, but generally, radioisotopes (eg, ¹²⁵ I), enzymes (eg, peroxidase and β -Using a reagent labeled with a label such as galactosidase) or a fluorescent dye (e.g. fluorescein) is more sensitive.

Pharmaceutical Compositions of the Invention The present invention encompasses pharmaceutical and immunogenic compositions comprising 4B5, either alone or in combination. Such pharmaceutical compositions and vaccines, either alone or in combination with other forms of therapy (eg, chemotherapy or radiation therapy), are for raising an immune response and for treating neoplastic disease. Useful for

The preparation of a pharmaceutical composition comprising the 4B5 antibody or a polynucleotide or polypeptide derivative thereof as an active ingredient is performed according to generally accepted procedures for the preparation of a pharmaceutical preparation. For example, Remington's Phar
medical sciences, 18th edition (1990), E.M. W
. Martin eds, Mack Publishing Co. See, PA. Depending on the intended use and mode of administration, it may be desirable to further process the active ingredient in the preparation of a pharmaceutical composition. Suitable processing may include sterilization, mixing with suitable non-toxic and non-interfering components, dividing into dose units, and enclosing in a delivery device.

A pharmaceutically acceptable liquid composition can be, for example, a polypeptide embodied herein in a liquid vehicle (eg, water, saline, aqueous dextrose, glycerol, or ethanol). It can be prepared by dissolving or dispersing. The compositions may also contain other medicinal agents, pharmaceutical agents (ph
It can include an armaceutical agent, an adjuvant, a carrier, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.

The pharmaceutical compositions of the invention are administered in a manner appropriate for the form of the composition. Representative routes include subcutaneous, intramuscular, intraperitoneal, intradermal, oral, intranasal, and intrapulmonary (ie, by aerosol). The pharmaceutical compositions of the invention for use in humans are typically administered by parenteral, most typically, intradermal, subcutaneous, or intramuscular routes.

Pharmaceutical compositions for oral, nasal, or topical administration may be in solid form,
It can be supplied in semi-solid form or in liquid form (including tablets, capsules, powders, liquids, and suspensions). Compositions for injection can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for solution or suspension in liquid prior to injection. For administration via the respiratory tract, preferred compositions are those that provide a solid, powder, or liquid aerosol (when used with a suitable aerosolization device). Although not required, the pharmaceutical compositions are preferably supplied in unit dosage forms suitable for administration in precise amounts. Also, the forms contemplated by the present invention are sustained release or sustained release forms, thereby providing a relatively constant level of active compound over an extended period of time.

[0158] Compositions embodied in the present invention may be evaluated for their ability to specifically recognize neoplasia. Thus, a test compound is prepared as a suitable pharmaceutical composition and administered to a test subject. Initial studies are preferably performed on small animals such as mice or rabbits, and then optionally on non-human primates, and finally on humans. Immunogenicity is preferably tested in individuals without a prior antibody response. Appropriate doses of the test composition will be administered in a suitable treatment regimen. It may be appropriate to compare different doses and regimes within the expected range. Such tests are within the skill of those in the art.

The compositions of the present invention are particularly suitable for administration to humans with neoplastic disease. Of particular relevance are melanoma, neuroblastoma, glioma, sarcoma, lymphoma, and small cell lung cancer.

The present invention encompasses a method of raising an immune response in a cancer patient that requires administering an effective amount of 4B5 to the individual. In this context, an "effective amount" is an amount sufficient to elicit an immune response (whether humoral and / or cellular). Preferably, the immune response involves the production of anti-GD2.

In a preferred embodiment, 4B5 is used for raising an immune response and / or for treating and / or alleviating advanced cancer. Suitable cancers are those that express GD2, which is expressed on the surface of tumor cells (eg, lung cancer and melanoma).
As used herein, “advanced” cancer means detectable metastasis, ie, the presence of a detectable tumor mass at a site other than the primary site of the tumor.
The clumps can preferably be detected by imaging techniques known in the art, such as X-rays or CT scans. An effective amount of 4B5 is administered to an individual with advanced cancer to raise, alleviate, or treat an immune response. Administration of an effective amount of 4B5 to an individual with advanced cancer may slow or slow the progression of the disease or ameliorate the disease, as compared to other patients who have not been so treated.

For some situations, including advanced cancer, individuals who have received 4B5
It may be moderate to severely immunocompromised due to either the nature of the previous treatment, the disease itself, or both. Thus, the time required to elicit an immune response and / or the number of 4B5 injections and / or 4B5 per administration
Can vary. For example, an individual may need a longer time to elicit an immune response once 4B5 has been administered. In this case, the individual, even if initially (
That is, even if no immune response is detected within the first month), it is recommended that the immune response be continued to be monitored. As another example, an individual may require more than the average number of injections to elicit an immune response.

The effective amount of 4B5 antigen-binding fragment to be administered depends on several factors, such as the route of administration, the condition of the individual, and the desired purpose. The term "therapeutically effective"
Means that the amount of antigen binding fragment used is sufficient to improve cancer. “Improve” means reducing the deleterious effects of cancer in an individual. Typically, when administered directly, a single dose will be about 10 μg to 20 mg, preferably 250 μg to 10 mg, more preferably 3 μg to 10 mg.
The amount is from 00 μg to 5 mg, still more preferably from 500 μg to 2.5 mg. Administration is typically performed weekly or biweekly until the desired measurable parameter (eg, reduction of disease symptoms) is detected. Administration may then be continued at an appropriate frequency as appropriate (eg, every two weeks or every month).

4B5 is typically injected every two weeks for four injections, then monthly as needed. The timing of the subsequent injection (ie, maintenance dose) depends, inter alia, on the condition and response of the individual being treated. Ab3 levels can be monitored, preferably by the diagnostic methods described herein, for example, to determine when maintenance (booster) administration should be given (typically about every three months).

The dosage range for the administration of 4B5 is large enough to produce the desired effect, wherein the symptoms of the malignancy are adverse side effects (eg, unwanted cross-reactivity,
Anaphylactic reaction). Generally, the dosage is
It will vary with the age, condition, sex, and degree of disease of the patient, and can be determined by one skilled in the art. The dosage may be adjusted by the respective physician in case of any complication. The dosage is about 0.1 m in one or more doses daily for one or several days.
g / kg to about 2000 mg / kg, preferably about 0.1 mg / kg to about 50
It can vary at 0 mg / kg. Generally, when 4B5 is administered in combination with a therapeutic agent, lower doses comparable to those used for in vivo immunodiagnostic imaging may be used.

Another method of administration is intralesional administration, for example, by direct injection into a tumor. Intralesional administration of various forms of immunotherapy to cancer patients does not cause the toxicity seen with systemic administration of immunological agents. Fletcher et al. (1987) Lymphok.
ine Res. 6: 45; Rabinowich et al. (1987) Ca.
ncres. 47: 173; Rosenberg et al. (1989).
Science 233: 1318; and Pizz et al. (1984) Int.
. J. Cancer 34: 359.

Regardless of eliciting and / or treating an immune response, or whether or not administration of 4B5 is indicated, one possible indicator of the efficacy of 4B5 administration is GD2 on tumor cells. Is the density of The density may vary between individuals and may vary widely over the course of 4B5 administration and / or over the course of the disease. As used herein, "density" of GD2 refers to either or both of: (a) the number of cells per total cell in a given biological sample having GD2 on its surface. (B) the amount of GD2 on the surface of each cell. The density (a) is
It is calculated by entering the number of cells in the sample that stained or otherwise indicate that GD2 is present divided by the total number of cells. This density is preferably greater than about 20%, more preferably greater than about 30%, more preferably greater than about 50%, even more preferably greater than about 70%, and even more preferably about 80% Greater, most preferably greater than about 90%. Accordingly, the present invention relates to a method for producing a composition comprising:
More preferably greater than 70%, even more preferably greater than about 80%,
Most preferably, it involves the administration of 4B5 to an individual having a GD2 density greater than about 90%.

Density (b) is the relative intensity of staining of cells (or G
(Intensity of any measurement indicating the presence of D2). For this density, one skilled in the art can make an empirical determination of the density. The density (b) depends on the normal tissue (ie, G
D2 deficient cells, or non-diseased cells), and the preferred range is about 1.5-fold, preferably 1.5-fold, relative to non-diseased cells, as detected by immunohistological staining density. Can be about 3 times, more preferably about 10 times higher expression. Unaffected cells can also be from the same individual.

This does not mean that individuals with lower densities, eg, less than about 50%, are not indicated for administration of 4B5. Without wishing to be bound by one theory, administration of 4B5 induces a series of immune responses that produce a more general response (eg, a cytotoxic T cell response) that is effective against GD2-associated tumors. It is possible that it can be triggered. However, lower densities may indicate that further treatment may be desired.

It is understood that density can also be used as an indicator of the extent of disease and response to 4B5 administration. For example, a sample taken from an individual at the beginning of 4B5 administration may exhibit a density of about 80% (ie, about 80% of the cells exhibit GD2). After receiving 4B5, samples taken from the same lesion could show only about 50% density, indicating that GD2-expressing cells are being destroyed. Similarly, if the intensity of staining of a sample from an individual receiving 4B5 decreases upon receiving 4B5, this indicates that GD2-bearing tumor cells are being destroyed.

4B5 is administered parenterally, preferably intradermally, for the purpose of eliciting an immune response or providing treatment against an individual having an advanced GD2-associated tumor. Other routes of administration include, but are not limited to, intramuscular, subcutaneous, and intradermal. 4B5 can be administered indirectly by treating cultured cells and then introducing these cultured cells into an individual.

Preferably, 4B5 is administered with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmaceutically effective substance. For example, an excipient may give form or consistency to the vaccine composition, or act as a diluent. Suitable excipients include, but are not limited to, solubilizing agents, wetting and emulsifying agents, salts to alter osmotic pressure, encapsulating agents, buffers, and skin permeation enhancers. Examples of pharmaceutically acceptable excipients are Remington's Pharmaceutical Science.
s (Alfonso R. Gennaro, eds., 18th edition, 1990).

Preferably, 4B5 enhances the presentation of 4B5, or otherwise
Used with adjuvants to enhance the immune response to B5. Suitable adjuvants are aluminum hydroxide, alum, QS-21 (U.S. Pat.
7,540), DHEA (US Pat. No. 5,407,684 and US Pat. No. 5,077,284), including precursors and modified forms thereof (eg, DH
EA-S, sulfonated form of DHEA)), β-2 microglobulin (WO9
1/16924), muramyl dipeptide, muramyl tripeptide (US Pat. No. 5
No. 4,171,568), and monophosphoryl lipid A (US Pat. No. 4,436).
No., 728; WO92 / 16231) and derivatives thereof, such as Detox ^™ , and BCG (US Pat. No. 4,726,947). Other suitable adjuvants are aluminum salts, squalene mixtures (SAF-1), muramyl peptides, saponin derivatives, mycobacterium wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, QuilA, cholera toxin B Subunits, polyphosphazenes and derivatives, and immunostimulatory complexes (ISCOMs) (eg, Takahashi et al. (1990) Nature 344: 873-87)
5 described above)). For veterinary use and for the production of antibodies in animals, the mitotic component of Freund's adjuvant may be used.

The choice of adjuvant depends, in part, on the stability of the vaccine in the presence of the adjuvant,
It depends on the route of administration, and the modulating ability of the adjuvant, especially if intended for human use. For example, alum has been approved by the US Food and Drug Administration (FDA) for use as an adjuvant in humans. 4B5 is
It can be administered in the form of a precipitate; for example, alum precipitate 4B5 can be used. QS
If -21 is used, preferably 100 μg is used for each dose preferably administered subcutaneously within about 30 minutes after mixing (with care to mix gently) with 4B5. If Detox ^™ is used, preferably 0.1
2 ml is used for each dose. These doses are preferably administered subcutaneously within about 30 minutes after mixing with 4B5. Adjuvant manufacturers generally provide recommendations regarding dosage, volume, preparation, and route of administration.

Alternatively, 4B5 can be encapsulated, for example, in liposomes. Liposomes suitable for packaging a polypeptide for delivery to a cell are known in the art.

4B5 can be heat treated before administration, and the heat treatment can be in the presence of an adjuvant (eg, alum). For example, 4B5 can be heated at about 40-60C, preferably 45C-55C, for about 5 minutes to 2 hours, preferably 15 minutes to 1 hour. More preferably, the heat treatment is for 30 minutes at 45 ° C. in a sterile vial in a water bath. Heat treatment can be performed anytime before administration. Preferably, the heat treatment comprises 7
Within days. Other heat treatment procedures can be used as long as the desired activity of 4B5 is not significantly impaired.

For the purpose of raising an immune response, 4B5 can be administered in an unmodified form. 4B5
Can be modified to improve its immunogenicity. Methods to improve immunogenicity are:
In particular, it involves crosslinking with an agent such as glutaraldehyde or a bifunctional coupling agent, or conjugation to a multivalent platform molecule. Immunogenicity can also be improved by coupling to a protein carrier, especially a carrier containing a T-cell epitope.

4B5 can be used alone or in combination with other agents that promote the desired activity / purpose. In this context, a "drug" can be any of a variety of substances. Further, “in combination with” means that it can be used simultaneously with, before, or after 4B5. The desired activity is any activity that facilitates, enhances, enhances, or modulates the desired purpose when using 4B5. Agents that can be used include cytokines, lymphokines, adjuvants, and drugs,
It is not limited to these. The drug may also be a substance that facilitates the delivery of 4B5 (eg,
Liposomes) or substances that facilitate delivery of 4B5 to a particular target (eg, cell receptors). For example, 4B5 can be administered with a cytokine such as GM-CSF.

To determine the effect of administration with 4B5, individuals were tested for antibodies to GD2 (
Either humoral or cellular immune responses, or combinations thereof, can be monitored.

To determine the level of GD2 antibody (Ab3) in a biological sample, for example,
Serum or plasma is obtained from the individual. If desired, the sample can be enriched for immunoglobulins before the assay is performed, although this is not usually required. If mouse immunoglobulin is to be used as an assay reagent, the sample is preferably pre-treated to remove anti-mouse immunoglobulin activity. This can be done, for example, by depletion of a mouse immunoglobulin column or by mixing non-specific mouse immunoglobulin into the sample and removing any immunoprecipitate that has formed.

To perform the assay, anti-GD2, which may be present in the sample, is treated with a limited amount of GD2.
Contact with antigen equivalent. This includes isolated GD2, nitrocellulose with GD2 immobilized by direct blotting or by transcription from a polyacrylamide gel, cells expressing GD2, membrane preparations from such cells,
Alternatively, it may be a fixed tissue section containing GD2. Alternatively, anti-idiotypes, especially 4B5, can be used.

[0182] Once the immune complex has formed, it is generally separated from the uncomplexed GD2 analog and the amount of complex present is determined. The complex can be separated by, for example, centrifugation to collect cells or sediment or to capture by solid phase. The amount of complex present can be determined by directly labeling the GD2 analog,
Alternatively, it can be measured by providing by incubating with a secondary reagent. Alternatively, a competition assay can be performed in which the sample is first incubated with the GD2 analog and then a non-limiting amount of a labeled anti-GD2 reagent, which can compete with the anti-GD2 that may be present in the sample. Suitable labels include radioactive, enzyme, fluorescent, and chemiluminescent labels. The standard curve consists of, in place of the sample, a solution known not to contain anti-GD2 and various relative concentrations of anti-GD2.
2 is created. Samples containing unknown amounts of anti-GD2 are generally assayed in parallel, and the relative amount of anti-GD2 contained therein is determined by comparison to a standard curve. A preferred assay for determining anti-GD2 levels using the 4B5 antibody is described in more detail in the following section.

The isoform of an anti-GD2 antibody can be determined in an immunoassay by including an isoform-specific reagent at either the separation or the labeling step. For example, anti-human IgG can be used to separate or detect antibodies of the IgG class present in clinical samples of human origin. The presence of specific anti-GD2 of the IgG class generally indicates a memory response. The presence of anti-GD2 of the IgM class generally indicates ongoing immunostimulation (eg, as may be due to the presence of a GD2-expressing tumor) or ongoing treatment with 4B5.

If desired, the anti-GD2 antibody detected in the biological sample can further include; for example, whether the anti-GD2 antibody is specific for a relevant epitope on GD2, in competition with anti-GD2 (Abl). By determining whether Ab1 and A
Competition assays for b3 are known and are not described in detail herein.

[0185] Anti-GD2 antibodies can also be tested to determine if they are cytotoxic. Complement-mediated cytotoxicity (CMC) is determined, for example, by using GD2-expressing target cells labeled with ⁵¹ Cr. Labeling can be achieved by incubating about 10 ⁶ cells with about 200 μCi of Na ₂ ⁵¹ CrO ₄ for 60 minutes at 37 ° C., followed by washing. The assay is performed by incubating the antibody (or a clinical sample containing the antibody) with target cells. The opsonized cells are then washed and incubated with a complement source; for example, guinea pig serum pre-adsorbed to remove endogenous antibody activity. After an appropriate incubation time at 37 ° C., the release of ⁵¹ Cr into the medium is determined and compared to the release of ⁵¹ Cr from non-opsonized control cells. ⁵¹ Cr release correlates with CMC activity.

Another way to characterize anti-GD2 antibodies is by testing their ability to contribute to the ADCC response (Cheresh et al., (1986), Cancer R
es. 46: 5112). Radiolabeled GD2-expressing labeled cells are incubated with anti-GD2 (in the form of heat-inactivated serum) and effector cells. Normal human peripheral blood mononuclear cells (PBMCs) are suitable effector cells, and are preferably used at an effector: target cell ratio of about 100. After about 4 hours at 37 ° C,
The percentage of ⁵¹ Cr released is determined as a measure of ADCC activity.

[0187] The cellular immune response in a subject who has received 4B5 can be quantified by performing a standard functional assay for specific T cell activity.

[0188] One type of assay measures T cell proliferation. In this test, peripheral blood mononuclear cells (PBMCs) are obtained from whole blood cells collected from treated subjects. For experimental animals, splenocytes can also be used. T cells are, for example, Ficoll (TM)
) Can be enriched by centrifugation on a gradient such as The cells are then cultured at various concentrations in the presence of GD2 or (more commonly) irradiated GD2-expressing cells. Preferably, the stimulator cells are autologous to the responder cells, particularly for histocompatibility class II antigens.

Another type of assay measures T cell cytotoxicity. In this test, enriched T to provide for lysis of ⁵¹ Cr-labeled GD2-expressing labeled cells prepared as described above.
A cell population is used. Preferably, the effector cells are autologous to the target cells, especially with respect to histocompatibility class I antigens. The T cell population is GD2
Alternatively, it can be primed with the relevant cell line. Then, T cells and approximately 10 ⁴ labeled target cells at various ratios; for example, in wells of a microtiter plate, are fit Serra. If necessary, the plate is centrifuged to initiate cell contact, and the cells are both cultured at 37 ° C. for 4-16 hours. The percentage of specific release of ⁵¹ Cr into the medium is determined by the labeled target (negative control) cultured alone and 0%.
. Target dissolved in a detergent such as 1% Triton ™ X-100 (
(Positive control).

Other relevant measures to determine the effect of 4B5 administration may include clinical trials as may be appropriate in determining the progression of a suspected type of cancer. Such tests include, but are not limited to, inflammatory indicators, mammograms, and radiation scintigraphy.

Another method in which 4B5 can be used is to assay for the presence of antibodies or other immune components that bind to 4B5 or GD2. Such components may be present in the immunocompatible host following therapeutic administration of 4B5, or may occur spontaneously due to the presence of a GD2-expressing tumor. Assays can be performed on biological samples, usually clinical samples.

In one embodiment of the present invention, 4B5 is used to detect the presence of anti-GD2, especially anti-4B5 idiotype, that may be present in a biological sample.
Samples may be suitably prepared by enriching for antibody activity, if necessary, prior to performing the assay. If the biological sample appears to contain antibody activity against non-idiotypic regions of 4B5 (specifically anti-mouse immunoglobulin), it is preferred to perform the assay to remove them or avoid their detection. An anti-mouse immunoglobulin antibody can be obtained from a sample by, for example, normal mouse Ig.
It can be removed by precipitation with G or adsorption using a mouse Ig adsorbent. Binding of anti-mouse immunoglobulin antibodies (particularly binding specifically to the Fc region) can be minimized by judicious choice of assay reagents. F (ab ') ₂ or Fab fragments of 4B5 and other mouse immunoglobulin reagents are particularly suitable.

After the sample is properly prepared, it is mixed with an excess of 4B5 functional equivalent under conditions that allow for the formation of a complex between 4B5 and any anti-GD2 that may be present. The amount of conjugate is then determined and the expected range of known amounts of anti-G
Compared to the complex formed using a standard sample containing D2. Complex formation can be observed by immunoprecipitation or turbidimetry, but is labeled with a radioisotope such as ¹²⁵ I, an enzyme such as peroxidase and β-galactosidase, or a label such as a fluorescent dye such as fluorescein. It is generally more sensitive to use the reagents provided.

[0194] Antibody assays can be performed in the liquid phase. For example, anti-GD2 can be mixed with label 4B5. Alternatively, anti-GD2 in a sample can be used to compete with labeled anti-GD2 for a binding site on 4B5. Generally, bound and unbound labels are separated to quantify the percent bound. Suitable separation methods include gel filtration chromatography and, optionally, in the presence of polyethylene glycol, precipitation with antibodies to immunoglobulins from the species from which the sample was obtained. Alternatively, the percentage of bound and unbound labels can be detected in situ, eg, using a fluorescent / quench label pair or an enzyme / inhibitor label pair. For example, US Pat.
, 996,345 (Ullman et al.).

As noted above, it is generally more convenient to perform capture assays using solid phase-bound reagents such as polyethylene test tubes, microtiter plate wells or magnetic beads. In a competitive capture assay, unlabeled anti-GD2 in the sample competes with labeled anti-GD2 reagent for binding to 4B5. 4B5
Can be attached directly to a solid support or can be captured later, for example, using anti-4B5. In this assay, the amount of label bound to the solid phase is inversely proportional to the amount of anti-GD2 in the sample.

In a sandwich capture assay, anti-GD2 is captured by 4B5 attached directly to the solid phase or via a secondary reagent. After washing, the anti-GD2 is replaced with a suitable species of anti-immunoglobulin or secondary 4B to which the label has been attached, directly or indirectly.
Detect using 5 antibodies. Alternatively, the anti-immunoglobulin can be attached to a solid phase,
Mark 4B5 is then used to complete the sandwich. The anti-immunoglobulin used is isotype-specific, and then the class of the antibody can also be determined. In this type of assay, the amount of label bound to the solid phase is positively correlated with the amount of anti-GD2 in the sample.

Other methods for measuring specific antibodies are known to those of skill in the art and are adapted for measuring anti-GD2 by using 4B5 as a target antigen. All of the methods so adapted are embodied in the present invention.

4B5 can also be used to measure cellular anti-GD2 activity, particularly the level of anti-4B5 idiotype. In a preferred embodiment, 4B5 can be used to identify anti-GD2 T cells, which are defined for the purposes of the present invention as lymphocytes that express T cell receptors that bind to the 4B5 idiotype. 4B
5 can be labeled and contacted with a population of cells suspected of containing anti-GD2 T cells. Alternatively, unlabeled 4B5 can be mixed with the cells and subsequently mixed with a labeled secondary reagent such as labeled anti-mouse immunoglobulin or protein A. Labels suitable for this purpose include radiolabels and fluorescent labels. The use of fluorescent labels also allows anti-GD2 cells to be separated from non-specific cells in a fluorescent cell analyzer and separator.

The present invention also provides a method of using 4B5 to remove a label (eg, radioactivity) from an individual who has received a labeled anti-GD2 antibody (Abl), for example, for radioscintigraphy or radiotherapy. Include. One problem common to the use of antibody-targeted radionuclides (ie, radioimmunotherapy) is the presence of excess Ab1 in the system, which limits the dose of radiolabeled antibody for treatment. In addition, effective imaging using radiolabeled antibodies is hindered by excess circulating radiolabeled antibodies, which often require several days to be cleared from the circulation and tissues. In these methods of the invention, 4B5 is administered at a specific time after administration of the labeled anti-GD2 antibody. The intent is to make 4B5 complex with anti-GD2, for example, at sites other than the tumor in the space within the circulation and stroma, thereby promoting its clearance. As a result, the level of the labeled moiety (eg, radioisotope) in the unaffected tissue is reduced, and the image of the tumor (compared to nearby tissue) is enhanced. Similarly, where a radionuclide is provided to a subject for irradiation of a tumor site, a reduction in nearby exposure of unaffected tissue is desirable. Thus, the present invention encompasses a method of treatment where a radiolabeled anti-GD2 antibody is administered at a treatment dose, followed by a molar excess of 4B5.

In any of these applications, there is a sufficient molar excess over labeled anti-GD2 to locate and bind any anti-GD2 that does not localize to the tumor site.
The amount of B5 is selected. The timing of administration and the amount of 4B5 will depend on the nature of the radiolabeled antibody, the type of radioisotope used and the condition of the individual. Preferably, 4
The molar ratio of B5 to anti-GD2 antibody is at least about 5: 1, more preferably about 25: 1 to 200: 1. Preferably, 4B5 is administered 5-24 hours after the individual has received the anti-GD2 antibody.

The present invention also encompasses a method of detecting the presence of an anti-GD2 antibody that binds to a tumor cell, the method comprising allowing the tumor cell to bind to the anti-GD2 antibody at 4B5 for a sufficient time. Intervening, treating, and detecting the presence of any complexes formed. The intent is to have 4B5 detect anti-GD2 previously attached to the tumor cells; alternatively, promote the binding of anti-GD2 to the tumor cells by forming a multivalent anti-GD2 / 4B5 immune complex. In the former example, 4B5 is provided using a detection label or a means to which the label can be attached. In the latter example, anti-GD2
Alternatively, either 4B5 is provided with the label.

[0202] For example, this strategy can be used to identify GD2 antigen-bearing cells in an isolated cell suspension. Cells can be used for anti-GD2 and 4B5 sequentially or simultaneously.
And washed, then the labeled cells are detected. Preferred labels for this embodiment include fluorescent labels (eg, fluorescein, rhodamine and Texas red). Optionally, labeled cells can be separated from unlabeled cells; for example, by sorting on a fluorescent cell analyzer or by affinity separation, using any solid phase positive or negative immunoselection techniques known to those of skill in the art. And can be separated.

For example, strategies can also be used to detect or image a tumor in an affected subject. Anti-GD2 and 4B5 are administered to the subject (usually continuously)
And accumulate at the tumor site. Suitable labels include radiolabels (eg, ¹¹¹ In, ¹³¹ I and ^99m Tc). The tumor can then be detected or visualized using standard techniques of radioscintigraphy.

The various compositions of the present invention may be used alone or in combination with other active agents that promote the desired purpose or provide the desired adjunct treatment. Suitable active agents include the aforementioned anti-tumor agents and biological response modifiers and Douill
and effector cells as described in Ard et al. (1986) Hybridomas (Supplement 1: 5139).

Using 4B5, a combination therapy design is possible. A therapeutic drug
4B5 in combination with effector cells and the same or different therapeutic agents
It may be desirable to administer before the administration of. For example, patients may initially
IFN-γ and interleukin-2 (Il-2) are administered daily for 5 days, and can be treated on day 5 by administering 4B5 in combination with effector cells, IFN-γ and Il-2.

Subjects suitable for treatment with 4B5 or a combination therapy comprising 4B5 include the pathology of any neoplasm, especially where carcinoma is suitable for treatment with a pharmaceutical composition of the present invention. Patients suspected of being at risk for effects. Subjects with a history of cancer are particularly relevant. Suitable human subjects for treatment include two groups that are classified according to clinical criteria. “Advanced disease” or “high cancer load”
or burden) have a clinically measurable tumor. Clinically measurable tumors are identified based on tumor mass (eg, palpation, CAT scan or X-ray; positive biochemical or histopathological markers may themselves be insufficient to identify this population) Can be done. Pharmaceutical compositions embodied in the present invention are administered for the purpose of eliciting an anti-tumor response in these patients, for the purpose of palliating the patient. Ideally, a reduction in tumor mass would result, but any clinical improvement would be beneficial. Clinical improvement includes a reduced risk or rate of progression or reduction of the pathological consequences of the tumor.

[0207] A second group of suitable subjects is the "adjuvant group", which is known in the art. These are individuals with a history of cancer, but were responsive to another mode of treatment. Prior treatments could include, but were not limited to, surgical resection, radiation therapy and traditional chemotherapy. As a result, these individuals have no clinically measurable tumors. However, these individuals are suspected of being at risk for disease progression, either near the original tumor site or by metastasis.

This group can be further divided into high risk individuals and low risk individuals. Classification is based on features observed before or after the initial treatment. These features are known in the clinical arts and are well defined for each different cancer. A typical feature of the high-risk subgroup is that the tumor has invaded nearby tissues or indicates lymph node involvement.

[0209] Another suitable group of subjects is a group that has a genetic predisposition for cancer but does not yet show the clinical characteristics of cancer. For example, women who test positive for a genetic mutation associated with breast cancer but are still of childbearing age may be treated prophylactically with 4B5 to prevent the development of cancer until preventive surgery is appropriate. May wish to accept.

The pharmaceutical compositions embodied in the present invention are administered to a patient in an adjuvant group or any of these subgroups to elicit an anti-cancer response. Ideally, the composition will delay or better reduce the risk of cancer recurrence (ie, improve the cure rate). Such parameters can be determined relative to other patient populations and other treatment modalities.

It will be appreciated that a crossover between these two groups of patients will occur and that the pharmaceutical compositions of the invention may be administered at the appropriate time. For example, 4B5 treatment can be administered before or during traditional treatment of patients with high tumor burden, and can be continued until the tumor is clinically undetectable. 4B5 treatment may be continued in patients who initially enter the adjuvant group but show signs of relapse. The attending physician has the discretion to determine how or when to use the compositions of the present invention.

In another embodiment, a 4B5 polypeptide can be conjugated to a carrier.
If the 4B5 polypeptide provides a binding site, but is correctly positioned to be too small and not immunogenic, the polypeptide can be bound to a suitable carrier. Many techniques for obtaining such linkages are known in the art and need not be described in detail herein. Any carrier that does not itself induce the production of antibodies harmful to the host can be used. Suitable carriers are typically large, slowly metabolized macromolecules (eg, proteins); polypolysaccharides (eg, latex-functionalized Sepharose, agarose, cellulose, cellulose beads, etc.); polymeric amino acids (eg, poly Glutamic acid, polylysine, etc.); amino acid copolymers; and inactivated virus particles or attenuated bacteria (eg, Salmonella). Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid and other proteins well known to those skilled in the art. As will be apparent to those skilled in the art, 4B5 polypeptides and the above-described recombinant forms of 4B5 (eg, fusion proteins) can then be fused to other amino acid sequences. For example, 4B5 scFv is a cytokine (eg, IL-2)
Can be fused.

[0213] The ability of a 4B5 polypeptide to bind to Ab1 and / or Ab3 can be assessed in several ways. In one test, binding of the 4B5 polypeptide to Ab1 is determined, for example, by radioimmunoassay (RIA), for example, by reacting a radiolabeled 4B5 polypeptide with Ab1 or Ab3 coated on a microtiter plate. Can be tested directly.

In another procedure, binding to Ab1 or Ab3 is determined by a competitive immunoassay. In one variation of this procedure, the binding of a labeled 4B5 polypeptide or a functionally equivalent fragment to Ab1 differs by A
b1, another Ab2, 4B5 or analog thereof, another 4B5 polypeptide, G
It is measured in the presence of an extract containing D2 or GD2, or other proteins. % Inhibition is calculated according to the following equation:% inhibition = In _{[1- (R T -R C)} / (R MAX -R C)] × 100% In another variation, the test fragment with putative 4B5 activity ,
It is tested for its ability to prevent binding between Ab1 and Ab2, or Ab1 and GD2. This test is more sensitive in some applications. Because
The lower affinity interaction between 4B5 and Ab1 can be too weak to form a stable bond, but if present at sufficient concentrations, will prevent the binding of another ligand-receptor pair. Because it may be appropriate. GD2 can be provided as a purified antigen or a GD2-expressing cell. Assays include Ab1 or G
This can be done by labeling either D2 or Ab2 and, if necessary, immobilizing the other members of the ligand-receptor pair on a solid support to facilitate separation. The test fragment is incubated with the labeled reagent, and the mixture is then presented to an immobilized target or test cell to determine whether the test fragment can inhibit binding. The degree of inhibition is
Correlates with 4B5 activity.

[0215] Various examples of competitive assays are known in the art to include the following. One test of 4B5 polypeptide activity is to measure the binding of radiolabeled Ab1 to semi-purified or purified GD2 in the presence of varying amounts of 4B5 polypeptide. The Ab1-GD2 mixture is then added to a plate coated with the 4B5 polypeptide and binding is compared to binding with labeled Ab1 alone. Preferably, this test is performed using a non-saturated amount of labeled Ab1 to detect changes in binding to small amounts of competitive GD2. In another competitive assay, GD2 positive target cells are grown as confluent monolayers in 96-well tissue culture plates. The binding of radiolabeled Ab1 in the presence or absence of the 4B5 polypeptide is determined. The degree of inhibition can be compared to the degree of inhibition of the intact 4B5 polypeptide or other 4B5 polypeptides.

[0216] A 4B5 polypeptide is considered to have bound Ab1 if inhibition was present when compared to a negative control (eg, an irrelevant anti-idiotype antibody that did not bind to Ab1).

In all of the above assays, the labeled molecule was labeled with a radioisotope (
That is, it will be apparent to those skilled in the art that ¹²⁵ I) and biotinylated molecules, and molecules for enzymatic detection, may be labeled in various ways, such as with non-radioactive labels such as fluorescent and chemiluminescent labels. is there.

The tests discussed above can also be used to compare the characteristics of various 4B5 polypeptide fragments. For example, a competitive assay may be performed, where:
A first 4B5 polypeptide is competed for binding to Ab1 in the presence of varying amounts of a second 4B5 polypeptide. Such tests may indicate the relative extent of binding affinity or other characteristics.

Another way to characterize a 4B5 polypeptide is to test its ability to generate an immune response. As used herein, "immune response" refers to either a humoral response or a cellular response, or both. As used herein, "ability to elicit an immune response" refers to any individual (including humans).

[0220] The ability of a 4B5 polypeptide to produce a humoral response can be determined by testing for the presence of antibodies that bind to the 4B5 polypeptide following administration of the 4B5 polypeptide. It is understood that this antibody (Ab3) was absent or less present prior to administration of the 4B5 polypeptide. Immunogenicity is preferably tested in individuals without a previous anti-4B5 response. Examples of suitable individuals include, but are not limited to, mice, rabbits, monkeys, and humans. For this test, the individual is administered a 4B5 polypeptide. The amount per administration and the number of administrations will vary depending on the individual. The required amount can be readily determined by empirical observations.

[0221] The presence of Ab3 indicates that the serum is first pre-incubated with autoimmune globulin to block antibodies to isotype or allotypic determinants, and then GD2 and / or 4B5 using, for example, ELISA or RIA. It can be determined by testing the serum for binding to the polypeptide. For example, different dilutions of pre-reacted serum are reacted with 4B5 coated on microtiter plates. Unrelated Ab2 is provided as a control. After washing, the Ab3-4B5 complex is labeled using, for example, ¹²⁵ I-labeled 4B5 in a homogeneous sandwich assay. The results from this assay are compared to the results obtained before administration of the 4B5 polypeptide. Or,
Binding to GD2 positive cells can be tested using immuno-flow cytometry.

The binding of Ab3 to GD2 can also be determined by immunoprecipitation using GD2 positive tissue samples, or by immunoreactivity or dot blot analysis.
In one method of dot blot analysis, a semi-purified extract of GD2 is blotted directly onto a nitrocellulose membrane. The filter is then incubated with Ab3 containing serum and the reaction is developed with enzyme-conjugated anti-immunoglobulin. If Ab3 binds to GD2, a positive blot should appear. To test using tissue samples, an immunoperoxidase assay can be used.

If desired, Ab3 raised by a 4B5 polypeptide can be further characterized. For example, a competitive assay can be performed to determine whether Ab3 shares the Ab1 idiotope. In this test, sera from individuals immunized with the 4B5 polypeptide are tested for inhibiting the binding of the labeled 4B5 polypeptide (or intact 4B5) to Ab1. Inhibition indicates that Ab3 and Ab1 contain at least similar binding determinants. Similarly, competition between Ab1 and Ab3 for binding GD2 (partially purified, purified, or on the surface of GD2 positive cells) is similar to A
It can be tested by co-incubating a fixed amount of labeled Ab1 with different dilutions of serum or Ab1 preparation containing b3 and GD2.

As will be apparent to one of skill in the art, Ab3 also has 4B using the assay described above.
5 can be used to characterize the polypeptide.

Another method of characterizing a 4B5 polypeptide is by testing its ability to elicit antibodies that are cytotoxic. The method for this determination is described above.
For determination of complement-mediated cytotoxicity (CMC), SKBR3 (target) cells (ie, cells expressing GD2) are labeled with ⁵¹ Cr. Label is about 200 μC
This can be achieved by incubating approximately 10 ⁶ cells with i Na ₂ SO ₄ at 37 ° C. for 60 minutes, and then washing. The assay is performed by adding and incubating serum that is expected to contain the antibody. Then, L
Guinea pig serum (or other source of complement) previously adsorbed on S174-T cells is added. Then, after an appropriate incubation period at 37 ° C., the extent of ⁵¹ Cr release is measured and compared to non-opsonized control cells. ⁵¹ C
Release of r correlates with CMC activity. Herlyn et al. (1981) Int. J.
Cancer 27: 769.

Another way to characterize 4B5 polypeptides is to use anti-GD involved in the ADCC response.
By testing the ability to raise 2 antibodies. Cheresh et al. (1986) C
ancer Research 46: 5112-5118. In this assay, cells cultured or expressing GD2 are labeled with ⁵¹ Cr and used as target cells. Normal human peripheral blood mononuclear cells (PBMC) are used as effector cells. Preferably, the ADCC assay is performed in the presence of heat-inactivated serum at an effector: target cell ratio of 100: 1 for 4 hours, although other suitable conditions may also be used. Then, the amount of ⁵¹ Cr released is measured.

[0227] The 4B5 polypeptides of the present invention can also be characterized by their ability to elicit a cellular response. As used herein, a "cellular response" is a response that involves a T cell and can be observed in vitro or in vivo.

One way to detect a cellular immune response is to assay for T cell proliferative activity. In this test, the cellular immune response is measured by the proliferation of peripheral blood mononuclear cells (PBM) incubated with the 4B5 polypeptide.
Peripheral blood mononuclear cells are isolated from blood after the required number of doses of 4B5 polypeptide and incubated with various concentrations of 4B5 polypeptide. If mice are used, T cells are obtained from the spleen. T cells are, for example, Ficol
by centrifugation in gradient, such as l ^TM, it is enriched. A non-specific mitogen such as PHA is provided as a positive control, and incubation with an irrelevant anti-idiotype antibody is provided as a negative control. Preferably, the stimulator cells are autologous to the responder cells, especially for histocompatibility class II antigens. After incubating the PBM for the appropriate number of days to allow growth, [ ³ H] thymidine incorporation is measured. In many cases, a suitable time is five days. If desired, a determination of which subset of T cells are proliferating can be made using flow cytometry. If necessary, spleen T cells were obtained from RL. 172 (anti-CD4 ⁺ ) or mAb. Either CD4 ⁺ or CD8 ⁺ can be pre-depleted prior to the proliferation assay by incubation with a monoclonal antibody such as 168 (anti-CD8 ⁺ ) and complement.

Another method for detecting a cellular immune response is to test for T cell cytotoxicity (CTL) activity. For this test, T lymphocytes (ie, an enriched T cell population) are isolated (typically from spleen cells) for use as targets in a standard ⁵¹ Cr release assay. Kantor et al., (1992)
J. Natl. Cancer Inst. 84: 1084-1091. ^{The 51} Cr release assay is described above.

Another method of characterizing a 4B5 polypeptide is to test its ability to ameliorate, delay, or reduce the extent of a GD2-associated tumor, of a GD2-associated tumor. Such tests may include indicators of inflammation, radioscintigraphy, or measurement of circulating GD2 levels (such assays are commercially available).

In summary, 4B5 polypeptides have many uses. 4B5 polypeptides can be used to induce an immune response in an individual, preferably an anti-GD2 response. They also detect and monitor Ab3 levels, or
can be used to purify b3. 4B5 polypeptides are also useful for the treatment of diseases associated with GD2, such as certain lung cancers and melanomas.

Thus, the present invention includes a method for inducing an immune response in an individual, comprising administering a 4B5 polypeptide in an amount effective to elicit an immune response. Preferably, the individual has a tumor associated with GD2. In this context, an "effective amount", whether humoral and / or cellular, is an amount sufficient to elicit a measurable immune response. An effective amount can be administered in one or more administrations.

[0233] The present invention also relates to 4B5 (ie, Ab3 and / or
Or a method for detecting an antibody that binds to Ab1). These methods are applicable, for example, in clinical settings for monitoring Ab1 or Ab3 levels in individuals, and in industrial settings, where commercial production of Ab3 is desired. These methods comprise contacting Ab3 and / or Ab1 in a sample with a 4B5 polypeptide under appropriate conditions to form a stable complex between Ab3 and / or Ab1 and the 4B5 polypeptide; and Detecting the stable complex, if any, formed. Many immunoassay methods are known in the art and are described herein.
As a further explanation, a test sample potentially containing Ab3 and / or Ab1 is typically mixed with a predetermined, non-limiting amount of a detectably labeled (eg, a radioisotope or enzyme) 4B5 polypeptide. Can be done. In a liquid phase assay, unreacted reagents are removed by a separation technique (eg, filtration or chromatography). In these immunoassay techniques, the amount of label associated with the complex positively correlates with the amount of Ab3 and / or Ab1 present in the sample. A similar assay can be designed where Ab3 and / or Ab1 in the test sample compete with the labeled antibody for binding to a limited amount of 4B5 polypeptide. Here, the amount of the label is negatively correlated with the amount of Ab3 and / or Ab1 in the sample. Suitable samples for measuring Ab3 and / or Ab1 levels are biological samples, including serum or plasma, preferably serum. Other samples include tissue samples.

In addition, the present invention also includes the step of contacting a biological sample containing Ab3 (and / or Ab1) with the 4B5 polypeptide, and obtaining the complex, if any, formed thereby. To purify Ab3 (or Ab1). Typically, a 4B5 polypeptide is associated with an affinity matrix for affinity column purification. Such methods are conventional in the art and need not be described in detail herein.

(Kit Comprising 4B5 Polynucleotide and / or Encoded 4B5) The present invention provides a kit comprising 4B5 polynucleotide and 4B5, preferably
Includes diagnostic kit. Diagnostic procedures using 4B5 polynucleotides and 4B5 can be performed by diagnostic institutions, laboratory laboratories, practitioners, or private individuals. The clinical sample is optionally pre-treated to enrich for the target being tested. Then
The user applies the reagents included in the kit to detect a change or alteration in the level of the diagnostic component.

Kits embodied by the present invention include kits that allow certain users to detect the presence of 4B5 polynucleotides and / or anti-GD2 antibodies (both Ab1 and Ab3). If desired, reagents, such as 4B5, included in the kit can be conjugated to a label to allow detection of any complexes formed with the target in the sample. In another option, a second reagent is provided that is capable of binding to the first reagent after the first reagent has found the target, thereby providing a detectable label. For example, labeled anti-mouse IgG can be provided as a secondary reagent for use with intact 4B5. Labeled avidin can be provided as a secondary reagent when the primary reagent is conjugated to biotin.

The kit can be used to test various biological samples, including liquid samples, cell suspensions, and tissue samples. Suitable assays using 4B5 that can be supplied in kit form include those described herein. Each reagent is supplied in solid form or in a liquid buffer, as appropriate for stock keeping and later replacing or adding to the reaction medium when the test is performed. Dissolved / suspended. Proper packaging will be provided. The kit may optionally provide additional components useful in the procedure. These selectable components include buffers, capture reagents,
Chromogenic reagents, labels, reaction surfaces, detection means, control samples, instructions, and information for interpretation include, but are not limited to.

The above description provides, among other things, detailed methods for preparing 4B5, along with polynucleotides encoding 4B5, 4B5 polypeptide fragments, and other derivatives. One skilled in the art can practice embodiments of the invention by reference to the sequence data for 4B5 provided herein. The following example,
It is provided for the purpose of illustration, not limitation of the invention.

Example 1 (Purification of Recombinant 4B5) 4B5 contained in a hybridoma cell culture medium or a lysate of 4B5-expressing cells
Antibodies can be purified by standard procedures known in the art (see, for example, “Method
s in Enzymology "(Academic Press, Inc.
) Was purified on a protein G Sepharose column. Fractions enriched for purified antibodies were collected and analyzed by SDS-polyacrylamide gel electrophoresis and visualization by staining. The results are shown in FIG.
5 matching the heavy (55 kDa) and light (23 kDa) chains of the immunoglobulin
The 5 kDa and 23 kDa proteins are recombinant or hybridoma 4B5
Was detected in samples containing Matches the molecular weight of the heavy / light chain dimer,
An additional band of approximately 110 kDa was also detected in the sample containing recombinant 4B5. This is most likely due to incomplete reduction of disulfide bonds bridging the two immunoglobulin chains.

Example 2 (Similarity of antigenicity between recombinant 4B5 and hybridoma 4B5 as determined by ELISA) To determine the ability of recombinant 4B5 to bind antigen 14G2A, ELISA
Plates were coated with 1 μg / ml 14G2A or mouse IgG as a negative control. Coated ELISA plate
Incubated at 8 ° C for 16-18 hours. The plate was washed with PBS-3% BSA.
For 1 hour at room temperature. They were then incubated for 2 hours at room temperature using either recombinant 4B5 or hybridoma 4B5 in PBS, or control human IgG or culture medium in PBS. The plate was washed and incubated with biotinylated anti-human IgG, and then incubated for 1 hour with streptavidin-conjugated alkaline phosphatase. After washing, p-nitrophenyl phosphate substrate is added to each plate,
After the incubation, the plates were read at 405n with an ELISA plate reader.
m.

The specific binding of the antigen 14G2A to the recombinant 4B5 is shown in FIG. These results indicate that recombinant 4B5 has antigen binding specificity that is indistinguishable from hybridoma 4B5 antibody.

Example 3 Antigen Specificity of Recombinant 4B5 and Hybridoma 4B5 Determined by Western Blot Analysis Equal amounts of antigen 14G2A and control samples, mouse monoclonal antibodies K914 and 9227 were first loaded on SDS-polyacrylamide gel Analyze by electrophoresis and then use recombinant 4B5 or hybridoma 4 under standard conditions.
Immunoblotting was performed using any of the B5 (for reference, see Methods
in Enzymology "(Academic Press, Inc.)
checking). Both recombinant 4B5 and hybridoma 4B5 are antigen 14
It reacted with G2A and did not react with the mouse monoclonal antibody. FIG. 4 shows the results.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the detailed description and examples should not be construed as limiting the scope of the invention, the scope of which is determined by the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of a general antibody structure.

FIG. 2 shows purification of recombinant 4B5 on a protein G Sepharose column. The 4B5 antibody produced by the hybridoma cells is shown in lanes 1 and 2. Recombinant 4B5 antibody is shown in lanes 3 and 4. Lanes 1 and 3 contain 3 μg of protein, and lanes 2 and 4 contain 6 μg of protein.

FIG. 3 shows the determination of antigenic similarity between recombinant 4B5 and hybridoma 4B5 by ELISA analysis. Black bars indicate binding of 4B5 antibody or control human IgG to antigen 14G2A. White bars indicate non-specific binding of 4B5 antibody or control human IgG to mouse IgG.

FIG. 4 shows the binding specificity of recombinant 4B5 to antigen 14G2A by Western blot analysis.

FIG. 5 shows a plasmid expression vector pNB2-4B5.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/39 A61K 47/48 39/395 48/00 51/00 C07K 16/00 45/00 16/42 47/48 C12N 1/15 48/00 1/19 C07K 16/00 1/21 16/42 G01N 33/577 C12N 1/15 33/68 1/19 C12N 15/00 ZNAA 1/21 A61K 37/24 5 / 10 37/66 G01N 33/577 43/00 33/68 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE , IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP ( GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ , DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT , UA, UG, US, UZ, VN, YU, ZW (72) Inventor Saleh, Mansole United States Georgia 30068, Marietta, St. Lyon Place 5408 (72) Inventor Nasov, Mark United States of America 92131, San Diego, Mira Lago Way 11734

Claims (44)

[Claims] 1. A substantially isolated polynucleotide sequence, wherein said polynucleotide sequence encodes an antigen binding fragment of an antibody, said fragment having the amino acid sequence of SEQ ID NOs: 3 and 6. Nucleotide sequence. 2. A substantially isolated polynucleotide sequence, wherein said polynucleotide sequence encodes at least five consecutive amino acid residues of SEQ ID NO: 3 or 6. 3. The polynucleotide of claim 1, wherein said coding sequence is within SEQ ID NO: 1. 4. The polynucleotide of claim 3, wherein said coding sequence is within SEQ ID NO: 4. 5. The polynucleotide of claim 3, wherein said polynucleotide encodes at least five consecutive amino acid residues of a CDR. 6. An isolated polynucleotide comprising a region of at least 20 contiguous nucleotides, wherein said polynucleotide is capable of forming a stable duplex with a polynucleotide consisting of SEQ ID NO: 2 or 5. Polynucleotide. 7. An isolated polynucleotide comprising a region of at least 20 contiguous nucleotides, wherein said polynucleotide comprises SEQ ID NO: 1 or 4
And a polynucleotide capable of forming a stable double strand. 8. The polynucleotide of claim 1, wherein said polynucleotide comprises a cloning vector. 9. The polynucleotide according to claim 1, wherein said polynucleotide comprises an expression vector. 10. The expression vector according to claim 9, wherein said expression vector is vaccinia. 11. A host cell containing the recombinant polynucleotide according to claim 7. 12. A pharmaceutical composition comprising the polynucleotide according to claim 1, 2, 3, 4, 5, 6, or 7, and a pharmaceutically acceptable excipient. 13. An immunogenic composition comprising the polynucleotide sequence of claim 1 and a pharmaceutically acceptable excipient. 14. A composition comprising an antigen-binding fragment of an antibody, wherein the fragment has the amino acid sequence of SEQ ID NO: 3 and the light chain V region has the amino acid sequence of SEQ ID NO: 6. 15. The composition of claim 14, wherein said antigen-binding fragment comprises a whole native antibody, a bispecific antibody, a chimeric antibody, a Fab,
F (ab ') 2, a single chain V region fragment (scFv) and a fusion polypeptide, wherein the fusion polypeptide contains an antigen-binding fragment fused to a chemically functional moiety. ,Composition. 16. The composition of claim 15, wherein said whole native antibody is a 4B5 antibody. 17. The composition according to claim 16, wherein said 4B5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and said light chain comprises SEQ ID NO: 6.
A composition having the amino acid sequence of 18. The composition of claim 15, wherein said moiety is a signal peptide, an agent that enhances immunological reactivity, an agent that facilitates binding to a solid support, a vaccine carrier, A composition selected from the group consisting of a biological response modifier, a toxin, a detectable label, a paramagnetic label, and a drug. 19. The composition of claim 18, wherein said factor that enhances immunological reactivity is a bacterial superantigen. 20. The composition according to claim 18, wherein the factor promoting binding to the solid support is selected from the group consisting of biotin and avidin. 21. The composition of claim 18, wherein said immunogenic carrier is selected from the group consisting of any physiologically acceptable buffer. 22. The composition of claim 18, wherein said biological response modifier is a cytokine. 23. The composition of claim 22, wherein said cytokine is tumor necrosis factor, interleukin-2, interleukin-4, interleukin-12, granulocyte macrophage colony stimulating factor and γ. -A composition selected from the group consisting of interferons. 24. The composition of claim 18, wherein said detectable label is a radioisotope, a fluorescent compound, a colloidal metal, a chemiluminescent compound, a bioluminescent compound, an enzyme, a substrate, a complement. A composition selected from the group consisting of an agent and an inhibitor. 25. A polypeptide comprising at least 5 consecutive amino acid residues of SEQ ID NO: 3 or 6. 26. The polypeptide of claim 25, wherein said five consecutive amino acid residues are from a CDR. 27. The polypeptide according to claim 25, further comprising an immunoglobulin C region. 28. A polymer peptide comprising the plurality of peptides according to claim 25. 29. The composition of claim 25, wherein said composition further comprises a pharmaceutically acceptable excipient. 30. The composition of claim 29, wherein said excipient is a liposome preparation. 31. An immunogenic composition comprising the composition of claim 25, wherein said immunogenic composition is a pharmaceutically acceptable excipient and an effective amount to enhance an immune response. A composition further comprising an adjuvant of 32. A method of treating a patient having a neoplasia, said method comprising administering to said patient an effective amount of a composition according to claim 25. 33. The method of claim 32, wherein the individual has a clinically detectable tumor. 34. The method of claim 32, wherein said method is a method for mitigating said neoplasia. 35. The method of claim 32, wherein a tumor previously detected in said individual has been treated and said tumor has not been detected clinically at the time of administration of the antigen binding fragment. A possible way. 36. The method of claim 32, wherein the method is a method of reducing the risk of a clinically detectable recurrence of a tumor. 37. The method of claim 32, wherein the antigen-binding fragment comprises subcutaneous, intramuscular, intraperitoneal, intracavity, intrathecal, transdermal, or intravenous injection. The method, wherein the method is administered by parenteral administration selected from the group. 38. The method of claim 32, wherein the antigen binding fragment is labeled with a therapeutic moiety. 39. The method of claim 38, wherein said therapeutic moiety comprises a radioisotope, an antitumor agent, an immunomodulator, a biological response modulator, a lectin and a toxin. The more choice, the method. 40. A composition comprising a substantially purified recombinant antigen corresponding to 4B5, wherein the composition comprises an H chain V having the amino acid sequence of SEQ ID NO: 3.
A composition comprising a region, and wherein the light chain V region has the amino acid sequence of SEQ ID NO: 6. 41. The composition of claim 40, wherein said antigen corresponding to 4B5 is present in an immunogenic amount, and further comprising an effective amount of said composition to enhance an immune response to GD2. A composition comprising an adjuvant. 42. A method for detecting an antibody specific for GD2 in a sample, the method comprising the steps of: a) under conditions allowing the formation of a stable antibody-antigen complex. Contacting the sample with the composition of claim 14; and b) detecting any stable complex formed in step a). 43. A kit for detecting or quantifying a polynucleotide, said polynucleotide comprising a polynucleotide encoding 4B5 or a fragment thereof in a biological sample, wherein said kit is in a suitable package. A kit comprising the polynucleotide according to claim 1. 44. A kit for detecting or quantifying an antigen corresponding to 4B5 in a biological sample, the kit comprising the antibody according to claim 14 in a suitable package.