CA1340311C - Screening for antibodies which bind carbohydrate epitopes of tumor-associated antigens, and uses thereof - Google Patents

Abstract

Cells which produce antibodies which bind carbohydrate epitopes of glycoproteins are identified by screening with mucinous body fluids in raw, or preferably partially purified, form. Fine differences in antibody specificity may be detected by further screening with mucinous body fluids treated to alter the carbohydrate epitope interest, e.g., removal of sialic acid from a sialosyl Lewis-a epitope. The selected antibodies may be used in immunopurification, immunodiagnosis and immunotherapy.

By this method, antibodies have been found whose binding to carbohydrate epitopes such as sialosyl Lewis-a is relatively insensitive to pH. Such antibodies are of particular value in immunological methods where pH is a consideration.

Description

13403t l ~CREENING FOR ANTIB4DIES
WHICH BIND ~PRQRyDRATE EPI~OPES OF TVMOR '~S~rT~ED ANTIGENS, AND USES THEREFOR

BACRGROUND OF THE lNVkNllON
Field of the Invention This invention relates to a method of screening monoclonal antibody-producing clones to identify those producing antibodies which recognize a desired carbohydrate determinant, such as CA
19-9. Such antibodies are useful in immunopurification, immunodiagnosis, and immunotherapy. By this method, we have identified antibodies suitable for immunodetection at physiological pH of antigens bearing the CA19-9 determinant.

Information Disclosure Statement Tumor-associated antigens are antigens which are present in the serum and tissues of cancer patients. Many ~uch antigens are also expressed in embryonic tissues, and, at low levels, in the tissue and serum of healthy individuals. Many of the tumor-associated antiqens are glycoproteins, glycolipids, or mucopoly~accharides.

The portion of an antigen to which an antibody binds is called the antigenic determinant, or epitope. An antibody raised against ~ glycoprotein may recognize a protein epitope, a .

13~0~11 carbohydrate epitope, or an epitope formed by the ~unctlon of the protein and carbohydrate moietie6. It is known that the terminal carbohydrate ~eguence of a glycolipid ~ay alco be found on a glycoprotein, thus, a glycolipid and a glycoprotein may bear the same carbohydrate epitope. See Rauvala and Finne, FEBS Lett., 97:1 (1979); McIlhinney, et al., Biochem. J., 227: 155 (1985).

Hakomori, ~MDnoclonal Antibodies Directed to Cell-Surface Carbohydrates", in Monoclonal Antibodies and Functional Cell Jines Proqress and A~plications Ch. 4 (1984) discusses the development of various anti-carbohydrate monoclonal antibodies.
He notes that t~ere are various difficulties in producing these antibodies. According to Hakomori, carbohydrate moieties in glycoproteins ~re only weakly immunogenic. While the carbohydrate chains in some glycolipids are said to be strongly immunogenic, others reportedly were weakly immunogenic, or perhaps not immunDgenic at all.

An ideal antibody reagent would always bind all to all antigens of interest, and only to those antigens: in practice, a modest degree of cross-reactivity is tolerated, which varies from one application to the next.

Only about se~en monosaccharides have been identified in the oligosaccharides of mammal$an glycoproteins and glycosphingolipids. These are sialic acid (acetylneuraminic acid), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), D-galactose (Gal), D-mannose (Man), D-glucose (Glu) and L-fucose (Fuc). Elycosphingolipids are composed of a base (e.g., ~phingosine), a fatty acid, and carbohydrate. The base i~ linked to the fatty acid via an amide linkage, and this substructure is called ceramide. The ceramide is glycosidically linked to the carbohydrate. Gangliosides are sialic acid-containing glycosphingolipids. The carbohydrate moieties of GSLs are the immunodominant portion of the molecule. See Yogeeswaran, "Cell 1~40~11 ..

Surface Glycolipids and Glycoproteins in malignant Transformation", 38 Advances in Cancer Research 289 (1983).

One of the first tumor-associated glycoproteins to be identified was carcinoembryonic antigen. See Hansen, US
4,180,499. As many as nine to twelve Fab antibody fragments may bind simultaneously to this 180,000 Dalton molecule.
Egan, et al., Cancer, 40: 458 (1977). The majority of the antigenic sites for which antibodies are available are polypeptide regions, however, antibodies which recognize carbohydrate epitopes of CEA are known. See Nichols, et al., J. Immunol., 135: 1911 (1985). Many anti-CEA antibodies react with binding sites which are dependent on the tertiary structure of the protein. Rogers, Biochim. & Biphys. Acta, 695: 227 (1983).

The carbohydrate structures of several erythrocyte glycoprotein and glycolipid antigens are known to behave as tumor and differentiation markers. See Feizi, et al., Nature, 314: 53 (1985).

One family of red blood cell surface antigens is called the "Lewis group." The Lewis-a determinant has been characterized as a trisaccharide, Beta-Gal(1-3)-[alpha-Fuc(1-4)]-GlcNac. Lewis-a determinants are found as terminal sugar sequences on both glycosphingolipids and on glycoproteins.
See Watkins, "Biochemistry and Genetics of the ABO, Lewis, and P Blood Group Systems," in 10 Adv. Human Genetics Ch. 1 (1980). Artificial Lewis-a determinant-bearing antigens have been prepared. Lemieux, US 4,137,401. Assays for Lewis-a antigens are known. See Steplewski, US 4,607,009.

Monoclonal antibody 19-9, produced by a hybridoma prepared from spleen cells of a mouse immunized with cells of the human colon carcinoma cell line SW1116, detects a serum antigen which appears associated with gastrointestinal and pancreatic cancers.
X

.

The ~ntibody 19-9) has been depo~ited ~s ATCC HB 8059.
Koprowski, US 4,471,057. The epitope of this ~ntibody is reportedly ~ carbohydrate with the ~ugar seguence NeuNAc-alpha(2 3)-Gal-beta(1-3)-tFuc-alpha(1-4)~-GlcNac-beta-(1-3)-Gal.
Magnani, et al., Cancer Research, 43: 5489-92 (1983). It should be noted that this "CAl9-9" epitope includes whst may be described as a sialylated Lewis-a with an additional galactose unit. For its biosynthesis, see Hansson and Zopf, J. Biol.
Chem., 260: 9388 (1985). It i~ known that the binding of 19-9 to CRC cell line SW1116 is abolished by pretreatment of the cells with neuraminidase. US 4,471,057. In SW1116 extracts, it apparently occurs as a ganglioside (with the structure sialosyl-Lea-beta(1-3)-Gal-beta(1-4)-Glc-betal-Ceramide) , but in serum, it is expressed as a mucin.

Mucins are glycoproteins of high molecular weight and high carbohydrate content. They ~re known to be secreted by the ceroviscous tissues found in the mouth, lungs, cervix and intestines. They are believed to provide a protective coating, shielding cells from osmotic and pH gradients and from physical trauma. A typical mucin has a molecular weight in excess of 500,000 daltons and a carbohydrate content of 60-80%. A typical mucin may possess as many as 200 oligosaccharide chains attached to a polypeptide backbone. Tumor-associated oligosaccharides on mucins include CA 19-9 (a.k.a. GICA)~ DuPan-2, CA 1 and YPAN 1.
Neuraminidase, an enzyme that ~electively cleaves sialic acid from oligosaccharides, alters the antigenic activity of many of these mucins. Rittenhouse, et al., Laboratory Medicine, 16: 556 (1985).

One problem with natural antibody-producing cell~ i8 that it is difficult to maintain them indefinitely in culture. In 1975, Kohler ~nd Milstein introduced a procedure for the continued production of monoclonal antibodie6 using hybrid cells (hybridomas). It entailed the fusion of spleen (antibody-.

134031 l producing) cell6 from ~n immunized ~nimal with ~n immortalmyeloma cell line in order to obtain lmmortalized ~ntibody-producing cell6. ln order to obtain ~ monoclonal ~ntibody which would recognize a colorectal cancer-associated ~ntigen, Koprowski immunized a mou6e with CRC cells. The hybridomas he eventually produced were 6creened for their Ability to produce antibodies which bound the immunizing cells. (It i6 also possible to obtain cell lines which continue to produce antibodies by other techniques, 6uch as optimization of culture conditions.) - Herlyn, et al., J. Immunol. Meth., 80: 107-116 (1985) describes a typical 6creening. Mice were immunized with intact tumor cells of various types, spent medium from tumor cell cultures, membranes of tumor cells, glycolipid extracts of tumor cells, and PEG precipitates from the 6era of cancer patients.
The hybridomas (12,818 in number) were first 6creened with the 6pent medium in which cultured normal and mal ignant cells had been grown. The more promising hybridomas (95) were then 6creened with the cells themselves. Finally, 40 clones were screened with cancer patients' 6era. One antibody, CO 29.11, was selected for detailed 6tudy. Its ability to bind to isolated sialylated Lewis-a glycolipid and to LRwi~-a was compared to that of the previously developed 19-9 antibody.

Herrero-Zabaleta, Bull. Cancer, 74: 387-396 (1987) reported the use of immunoprecipitated (with antibody 19-9) components of the fluid of a mucinous ovarian cyst for mouse immunization.
Splenocytes from the immunized mouse and myeloma cells were fused to obtain hybridomas. The hybridomas were 6creened for the ability to produce a pattern of immunoperoxidase 6taining on paraffin 6ections of esophageal mucosae which was 6imilar to that generated by 19-9. Only the 6upernatants of five of the 150 hybridoma wells 6howed reactivity with this material and only one had a pattern fiimilAr to that of 19-9. The antibody (121 SLE) was compared with 19-9 for it6 ability to react with ovarian . . .

13 4 0 31 l ~ucinous cyst extr~ts. Both were reactive. In ~ddition, s~mples of the cyst wall were fixed ~n ethanol, e~bedded ~n paraffin, and cut into ~ections. The ~ections were then deparaffinized and incubated with neuraminidase. Both ~ntibodies were tested for their ability to bind to the neuraminidase-treated 6ections and neither exhibited ~ignificant reactivity. mis is a very time-consuming procedure. Herrero-Zaba~eta did not use neuraminidase-treated mucinous cyst fluids in his study, and the his unpurified cyst fluids were used only for characterization and not for screening.

Xortright, W0 87/01392 proposes imprDving the binding of a monoclonal antibody to a human carcinoma tumor antigen by removing sialic acid which apparently sterically hindered formation of the immuno-complex. Schauer, TIBS 357 (Sept. 1985) discusses the dual role of sialic acid i~ both masking and accentuating antigenicity, ~nd sets forth the structures of the natural sialic acids.

A heterogeneous immunoassay is one in which the antigen of interest i6 bound into an antigen-antibody complex, and this complex is physically separated from the remainder of the sample.
There are two basic types of heterogeneo~s immunoassay. In a sandwich assay, the sample antigen is bound by both an immobilized antibody and a labeled anti~ody, thus forming a ternary complex. For this to be possible, the antigen must bear at least two epitopes sufficiently far apart to permit the antigen to be simultaneously ~ound by bDth antibodies. See David, US 4,376,110. Sandwich assays are, unfortunately, prone to displaying a "high dose hook effect." ~ragle, US 4,595,661.

In heterogeneous competitive immunoassay~, the sample antigen competes with a known quantity of ~it antigen for the binding 6ites of a kit antibody. Either the kit antigen may be labeled and the kit anti~ody immobilized, or v~ce versa.

, 1340311 Competitive inhibition assay~ are generally t~nqht to have the disadvAntage of a ~hort dynamic 6tandard curve range a6 compared with sandwich assays. Moreover, since only one blnding event i~
required to produce a signal, the assay may be le6s discriminating.

The commercial assays for CA19-9, marketed by Centocor, Abbott, Commissariat a L'Energie Atomique, Hoffman-LaRoche, Inc., Sorin Biomedica and FujiRebio, are all ~andwich assays.

Delvillano, Jr., Wo 84/00758 describes a forward sandwich immunoassay for CA 19-9. Delvillano states that with regard to detecting CAlg-9 antigen with the 19-9 antibody, the preferred pH
was 2.5-6.5, and especially 4.5. Table 4 ~hows the effect of buffer pH on CA 19-9 detection. It is generally known that pH
may alter the binding affinity of an antibody, and it has been ~uggested that the pH of an incubation may be selected to increase specificity. Mosmann, et al., J. Immunol., 125: 1152 (1980). It is also known that acidic pH may dissociate existing antigen-antibody complexes in ~erum samples. Thomson, et al., PNAS (ffSA) 64: 161 (1969).

~ oprowski, US 4,471,057 did describe a competitive immunoassay for a "monosialoganglioside" bearing the epitope recognized by antibody 19-9 (ATCC HB 8059). A serum sample was incubated with this antibody, and the mixture was then brought into contact with a surface having attached thereto a monosialoganglioside antigen from a known human CRC cell. The assay was commercially impractical, as it included three overnight incubations, two washes, and an indirect signal system.
Significantly, none of the aforementioned commercial assays for CAl9-9 are competitive assay~.

Adachi, US 4,389,392 de~cribes a method for determining the level of tumor-associated glycoprotein or glycolipid in a ~ample .. .. .. . ..

1340~1 L

by incubating the eample with a lectin ~nd ~easuring the ~mount ~f bound or unbound lectin. Pukel, US 4,507,391 diecloses ~n immunoassay for a GD3 ganglioside. ~imilarly, Irie, US 4,557,931 discloses an assay for GM2 ganglioside.

No admission is made that any of the foregoing references constitute prior art or pertinent prior art, that the publications accurately reflect the actual experimental work of the authors, or that the dates of publication are exact.

SUMMARY OF THE lNv~NllON
one of the greatest advantages afforded by monoclonal antibodies i6 that a pre-defined immunogen is not required in order to produce antibodies of a predefined specificity.
Increasingly, the problem of understanding the exact binding specificity of an antibody has become the focus of much study.
To conduct these studies a significant guantity of ~ntibody is usually required in a purified state: moreover, pure, well-defined antigen is required but is typically expensive to obtain if available at all. The more detailed the understanding of the specificity of an antibody becomes, the easier it is to use it as a reagent or pharmaceutical agent. Although the best immunization and screening procedure for a particular analyte is likely to be for different classes of antigen, a general method for determining which antibodies are useful to study or in order to identify those of a predefined specificity manifested in different classes and sub-classes is useful. For example, IgMs have been classifically been thought of as good agglutinating reagents while IgG's have typically been desired for sandwich assays. Moreover, some subclasses are better suited for "antibody dependent cellular cytotoxicity" - based therapy and some antibodies are more easily purified than others.

Typically, the initial screen of a serie6 of monoclonal antibodies is to determine whether or not it reacts with the .

- 1340~11 , ~munogen. ~hereafter, a ~reat number of different combinations of ~creening procedures have been employed ~epending on the specific purpo~e to which an ~ntibody i6 reguired (for example see Weltman U.S. Patent 4,689,311). Thereafter, the method of production, screening, purification must bè developed ~nd clinical analysis, and studies related to the exact chemical specificity of the antibodies must be conducted.

It is particularly desirable to ~ave a method for guickly making the decision for production in the case of a ~earch for an anti-carbohydrate antibody. The definition of fine ~pecificity of an anticarbohydrate antibody to various components of the epitope is more important than in other antigens in view of the microheterogeneity of carbohydrate-bearing glycoconjugates (for example, Lewisa, CA 50, and CAl9-9 may be found on the same mucin molecule). Therefore, while one would attempt to eliminate cross-resctivity whether an antibody was carbohydrate or not, other classes of molecule do not have as many repeating ceguences and related 6tructures.

There is considerable interest in identifying monoclonal antibodies which are specific for particular carbohydrate determinants, particularly those containing a terminal sialic acid. However, the screening of hybridomas to identify those producing the desired antibodies is a laborious and expensive process.

Each of the ccreening materials previously known to the art has its disadvantages. It is known that there may be sntigenic differences between tumor cells grown in vitro and those propagated in the human body. See Freifelder, MOLECULAR BIOLOGY
(2d ed. 1987); Freshney, ed., ANIMAL CELLS IN CVLTURE: A
PRACTICAL APPROACH (1986). Consequently, antibodies which bind well to cultured tumor cells, or to their supernateF or membrane sonicateF, will not necessarily bind to a tumor product naturally 13~0311 ~ . .

expressed at high level~ in a patient'~ body fluid~.

A particular antigen may be fully purified from a ~elected tumor cell, or a ~ynthetic antiqen with the ~ame epitope may be prepared. Purification and 6ynthesi6 are expen6ive, and availability of substantial quantities of the purified or synthetic antigen is likely to be a problem.

The patient'~ 6erum, in raw or partially purified form, may also be used as a 6creening material. However, ethical considerations limit the amount of blood which may be withdrawn from an individual, particularly one 6uffering from a life-threatening disease. Moreover, many clinical tests make use of serum samples, so there is a high demand for this material.

For our purposes, we found that the pathological fluids from the thoracic and peritoneal cavities, which are aspirated in the course of normal treatment of the patient snd available in large volumes, very useful. Both of these cavities are lined with membranes which 6ecrete 6mall amounts of 6erous fluids into the cavities. Thus, peritoneal fluid (ascites) is normally ~ecreted by the peritoneum. Under pathological conditions (inflammation or cancer) large amounts of serous or mucinous fluids can be found in these cavities, secreted either by the inflamed membranes of the cavity or by malignant or benign cells in the cavity.

We have found that the mucinous fluids from the thoracic and peritoneal cavities of cancer patients, or from benign or malignant cyst6, make excellent 6creening and immunizing materials. Often, as a result of the cancer, these fluids are overproduced and accumulate. It i6 usually clinically desirable that the excess -fluids be removed from the body, while the removal of blood i6 usually undesirable. Upon removal, these mucinou~ fluids are usually discarded. The~e fluids do not .

usually a ~ umulate in a healthy patient. When the accumulation is responsive to cancer, it i6 reasonable to ~Ypert that these fluids may be Ynriched for tumor-a6~0clated ~ub~tances. The glycoprotein antigens, and especially muci~ antigens, are of particular ~ntere~t.

Surprisingly, the glycoproteins, though of nonserological origin, are of value for the screening of antibodies in ~erodiagnostic assays. (N.B. We will 6peak interchangeably of ~creening antibodies and screening clones: it should be understood that it is not the clones themselves but rather their antibodies which ~re tested.) Moreover, the mucinous fluids may be taken from a patient who i6 6uffering from a different cancer than the one which ic to be diagnosed.

It ~hould be noted that materials ~uitable for use in the characterization of a ~elected antibody are not necessarily ~uitable for use in the 6creening of hundreds or thousands of antibodies. Screening material6 must be available in large quantities at a relatively low cost. They must also be easy to handle.

In a preferred embodiment, these mucinous fluids are partially purified to obtain a high molecular weight (usually greater than 20~,000 daltons, and preferably greater than 2,000,000 daltons) fraction. The partially purified fractions will mainly comprise m~ins, with perhaps a few other high molecular weight materials such as IgM aggregates and antibody:antigen complexes.

Thus, in one embodiment of our invention, a body fluid, accumulated by the body as a conse~uence of a cancerous condition, i8 used a6 a screening material. ~referably, this fluid is ~ mucinous fluid such a6 a6cite6 fluid, pleural effusion, or cystic fluid, and de~irably, this fluld is partially purified so that it is enriched for mucins. The purification may be by size, since mucins are of high molecular weight, or by affinity, using a lectin or antibody affinity column, In the case of screening for antibodies which recognize the sialosyl Levis-a (SLA) determinant, it is desirable to use both the F1 and F2 fractions described hereafter as screening materials since these may include distinct species of SLA-bearing mucins.

Particular mucinous products may serve as positive or negative "screens", depending on the extent to which their component mucins contain the carbohydrate determinant of interest. The use of a negative screen allows one to refine the selection so as to limit the selection to antibodies which are "specific" for the determinant of interest. It should be noted that the term "specificity" is a relative one.
Generally, an antibody whose affinity for an interfering species is not more than 5% of its affinity for the analyte of interest would be considered essentially specific; in certain contexts, a higher level of cross-reactivity could be tolerated.

Besides their use as screening materials, these mucinous fluids or partially purified mucins are also useful as immunizing agents in the preparation of the antibody-producing clones. Preferably, a combination of mucins or mucinous fluids from different patients is employed.

In a preferred embodiment of the invention, the antibodies are screened to determine whether they bind to a particular carbohydrate structure of a selected mucin. The native mucin is used as a positive screen and the mucin in a treated form, wherein its carbohydrate structure is changed, is used as the negative screen.

~r . .. . . , . . . . .. _ . . .

Preferably, the nature of the modification i6 the removal of one or more of the component 6ugar6 ~and e~pecially the terminal ~ugar) of the carbohydrate determinant of lntere6t by glycosidase. However, the modification could be the addition of one or more 6ugars to the terminus of the carbohydrate chain by a glycosyltransferase, or the modification of one or more of the suqars by an epimerase or an isomerase. Moreover, it is not necessary that the modification be made enzymatically, though the use of enzymes is preferred.

In the case of a method of ~creening for antibodies 6pecific for sialylated Lewis-a determinants, it is preferable that the modification be the removal of sialic acid. This may be effected by any 6ialidase, but preferably by neuraminidase. The 6ialidase may be one which removes any ~ialic acid, or it may be 6pecific for a particular sialic acid variant (such as N-glycolyl or 0-acetyl sialic acids, 6ee Schauer, supra) or for a 6ialic acid linked in a particular manner to other 6ugars.

While the foregoing example is directed to the identification of antibodies which bind to sialoside epitopes of tumor-associated glycoprotein antigens, the method is of more general applicability. Thus the 6ensitivity of the binding of anti-carbohydrate antibodies to other sugar modifications may be determined. For example, afucosyl mucins may be produced using alpha-L-fucosidase and agalactosyl mucins may be obtained using alpha- or beta-galactosidase or endo- or exo-galactosidase.
These may then be used as negative screens in a manner analogous to the use of the asialomucins described previously. In general, the negative 6creen is preferably a glycoprotein deficient in one of the constituent ~ugar6 of the desired epitope but presenting the remaining 6ugar6 of that epitope in the appropriate ~equence.

It i6 al60 preferable that the antibodies be further ccreened in a hapten inhibition a66ay, the hapten being an 1340~11 oligosaccharide or glycolipid bearing the epitope of interest.
The antibodies additionally may be screened with products obtained by treating the mucins of interest with, as appropriate, an N-glyconase or O-glyconase, or some other deglycosylating agent. This will strip the carbohydrate from the protein backbone of the mucin. That protein may be used as a negative "screen", while the free carbohydrate may be used as a positive "screen".

One may also use traditional screening materials, such as normal or cancer patients' sera, or purified antigens of a known character, in combination with the novel screening materials of the present invention. Most immunogens from human sources contain varying amounts of human serum albumin (HSA), therefore, a negative screen using HSA is usually appropriate.

Selection is not necessarily an absolute process. If few or no antibodies are found which rigorously meet the selection criteria, the criteria may be relaxed. For example, if a panel of several negative screens are used, one might pass an antibody which binds to only one of the negative screens.

Once antibodies are identified that bind to the desired epitope, these antibodies may be used in screening for further antibodies of interest, based on whether they compete with the candidate antibody for a particular epitope.

Certain apparatus may be used to facilitate the screening process. Essentially, this apparatus comprises a support means to which both the positive screen glycoprotein (bearing the unmodified carbohydrate epitope) and the negative screen glycoprotein (bearing the modified carbohydrate) are attached at distinct, known locations. Typically, it will take the form of a microtiter plate, with the positive screen immobilized in a first series of wells and the negative screen immobilized in a second X

.... . . . .. . ..

13~o3ll ~eries ~f wells.

In another preferred embodiment, the ~creening i~ carried out at a phy~iological pH. Antibodies which bind the tumor-associated antigens at physiological pH are more likely to be suitable for use in vivo. e.g., in immunotherapy and immunoimaging.

It is especially preferable that the antibody be screened for its ability to bind the mucin at a wide ran~e of pH values, both below and above physiological pH. An antibody whose binding is essentially pH-insensitive is more likely to be of value in an enzyme immunoassay for the antigen. Enzyme-substrate reactions are often pH dependent; an assay based on an antibody which is essentially insensitive to pH over the pH range of enzyme activity may be conducted at whatever pH is optimal for the enzyme.

It is contemplated that the antibodies identified by the proposed ~creening method will be used in immunopurification, immunodiagnosis (including serum or urine diagnosis, histoc~emical studies, and in vivo immunoimaging), immunotherapy, and other immunological methods.

For the purposes of this application, the term "CA 19-9" is used to refer to one or more tumor-associated antigens bound by antibody 19-9 (secreted by hybridoma HB 8059), and apparently a mucin in body fluid. Like antibody 19-9, our preferred antibodies appear to bind a sialosyl Lewis-a determinant of this mucin. The term anti-(CA 19-9) antibody refer~ to any antibody which binds this mucin, including Xoprowski's 19-9 antibody and our own preferred antibodies, whether or not they are cross-reactive. The Centocor CAl9-9 RIA, which employs the 19-9 antibody for the detection of CA 19-9 antigen, is referred to ..... .

hereafter as the ~conventional a~ay. n By means of this method, we have identified ~ 6eries of particularly useful ~ntibodies for the sialylated Lewis-a determinant. The bindinq of these antibodies to that determinant is relatively insensitive to basic and neutral pH, unlike the anti-CAl9-9 antibodies known in the art. In addition, by thi~
method, other anti-~ialoside antibodies have been generated which essentially do not bind to 6ialosyl Lewis-a, such as our B32.2 discussed hereafter. Our preferred nti-CA 19-9 antibody i~
B25.10.

Preferably, an antibody is 6elected 6uch that the percentaqe of antigen bound by the antibody within a pH range of 4-9 is always at least about 66% of the percentage of the antigen bound at the pH within the range of 4-9 at which the binding is maximized. This invention is not limited to the detection of CAl9-9.

Competitive immunoassays employing these antibodies appear to be able detect cases of cancer which are overlooked by ~ssays employing the conventional 19-9 antibody. This may reflect the existence of CAl9-9/bearing mucins which present the CA 19-9 epitope only under basic or neutral conditions. That is, certain mucins may be subject to p~-dependent secondary or tertiary conformation changes. Another possible explanation is that there are CAl9-9/bearing mucins whose epitopes are ~o distributed that they cannot participate in the ternary complexes necessary for the success of the sandwich assay format.

Other advantage~ of the present invention will be apparent to those in the art after perusal of the specification, claims and drawings presented herewith. The appended claims are hereby incorporated by reference as a further enumeration of the preferred embodiments.

.

ret~iled Descr~tlon o~ the I~rLnth~n P~Da~ lh~ Ant j~Pnc We have lound lhal the body ftuids of cancer palienls, or f~ lions thereof contain si~ni~icant bvels of rnucins bearin~ CA 19-9 and olher~urnor a5soc ~'ed a,lti~en'~ delerminanls. These body lluids may be oblained by paracen~e~s of Ihe atxk ..,inal cavity to remove asches tluid, of the thoracic cavhy to remove pleural et~usions, or of olher mucinous ~luid-bearin~ structures such as ovarian cysls e. 9., ~ladenol"as.

The lollowing table describes a sefies ot body 11uids ob~ai"ed which have been assayed tor their content ol various turnor markers.

Cancer ~e r~ined by ~an~i~y (m~ CA 19-9- ~ CA 125t S~Qt t (U/m~ ~UVmt) (Ulmt) Ovarian Carc. p750 26 0.75150 000 0 2 Ovarian Carc. p750 24 1.07 100 0 3 Ovarian Carc. p248029,866 650 600 2 225 4 Ovarian Carc. p1475 44 1.525,600 0 Ovarian Carc. p3185 31 1.522 400 0 6 Ovarian Carc. t 8 39 0 4071 0 7 Ovarian Carc. p 36 53 1.520 793 0 7a Ovarian Carc. p500 62 2.7524 100 0 8 Gastroesophogeat Carc. p4690 39 19.252 200 0 8a-1 Gastroesophogeal Carc. p5000 35 111 100 8a-2 Gastroesopho3eal Carc. p5000 51 18.5 0 0 8a-3 Gastroesophogeal Carc. p1250 38 183 0 8b Gastroesopho~eal Carc. p2000 48 40 447 0 8c Gastroesopho~eal Carc. p3500 42 10.5669 0 9 Ovarian Carc. p1700 34 1.252 500 0 Mucinous ovarian Cy~a~,-o-"a 685 62,000 9 0004,000 6 125 10a 1st dau~htercyst 1754,000 50 00013 5517,750 10b 2nd dau~htercyst 1234,000 34,2506 0398,000 11 pleuralcar,;no",a 540 33 13.25417 0 12 Lun~ meso~l,e'ic ,.a p5340 25 12 0 12a Lun~r"esotl e'ic "a p4100 16 0 0 12b Lun~ ",esotl,e'ic."a p7000 14 0 0 0 12c Lun~ mesothelioma p7~50 12 0 0 0 - 1340~11 13Ovarian carc.1~ 5190 32 2,400 o 14 Ovarian anap6slic p2940 34 2.0 S,200 o Cobreclal carc. p6001,818 1,006 1,899 250 15a C~ r~lal carc. p26003,208 1,375 1,851 300 15b Cobrec~al carc. p20001,560 1,175 3,225 500 15c Cobrec~al carc. p25002,138 ~50 1,905 550 16 Cobreclalcarc. p1190 92 1,575 3,200 0 17 Unknown Primary p17021,717 6500 1500 3,300 17a Unknown Primary p4002,132 225 0 425 18 Breas~ Adenoca,.,;nofi.a p1100 32 1000 4,100 0 18a Breas~ Adenocarcinoma p25 0 26.5 183 0 18b Breas~ Adenocarcinoma p27 0 39.5 447 0 18c Breast Adenocar(.;r~".a p41 100 36.5 405 100 19 Primary unknown carc. t1,1005,745 1500 2889 475 19a Primary unknown carc. t1,0002,598 1125 1335 300 Renal cell carc. p7,140 41 0.25 2,800 0 21 ? 115 31 3.75 1635 100 22 Ovarian serous cysi ~rnain) 215 51 .75 79,500 0 22a Ovarian cyst (daugh~er) 50 63 0 66,200 0 23 ? 3,400 41 11 159 0 24 Colorec1al Carc. ~2,3003,77351,000 495 250 24a Colorecial Carc. p3,0002,19235,750 159 250 24b Colorec~al Carc. p2,0002,22926,750 0 25 Prostale Carc. - 14,500 38 7 1041 0 25a Prosta~e Carc. l2,500 37 11 1947 0 26 Endo",el,ial Carc. p 4,000 31 .5 1389 0 26a Endome~rial Carc. p3,400 30 1.0 1929 0 26b Endo",.:lrial Carc. p3,750 44 .87 1665 0 27 No~ Diagnosed(pleural) 250 14 .87 0 0 27a No~ Diagnosed(pleural) 500 33 .5 0 0 27b No~ Diagnosed (pleural) 24 .75 0 28 Ovarian cysi ~luid 550431,417 500 82,300 8,000 29 Pancrea~ic carc. p250 41 1.0 1,707 0 No~ available 600 669 2,500 0 31 Meiac~tic. ca,.,;nor, a (?) pf200 15 11 5883 32 Benign Ovarian cys~adenoma 50 23 .25 17,973 33 Benign pf10 56 1.75 0 34 Undiagnosed pf400 17 2.25 27 No~available 800 50 7.0 825 36 pf650 59 .25 3,165 37 Ovarian Cys~ pf4000600,000 700 20,000 38 570 30 .5 675 39 Ovarian carc. p3,625 25 .5 500 Breas~ carc. 1,275 19 150 1821 40a Breas~ carc. 1,845 18 122.5 789 41 Ovarian cys~ p2,695 35 1.0 7,000 41a Ovarian carc. 3,000 17 1.25 6,000 41b Ovarian carc. 2,120 870 .87 2,031 41c Ovarian carc. 550 29 1,281 42 Primary unknown carc. p5,595 22 .25 1,461 43 Breas~ 195 13 44 Cobrec~alcarc. p1,6502,450 1.0 2,163 Ovariancarc. 1,1001,800 30 41,500 ~ 1340311 pf = Pleural effusion p = paracentesis t = Thoracentesis * Centocor Units ** Hoffman-LaRoche Units t Centocor Units tt Stena Diagnostics Units Purification of Body Fluids In order to determine the elution patten on Sephacryl S
500* of some of these fluids, and to make possible the use of different elution pools to coat microtitre plates for screening, the following procedure was used. The body fluids obtained from patients at a local cancer centre are brought up to 3 % phenol for sterilization purposes and then clarified by centrifugation for 15 min. at 17,500 g in a high speed centrifuge. The supernatants are then pumped onto a 5X75 cm. Sephacryl S-500* column equilibrated in 10 mM phosphate and 150 mM NaCl, pH 7.2, containing 1 mg/mL of NaN3. The column is eluted with the same buffer at a flow rate of approximately 180 mL/hour, and monitored at 280 nm.
Fractions, 12 mL each, were collected and screened for CA 19-9 activity. The first active peak, which occurs in the void volume, is pooled and labelled F1. The second peak of CA 19-9 activity, included in the column but eluted earlier than the main protein peak (as determined by absorbance at 280 nm) is pooled as F2. Several other body fluids have since been fractionated by the same method and a similar elution pattern was observed.

The F1, F2, or a pool of the two fractions of one or two CA 19-9 positive body fluids are coated on a microtitre plate at about 100 U/Well to become the positive control for screening culture supernatants of hybridomas. The F1 of a CA
19-9 negative body fluid is coated on the screening plate at approximately the same dilution as the F1 fraction. Then neuraminidase treated (see below for methodology) CA 19-9 * trade-mark X

positive F1 (the positive control) is coated on the screening plate. Finally, some of the wells, of the microtitre plate are coated with HSA. The plate is then blocked with 1~ BSA in PBS for at least 1/2 hour. Hybridoma supernatants which react with the positive control body fluid and none of the others will have the desired reactivity pattern.

To cleave off the terminal sialic acid groups of the partially purified Sephacryl S 500* mucin fractions, the following procedure is used. 10 mL of PE- F1 are incubated overnight at 37~ C with 1.25 mL of neuraminidase-agarose (1.25 U of neuraminidase activity) while tumbling. It is convenient to use fractions which will contain approximately 3000 U/mL of CA 19-9 activity. The neuraminidase-agarose gel is then spun down, and the supernatant is dialyzed overnight against PBS to remove any free saccharides. Chemical estimation shows the reduced content of sialic acid and at least 98% of the CA 19-9 activity in a binding assay is abolished with the asialo mucin preparation.

Preparation of Anti-(CA 19-91 Antibodies Immunization: Several immnunogens have been used, the best in our hands has been the body fluids of this invention.
Partially purified ascites mucins (PE3, PE10, PE17, and PE28) rich in SLA activity isolated from Sephacryl S-500* void volume peak were used at doses of 5,000 - 20,000 U/immunization. Combinations of partially purified mucins of PE3, PE10, PE17, and PE28 have been adopted in various immunizations exploiting long and short-term protocols. See Table VI.

Fusion: Fusions are done using SP 2/0 cell line and according to published protocols. (G. Gafré and Ceasar Milstein (1981) Methods in Enzymology 73:3).

Screening Strategy Our screening strategy for new clone identification is based on the following * trade-mark procedure.

Hybridoma supernatants are reacted on plates coated in different rows of microtitre wells with a judicious selection of purified CA 19-9 positive and negative mucins (such as PE
1, PE 3, PE 10, PE 17, and PE 28). The wells are then blocked with BSA. Then 100 uL of each different hybridoma supernatant is added to each of the different antigen coated wells such that antibody from each of the sample hybridoma antibodies contacts one well of each antigen. The mouse monoclonal antibodies bound are detected by an appropriate dilution of goat anti-mouse immunoglobulin conjugated to HPRO (Tago, Immunochemicals) and the color is developed by an appropriate peroxidase substrate and chromogen. PE 1 is an CA 19-9 negative mucin while PE 3 and 17 are positive mucins.
Treatment of PE 17 with neuraminidase abolishes the reactivity of the mucin in the conventional assay. Additional CA 19-9 positive body fluids such as PE 10-f1 and asialo- PE 10-f1( PE
10-f1-NA) may be added to increase the resolution of the screening procedure.

Thus, we have an improved screening procedure which would allow others to produce and select SLA reactive clones. More generally this procedure can be adapted to identify any of the cancer-associated sialosides given the appropriate selection of body fluids and derivatized antigen species generated therefrom. In general, we have used PE1, PE3, PE 17, asialoPE17, and then HSA as an additional negative control.
These results are displayed in Table II.

Table II

Clone # PEl PE3 PE17 HSA asialoPFI7 Tumour Tissue Reactivitv B4.20 - ++ ++ - _ B24.7 - + ++
B24.8 - + +++
B25.7RI - ++ ++ - - 3/4+
B25.7R2 - ++ ++ - - 3/4 B25.9R5 - ++++ +++ - - 2+
B25.6R3 - ++ +++ - - 3+
X

~ 13 1 0 311 ( B25.1OR1 - ++ +++ . . 3+
B25.11R3 - + ~+ . , 3+
B25.12R1 - ++ + . . 1+
B25.16R1 - ++ +,++ - - cells6n~oosa B25.17R4 - + +
B25.18R1 - ~+ ++ . . o B25.19R5 - ++ ++ - . o B25.21R5 - ++ +++ - - cells6n~cosa B25.22R3 - + ++ . . o B25.23R1 - + + - - 2 B28.2R1 - ++++ ++++
B32.2 - + +
B32.4 - ++ +
B32.9 - + +
B32.3 . + +
B34.1R7 - + +
B37.4R1 - ++ +
B37.23R11 - ++ +++
B37.35R1 - ++ +
B37.43R3 - +++ +++
B45.1 - +++ +++
B45.2 - ++ ++
B45.3 - +++ +++
B45.7 - ++++ +++
B45.13 - ++ +
B45.16 - ++ +++
B45.18 - + +
B45.24 - +++ +++
B67.2 - ++ +++
B67.3 - ++ +++
B67.4 - ++++ +++
B67.5 - ++ +++
B67.6 - ++ +++
B67.7 - + +++
B67.8 - ++ +++ - +++
B67.9 B67.10 - + ++
B67.11 - + +++
B67.12 - + +++
B67.13 - ++++ ++++
B67.15 - ++ +++
B67.16 - ++ +++
B67.17 - ++ ++++
B67.18 - + +++
B67.20 - + +
B67.21 - + +
B67.23 - ++ ++++
B67.24 - ++ ++++
B67.25 - ++ ++++
B67.27 + +++~ +++
B67.28 - ++ ++++ ~ +
B67.45 . . .

In the coding system, B is simply a coding device, the first number indicates the fusion number, the post-decimal number indicates the clone, and the number after the R
indicates the reclone number. Typically, we choose clones which are PE3(+), PE17(+), PE1(-), asialoPE17(-), and HSA(-).
Asialo-PE 3, PE 10, PE 28 and other partially purified body fluids have also been successfully used as part of the screening procedure. The above clones have been recloned and the screening repeated on the reclones. Sometime, as in the case of B28.2, recloning assists in resolving the specificity of the antibody (i.e., in the B28.2 case such that the reactivity with asialo PE 17 was recloned out). Selected reclones are expanded and ascites are prepared.
Immunoglobulins are purified by standard affinity or ion exchange methods.

For each antibody we have utilized further, a preferred method of purification was determined. We have used variations of several systems. These methods may not be optimal and other methods may be used depending upon the character of the antibody.

For example, for purifying IgG from ascites, protein A, immobilized on agarose gel, is used for affinity chromatography. The ascites is loaded onto the column at pH
8.5; the bound IgG is then eluted by means of a stepwise pH
gradient, between pH 6.0 and 2.5, depending on the IgG
subclass of the antibody (See Ey, et al., Immunochem. 15:429 (1978)).

In contrast, we have generally purified IgM antibodies on a hydroxyapatite column, followed if necessary by a Bakerbond Abx* column on HPLC. The hydroxyapatite column was equilibrated in 10 mM potassium phosphate, pH 6.8. The ascites, clarified by centrifugation was diluted 1/2 in the 10 mM potassium phosphate buffer and loaded on the column. The monoclonal was subsequently eluted with 90 mM potassium phosphate, pH 6.8, followed by gradient from 90 - 500 mM
potassium phosphate. The purification was monitored by absorbance at 280 nm, and the peak fractions are tested by agarose gel * trade-mark X

.

electrophoresis and ELISA.

If insufficient purity was achieved after the hydroxyapatite column, the partially purified monoclonal was diluted in 1/2 in 100 mM MES, pH 5.6 and loaded on a 5u Bakerbond Abx* HPLC column equilibrated in 25 mM buffer.
Elution is achieved by a gradient from 25 mM MES, pH 5.4 to lM
sodium acetate, 20 mM MES pH 7.0 over 60 minutes. After dialysis against PBS, the biological activity and purity of the monoclonal antibody is again verified by an ELISA assay and agarose gel electrophoresis and ELISA assay. Other purification methods may be used, depending on the antibody.

Using this system, we readily identified clones which were satisfactory for the purpose of making assays for (CA 19-9)-like determinants.

Hapten Screening Clones identified by the above procedure are then typically studied for their hapten inhibition characteristics.
Polystyrene tubes were coated with a fraction of PE 3 at 50 units per tube, diluted in 300 ul of PBS, overnight at room temperature and additional protein binding sites were blocked with 1% BSA in PBS for one hour at room temperature. Then 50 uL of the sialosyl Lewlsa or Lewisa hapten diluted in normal pooled serum was added to each tube. 250 ul of the radiolabelled antibody (the specific activity is indicated in Table II). The mixture was incubated overnight at room temperature on the benchtop. Tubes were then washed with 2.0 mL of distilled water and counted in a gamma counter (Packard). Normal pooled sera was used as the calibrator for 100% bound or 0% inhibition and percent inhibition or percentage bound (respectively) was calculated. Clones which were reactive with sialosyl Lewlsa but showed little or no reactivity with Lewlsa were chosen for further study.

* t rade-ma rk X

1340~1 L

H~ten Inhibnion of ~ nn~ n~l Antihn.ly Rin~i~ t~ ~nr~r An~;~en ,~/r înhibiti~ n MDnoc'hn~l~ Ah (~ lhe) Input nPM ~K Roun~ ~;ql"~;yl L~wie~ L~

B25.19R6 0.45 54,949 41 52(10) 3.5(10) B25.16R1 0.60 61,226 41 45(20) ~i(20) B25.10R2 0.48 32,177 23 36(40) 5(40) B25.7R1 0.36 47,718 43 51(20) 2(20) B25.17R410.78 28,467 27 22(40) 1(40) B28.2R1 1.90 50,676 40 17(40) 4(40) Under similar conditions the Centocor anti-(CA 19-9) a,ltiL,ody binding was inhibited by 50% at 8 g~lube of sialosyl Lewis~ hapten but no~ by a Lewisa haptens even at 50jl~/tube.

At this poin~, clones which react with a sialosyl Lewisa hapten but nol by Lewisa hapten in competition experiments are chosen. Sialosyl Lewisa haptens similar 10 lhose c~"-"-er~ally available (a sTabsyl Lewisa hexAeaccharide obtained ~rom human milk by Biocarb, Lund, Sweden) can be used in these experiments with analogous results.

A more elaborate hapten screenin~ e~ ~,eri,--e,ll yielded similar results. Polystyrene tubes (Sarsted) were coated wdh a 1rac~ion ol PE 37 a~ 200 units per tube, dilu~ed in 300 ~11 o~ PBS, overni~h~ a~ room tempera~ure. Then, the additional prolein binding sdes were blocked with 1 % BSA in PBS 10r one hour at room temperature. 50iUI PBS, 1 % BSA wi~h or without 4 X 10 5 M o1 the app-upriate hapten plus 50111 PBS, 1 % BSA contaTnlng 5 ng of 1251 labelled r~nochnal antibody were added lo 1hese tubes and 26 13 4n3 incubated for 3 hours at room temperature on shaker at 200 rpm. The tubes were then washed twice with double distilled water and counted. The percentage bound was determined by the following formula: % Bound = (CPM Bound with Hapten/CPM Bound in the absence of Hapten) X 100.

Table IIIA
Hapten Inhibitions MAb SLA CA50 Lea X Y SLX NANA-lactose B25.10R3 55 0 0 0 0 NA 0 B32.2R2 12 0 0 0 0 0 0 B67.~R12 89 0 0 0 0 NA 0 B67.7R22 96 0 0 0 0 0 0 B67.17R12 98 0 0 0 0 0 3 The critical test of the specificity of the antibodies was determined by utilizing a family of closely related haptens.
HB 8059 and most of the antibodies were strongly inhibited by the SLA haptens (synthetic tetrasaccharides or natural hexasaccharides). Neither the asialo hapten (Lewisa) or a afucosyl hapten (CA 50) gave significant inhibition.
Moreover, this was true for X, Y, Sialosyl LewlsX (SLX), and NANA-lactose. B32.2 was the only one which was not significantly inhibited by any of the haptens.

Determination of pH Optimums DelVillano et. al claim in the PCT WO 84/00758 that higher signal to noise ratios could be obtained by running an immunoassay for CA 19-9 at an acidic pH. In order to test this supposition, a number of our antibodies were tested by the following method.

Polystyrene tubes were coated with a fraction of PE 3 at 50 units per tube, diluted In 300 ul of PBS, X

13~0311 -overnight at room temperature and additional protein binding sites were blocked with 1% BSA in PBS for one hour at room temperature. Either 2 ng. or 10 ng. of 1251-labelled monoclonal antibodies HB8059, B25.10, B25.16, B28.2, B67.4, B67.7, and B67.17. were incubated for two hours at room temperature on a shaker in these tubes in phosphate buffers with different pH's. Table IV shows the results:

Table IV

pH Sensitivity of a Selection of Anti-Sialosyl Lewisa Clones ~ Bound Antibody pH4 pH5 pH6 pH7.1 pH8.1 pH9.1 B25.10 (10 ng/tube) 61 62 66 64 62 55 B25.10 (2 ng/tube) 57 61 63 63 61 53 B25.16 (10 ng/lube) 30 29 28 28 25 20 B25.16 (2 ng/tube) 30 29 27 27 23 19 B28.2 (10 ng/tube) 51 48 56 56 48 54 B28.2 (2 ng/tube) 51 48 55 56 48 56 B67.4 (10 ng/tube) 65 63 64 62 58 58 B67.4 (2 ng/tube) 65 61 64 62 59 56 B67.7 (10 ng/tube) 43 39 40 38 33 29 B67.7 (2 ng/tube) 43 39 39 37 31 28 B67.17 (10 ng/lube) 57 58 59 56 44 39 B67.17 (2 ng/tube) 57 60 60 57 47 37 HB 8059 (10 ng/tube) 64 46 33 17 11 8 HB 8059 (2 ng/tube) 61 51 36 17 11 9 The anti-SLA antibodies of the present invention exhibit comparable binding to CA 19-9 mucin X

coated tubes over wide range of pH. This result is in sharp contrast to HB 8059 whose optimal binding is at a pH of about 4. Increasing the pH for this antibody drastically reduced the amount bound as well as the affinity.

Serum Sample Screening We collect and maintain a cancer serum sample bank cross referenced to patient histories collected in accordance with accepted practice of patient consent and confidentiality. All serum assays herein disclosed have used samples from this bank.

After the hapten specificity was determined on the antibodies of Table III, three different sets of serum samples for which CA 19-9 values were available were chosen randomly on the day of experimentation. In Table V, various antibodies selected according to the present screening method were tested for their ability to correctly discriminate normal and cancer sera.

Table V
B25.10R2 Clinical Screen Normal Sera Patient #Conventional RIA B25.10R2 (Units/mL) (% Bound) 287 17 97.7 288 13 95.3 290 10 97.9 291 ND 96.6 292 34 87.5 293 ND 98.1 296 21 88.8 298 8 87.8 299 51 71.2 300 58 79.3 X

, t~neer ~:era Patient ~ ~',A 19-9 RIA R~.lDR~
~UnltslmL) ~% Bouna7 ~02 186 ~0 938 237 23.9 1576 77 64.1 1607 1285 13.3 1835 65 67.1 1839 . 26 52,4 1861 21 71.9 1891 21 81.9 1971 6 58.9 1972 12 86.1 1980 44 73.4 1884 14 89.3 ~,7R1 and R32,2R1 Clini~l Screen ~ormal Sera Patient #' CA 19-9 ,R2~.7R1R25.7R1 R32.2R1 (UnitslrnL) % BoundUnits/mL % Bound (Arbitrary) Cancer Sera Patient # CA 19-9 R25,7R1,R2~.7R1 p32.2R1 (Uni~s/mL) % BoundUnits/mL % Bound (Arbhrary) 336 t 0 60 25 64 .. . .. ....

7547786 10.82925 13 16071285 6.12056 15 Inhibition reactions were performed using polystyrene tubes as coated in the hapten inhibition experiments.
Radiolabeled anti-(CA 19-9) antibody in 200 ul of PBS at pH
7.2 in 1% BSA was added to each tube together with 100ul of sample or standard. The reaction mixture was then incubated for 24 hours on shaker at room temperature shaking at 200 rpm.
The tubes were washed twice with 2 mL of distilled water.
Bound activity was measured in a gamma counter (Packard).

The sandwich assays performed with polystyrene tubes coated with 1 ug of antibody in PBS overnight at room temperature. The tubes were then blocked with 1% BSA in PBS
for two hours at room temperature. 100 ul containing 2 ng.
(approximately 50,000 CPM) of radiolabeled antibody and 100 ul of either sample or standard solution is added. The mixture was incubated overnight at room temperature on a shaker, washed twice with 2 mL of distilled water, and counted.

All of the tested antibodies exhibit reduced binding to mucin coated tubes in the presence of cancer sera but not normal serum samples. Those serum samples exhibiting the highest CA 19-9 value as determined by a commercial assay also exert the maximum inhibition of the binding of the antibodies.
This is true also for B32.2 which is only weakly inhibited by the SLA hapten which is only weakly inhibited by a study of specificity determinations.

B25.16 and B25.10 were selected for further studies.
One-hundred-seventy-four serum samples were chosen randomly from the bank and the assayed by the method expressed in the procedure of the B25.10 assay section. One-hundred-seventy-four samples were tested in a 3 hour simultaneous, solid-phase, competitive radioimmunoassay format using either B25.10 or B25.16 as the tracer antibody. A

-- . ... . .

1340~11 ~ ~ ~ 31 correla~ion coetlicien~ ot 0.95 was de~ermined ir 1icr ~o the ciinical ~-.~a~ y ot ~he two an~i cd es R?~ 10 Cont~inin~ Simuh~nPrtls. ~:o~j-ph~ce. ~ e~nive Inh~ hn R~r~jpinnn~ e~y As one e,~ od;.--enl o1 1his invention, CA 19-9 was measured usin~ a simultaneous, solid-phase, compe~i~ive inhibhion raJ -. ~-",unoassy utilizin~ " onGcl~,nal antibody syn~hesked by the hybridoma clone B25.10. Polystyrene 1ubes coa~ed w-nh CA 19-9 epil~pe bearin~ anti~en are incubated whh standards (10~ 40, 75, 150, and 300 lU~iL) made Irom PE 37 in a human serum malrix wnh 1 m~/mL sodium azide, control (also in a human serum ma~rix), or a serum sample to~ether whh 125-lodine-labeled B25.10 monoclonal an~i-CA 19-9 antibody. Durin~ this incuba~ion CA19-9 bearin~ an~i~ens will bind the labeled antibodies thereby preven~in~ the bindin~ o~ these -abeled antibodies to the solid phase.

Unbound material is removed by washl)~ the tubes, and r d-;activity is measured in a ~amma scintillation counter. From the standard curve derived, the CA19-9 inhibition unhs (IU) o~ the un~, ~v,ns and the control can be detemmined.

The B25.10 Simllh~neous. Solid-Dh~se. Corrretitive Inh-~ition P~dioimmunoassy Procedure A. Pipette 25 1110~ each cal br~or, control or patient senJm sample to the bottom of the appropriate tubes. The user should note that it is Important, due to small sample size utilized, that the sample be pipened directly 1O ~he bottom o~ the tube. Failure to do so will adversely af~ect assay prec;sion. Use a fresh pipetle tip lor each sample.
B. Pipette 200 jll o~ the '251-labeled B25.10 antibody into each tube. For lar~er number of samples a repeater pipet is very convenient ~or this step.
C. Thorou~hly mix the contents of the tubes by vi~orous shakin~ or ~entle vortexin~
D. Cover all tubes with ParafilmOED or equivalent.

131q~1'l !

E. Pbce ~ubes In a water bath at 37~ 2~ Centi~.ade and incubate 10r three (3) hours ~1ive ~5) minutes.
F. Aspirate the tubes and wash two ~2) times with 2 mL of distilled water. An O~dord oipener, Comwall syrin~e, or equivalent device b tL~g~Gt~d 10r this step. Ensure complete removal of ~11 Iiquld atler each wash.
G. Measure ._ k ~c~ivity of the tubes in a gamma scintillation counter.

RESULTS
Resuhs were obtained by the lo lo~ procedure:
A. The standard curve is constructed by pbttin~ the mean 1251 cpm for each standard (Y-axis) a~ainst the concentration . The curve is then drawn by a best fittin~ curve~ method. Gamma counters outfit~ed with automa~ic data reduction p,ogra"~ (preferably b~n-iog) may also be used.
B. The concentrdtion ot the control and the patient ~enum samples are determined from the standard curve.
C. Samples readin~ over 300 unnstmL were diluted and reanalyzed 10r more accurate results. Dilute the sample 1t10 with the 0 unitstmL sland.,.d. To calculate the resuh atter dilution muhiply by 10.

Analytical Sensnivity The minimal detectable concentration of CA19-9 was determined to be 3.0 IU. The minimal detectable concentration is detined as that concent,dtion of CA19-9 equivalent to 2 SD from the 0 1 UtmL
standard.

Dilution Linearity Serum samples containin~ hi~h levels of CA 19.9 were diluted serially w1~h the kh diluen~ and reassayed. When expected vs actual results were analyzed by linear re~ression all ~howed a correlation .

r ( 1 3 4 0 3 1 1 coeflkien~ 20.990.

E~tpected Values 1. Normal Sera: CA 19-9 levels In sera from normal individuals (blood bank donors)were detemnined by ~his assay. It was de~ermined 1ha~ 98.6% of 1hese normal ~era yielded CA 19-9 levels bss 1han 60 lU/mL.
The dis~ribu~lon of 1he values in 1hese normal sera is ~hown in Table Vl.

Table Vl ni~trihution of 361 Norm~l sera In~erval # of Samples lUtmL in Interval ~70 3 2. Malignan~ Sera: Sera from 319 pa~ients diagnosed with various cancers were 1es~ed in 1his assay.
The resul~s shown in Table Vll indicated 1ha~ 1he B25.10 detected a high proportion of samples ol pancrea~ic cancer.

, T51hl~. Vll nie~rih ~tir~n nt CA 19-9 VP~ IPC in ~:Pn~m 1rom PJ-tiPnt~ h ~ n~ cpac~

r~ Inc~Ory) N 0-6060-120 ~120 lUtmL lU/mL lUtmL

Colorectal Cancer Limited Disease 25 84.012.0 4.0 Extensive Disease 43 41.9 18.6 39.5 Total 68 ~7.416.2 26.4 Gastric Cancer Llm-ned Disease 27 '77.818.5 3.7 Extensive Disease 13 23.1 46.2 30.8 To~al 40 6û.0 27S 12.5 Pancrea~ic Cancer Limlted Disease 9 Z~ 55.6 22.2 Extensive Disease 8 125 75.0 12.5 Total 17 17.664.7 17.6 Breast Canoer Llmited Disease 70 84.0 7.1 11.4 Extensive Disease 124 S7.7 19.4 12.9 Total 194 ~~7 15.0 12.3 ~ 1340~11 Comparison of CA 19-9 Assay Results The cut off for the assay was selected by applying traditional mathematical criteria to a group of test results on apparently health blood donor specimens (normal values).
Our inhibition unit value ranking at the 99th percentile was taken as the upper limit of these normal values (which was defined as 60 IU). This is identical to the method by which the conventional assay cut off of 37 U/ml was originally determined. Although our assay and the conventional assay employ technologically different units, the method employed to determine the upper limit of normal for the two kits was the same. The only difference then is that the same decision criteria was applied to data derived by using two completely different assay formats.

Clinical Sensitivity and Specificity Although the conventional assay and our unit values showed good numerical correlation, they are not directly comparable because they are measured in two different assay formats. Nevertheless, one can sort results with respect to categories of above (positive result) or below (negative result) the upper limit of normal. Since identical mathematical criteria were applied in determining the upper limit of normal for both kits the "positive" vs. "negative"
results are directly comparable. These results can also be used to compare relative clinical sensitivity and clinical specificity for the two kits.

Using the published conventional assay cut off of 37 U/ml and our 60 IU/mL cut off, Table VIII illustrates the increased clinical sensitivity of our test, especially with respect to Gastric and Pancreatic cancer. Upon some further analysis, these results can be compared with a third qualitative variable; that is, the physician's judgment on diagnosis during patient follow up. These physician judgments were not based on tumor marker results but by consideration of a number of other clinically accepted decision criteria. These diagnosis are the results of physical examination, x-rays, other visualization methods and X

,.

categorization of other blood chemistries as above or below the upper of normal (positive or negative). Unfortunately, we do not have enough data on pancreatic and gastric cancer patients to test the greater specificity of the assay.
Nevertheless, we do have a considerable number of serum samples from a population of colorectal cancer patients who have been followed after primary treatment and subsequently diagnosed by standard criteria to be free of disease (FD), disease stable (DS), disease regressive (DR), or disease progressive (DP).

Conventional Assay Using HB 8059/Our Assay Using B25.10 Gastric Colorectal Pancreatic Colorectal Colorectal Total Active Colorectal Cancer Cancer-DP Cancer Cancer-DR Cancer-DS Cancer* Cancer-FD
+/+ 10 17 11 3 5 46 -/+ 5 3 3 0 1 12 2 +/- 0 1 0 2 1 4 8 Correlation R= 0.96 0.84 0.99 0.99 0.83 0.86 # of Samples 40 39 17 13 16 125 80 In a comparison of assays using the HB 8059 antibody and our B25.10 antibody, we found both capable of detecting elevated serum levels of CA 19-9 antigen (see Table VIII).
Generally there is good correlation for all types of cancer and their subgroups. However, for overall active disease, the B25.10 assay is capable of identifying about 8% more cancer than would be negative in the assay employing HB 8059. On the other hand, only about 3% of active disease patients are detected by HB 8059 that is not detectable in the B25.10 assay.

Affinity Constant Determinations of Anti-CA 19-9 Antibodies on a Solid Phase Coated with Multivalent Mucin V

, ,~ .. ~ _ .. . . ..

13~0311 Fx~erim~nl~l se~l~

Mucin-coaled tubes were ~ ~ar~ d as above. 100 ùi/tube Ol 12511abeled n-Dnocbnal antibody at rnolar concen~ral;ons typically ranpin~ for these aut;L ~ d-~ s ~rom 10-8 1O 10~'~ M were added. 50111 PBS
with 1% BSA was used 1Or the assays conducted at pH 7.2. For the assay done at pH 4.5 0.1 M cn~ale containin~ 1 % BSA was used as the buffer. The ant Lr ~ es were incubated ovemi~ht al room temperature on shaker at 200 rpm. The inpul was coun~ed and the tubes washed twice with double distilled water.

Ka values were de~ermined by double ,~c;~,r-,cal plot ot 1t[bound] versus 1~1free]. (Ka - X in~ercepI) Iable IX
il~Vall les MAb pH 7.2 pH 4.5 B25.10R3 1X109 NA
B67.4R12 3.1 X 108 NA
B67.7R22 0.6 X 108 NA
B67.17R12 3.8 X 108 NA
HB 8059 1.15 X 108 1 X 109 The affini~ies (or more correc~ly avidity) of the various antibodies were de~ermined by experimen~s measurinp their bindin~ to mucin coated tubes. B25.10 exhibi~ed the best p~pe,lies a~ pH 7.2 and this rneasurement was comparable to the pH 4.5 Ka ~or HB 8059. The laner antibody has a b~old lower a~ini~y at physk!c~ic-l pH.

Hyi.,ido".a B25.10R3 was deposRed on February 3 1988 with the Amencan Type Culture C~lle~bn under the Bud~pes~ Trea~y and received Ihe desi~r dtion ATCC HB 9636. The makin~ of this deposit should not ibe deemed license to rnake use, or sell the hyl"iJ~,".a or its ~.~tiLod~r.

~ . .

REFERENCES

1. G. Yo~ees~r~ a,~n ~1983) Adv. Cancer Res 38,289.

2. S. Hakornori and Kanna~i (1g83) J.Na~I. Cancer Ins~. (USA) 71, 231 3. J. L. Maanani, B. Nilsson, M. Brockhaus, D. Zopf,Z. ~IeFle;r~ , H. Kt~p-o~jki and V. Ginsburg ~19B2) J. Biol. Chem. 257, 143~5.

4. J. L. Magnani, Z. Steplewski, H. Kopro.~iki and V. r3insburg (1983) Cancer Res.43,54B9.

5. M. Herlyn, H. F. Sears,Z Step!L.~ nd H. Ko~.. ~i (Ig82) J. Clin.1rrununol. 2,135.

6. B. C. Del Villano, S. C\_.u-an, P. Brock, C. Bucher, V. Ln~, M. McClure, B. Rake, S. Space, B. Westrick, H. Schoernaker and V. R. Zurawski Jr. ~1983~ Clin. Chem. Z9 ~49.

7. R. E. Ritts, B. C. Del Villano, V. L. W. t;o, R. Herb.errnan, T. L. Klu~ ~nd V. R. Zurawski (1984) Int. J.
Cancer 33, 339.

8. M. K. Gup~a, R. ArciaDa, L 400ci, R. Tubbs, P~. ~uA~~wski arfd ~ . DeQdhar (1985) Cancer 56,277.

9. W. M. S~einber~, R E;e~and, K. K. Anderson, J. ~lenn, S. H. Kunzrnan, W. F. Sindelar and P. P.
Toskes (1986) Ga~ t~-DJ~ 43.

1 0. H. Tornoda and M.'F~nusawa (1986) Jap. J. Suro. Ui, .

e %
~ nm~ Irr~nrtanl Tumnr-Acc- cl ~ed ''alyla~ed t ~ C~ I,arltltr e Tnvial Na-ne ~:tructure- Antihndy ~Tumnr Accnci~linn ) L~ctn Series (TyDe l~

Sialosyl Lowisa (2~3) GalB1 _ 3GlcNAcB1 ~ 3Gal1 _4Glc_Cer 19-9;C50 (Colorectal) t2,3 t 1,4 B25.10 NeuA~ Fuc~

Sialosyl Lewisa ~2_6)GatB1 _ 3GlcNAcB1 _ 3Gal1 _ 4Gtc _ Cer (colorectal) t 2,6 t 1,4 NouAc~ Fucr Di-Sblosyl Lewisa NeuAa~ FH7 (Colon, ~astric, pancreas, l 2,6 lung) .GalB1 _ 3GlcNAcB1 _ 3Gal1 _ 4Glc ~ Cer t 2,3 t 1,4 NeuAcct Fuc~

SialylLacto-neo1ert,aosyl GalB1 _ 3GlcNAcB1 ~3Gal1_4Glc_Cer ceramide t 2,6 (Colon; Terato) Neul~

Sialyl Lacto-neG tert~G .yl GalB1 _ 3GlcNAcB1 _ 3Gal1 _ 4Glc _ Cer K4 c~ra.":~e t2,3 (Colon; Terato) Ne~uA~~

Di-sialylLe k n~etln. -syl NeuADa FH9 ce, . ~ J ~! 1 2,6 (Colon) GalB1 ~ 3GlcNAcB1 ~3Gal1 _4Glc_Cer t2,3 Neu~m 134~311 ~ , ( Sefies ~TvDe 11) Siabsyl LowisX (2_3) GalB1 _ 4GlcNAcB1 _ 3Gal1 _ 4Gk _ C~r CSLEX1 (G~ s~ ~al) t2,3 t1,3 NeuA~ Fur ~

Sialosyl LowisX (2_6) GalB1 _ ~ NAcBl _ 3Gal1 _ 4Glc _ Cor (C~ al) t 2,6 t 1,3 NeuAm Fuc~

Dimeric ciabsyl LowisX (2_3) GalB1 _ 4GlcNAcBB1 _ 3 GalB1 _ 4GlcNAc131 _ 3Gal1 _ 4Gk _ Cer FH6 (Ga,l,,ir.l~s1'~al) t2,3 t1,3 t1,3 NeuAcs~ Fur~ Fuc~

Trimork sialosyl L WISX (2_3) GalB1 _ 4GlcNAcB1 _ 3 GalB1 _ 4GlcNAcB1 _ 3Gal1 _ 4Glc _ Cer (Ga~ .'' ,al) t2,3 t1,3 t1,3 NeuAc~ Fuc~ Fuc~

Ganalio Series FucosylGM1 GalB1 _ 3GalNAcB1 _ 4GalB1 _ 4GlcB1 _ Cer F12 (Small-cell lun~) t 2,3 t 2,3 Fum NeuAm GM2 GalNAcB1 _ 4GalB1 _ 4GkB1 _ Cer t2,3 NeuAm GM3 GalB1 _ 4GkcB1 _ C~r t2,3 NeuA~

~ , , 13~0311 GD2 ~slNAcB1 _ 4GalB1 1 4GlcB1 _ C~r LS5 t2,3 2N~

GD3 GalB1 ~ 4GlcB1 ~ C~r ' 4.2;R24 t 2.3 NeuAc~8 ~ 2 NeuAc~

GD3 (9- 0 ~ yl) GalBl ~ ~8kB1 _ C~r D1.1 ~Melanoma) t 2,3 Neul' 8 ~ 2NeuAca(9-0-Acotyl) These basic structures can have sliah2 variations such as the seen wnh 9 - O - Ace~yl GD3 or N - glycolyl TABLE XI
Fusion Process ) ~uslon Code Ho~l Slraln Fuslon i9Oo~ter iPoute . Amount Antlgen Adluvant Partner Inlerval B3 rbl/dn 1 ip 100 Slam 99 CFA
7 ip 100 Slam 99 CFA
ip 100 Slam 99 Pi3S
ipiv200 Slam 99 Pi3S
Ipiv200 Slam 99 PBS
hx/ny 156 Ip . 500 PE-3 PtlS

B4 rbl/dn 1 ip 100 PE 3 CFA
9 ip 100 PE 3 CFA
6 ip 800 PE 3 SAL
ipiv400 PE3 SAL
lox/ny 1 ipiv400 PE 3 SAL
BS rbl/dn 1 ip 100 PE 311 CFA
7 ip 100 PE 311 CFA
6 Ip 800 PE 311 SAL
ipiv400 PE 311 SAL
lox/ny 1 ipiv400 PE 3il SAL
B6 rbl/dn 1 Ip 106 SW 1116 CFA
33 ip 106 SW 1116 CFA
31 ip 106 SW 1116 CFA
28 ip 106 SW 1116 CFA
39 ip 106 SW 1116 CFA
44 ip 106 SW 1116 FA
fox/ny 29 ip 106 SW 1116 IFA
B7 rbl/dn 1 ip 500 PE 10 (pel) crA
7 ip 500 PE 10 (pel) CFA r.~.
7 ip 800 PE1011 Pi3S
1 ip 800 PE1011 Pi~S ~ _.
Iox/ny 1 ipiv800 PE1011 Pi3S C~
B~ rbl/dn 1 ip 100 PE1011 CFA
28 ip 100 PE1011 CFA
SP2/0 34 ip 100 PE1011 CFA

Fusion Process J
E~13 rbl/dn SP2/0 ip 108 Le-a Fii-3Cs P-3S
R1~ rbl/dn ' ~P '~~ Pe 3(raw) CFA
21 ip 100 Pe-3 CtA
29 ip 100 Pe-3il iFA
34 ip 10û Pe-1011 tA
SP2/0 45 ip 100 Pe-17~1 iFA

Bt5 rb~/dn ~ ip 100 P~-3(ra~) CFA
22 Ip 100 Pe-3 CFA
29 Ip 100 Pe-3il CFA
34 ip 100 Pe-1711 CtA
42 Ip 100 Pe-1011 CFA
SP2/0 14 Ip 100 P~-2711 CFA
~t6 rbf/dn 1 ip 20 Pe-3~ra~) CFA
22 Ip 20 Pe-3 CFA
29 ip 20 Pe-3~1 iFA
34 ip 20 Pe-10~1 iFA
42 Ip 20 Pe-1711 itA .
SP2/0 16 ip 20 Pe 10~1 FA
~t7 rbl/dn 1 ip 500 Pe-10~pel) CFA
29 Ip 100 Pe-1011 CFA
ip 100 Pe-1011 iFA
ip 100 PR-1Ot1 FA
SP2/0 31 Ip 300 3.10.1711 FA
~11J rb~/dn 1 Ip 20 Pe-3~ra~) CFA
22 ip 20 P~ 3 CFA
29 ip 20 Pe-311 i-FA
34 ip 20 Pe-1711 li--A j_ 42 ip 20 Pe-10~1 iFA C
SP2/0 24 Ip 1000 serumt1606 itA ~r~
l~t9 rb~/dn 1 Ip 5 Pe-lO~pnl) CFA
29 ip 100 Pe-1011 CFA
Ip 100 Pe-10~1 iFA ~_.
42 ip 100 Pe-1011 iFA
29 Ip 500 Pe 3il iFA
SP2/0 ~ iv ~000 P~-~7~1 Pi3S

TABLE X~
Fusion Process ~2G rbf/dn 1 ip 100 PE 311 CF~
36 ip 800 PE 311 P13S
ipiv 400 PE 311 Pi3S
ipiv 400 PE 3il Pi3S
~ 116 Ip 400 PE 311 PBS
SP210 0 iv 2107 P4 Spleen MED
~25 -3ab/c 1 Ip 2500 PE 3il CFA
Ip 2500 PE 311 CFA
6 ip 10~ PE 311 Pi3S
0 ip 7250 PE 1711 Pi3S
2 ip 104 PE 311 P13S
0 iv 7250 PE 1711 Pi3S
ip 104 PE 3il PlS
SP2/0 0 iv 7250 PE 1711 Pi-3S
~26 rbl/dn 1 Ip 100 PE 1711 CFA
41 Ip 100 PE 1711 CFA
29 Ip sno PE 3tl FA
41 Ip 2500 PE 1711 iFA
SP2/0 7 Ip 5000 PE 28/50 i-FA
Ig28 i3aibtc 1 ip 2500 PE 3il CFA
8 Ip 2500 PE 311 CFA
6 Ip 1oooon5oo PE 3~i/PE 1711 P-3S
2 ipiv 10000/7500 PE311/PE 1711 Pi3S
t ipiv 10000/7500 PE3~1PE 1711 Pi3S
SP2/0 26 ip 2000 PE 28150 ~A
~29 rb~/dn 1 Ip 100 PE 1711 CFA
41 ip 100 PE 1711 CFA _.
29 Ip 500 PE 3il iFA C~
41 ipiv 2500 PE 17n IFA ~, SP2/0 14 ipiv 2500 PE 28t50 tA O
t~O CAF1 1 Ip 5000 PE 1711 CFA
29 Ip 5000 PE 1711 CFA
SP2/0 39 Ip 45000 PE 3tl SAL
S32 CAF1 1 Ip 5000 PE 3tl CFA
29 Ip 5000 PE 3~1 CFA

TABLE XI
Fus*n Pr~cess SP2/0 45 iv 8750 PE 1011 PBS
R33 CAF1 1 Ip 5000 PE 3~1 CtA
29 ip Sooo PE3fl CFA
SP2/0 49 iv2000/1300 PE 1711/PE 28 PBS
~3~ rb~/dn 1 ip .Sml PE 10 (pul) CtA
29 ip 100 PE 10~l CFA
ip 100 PE 10~l iFA
42 ip 100 PE 1711 iFA
29 ip 500 PE 311 iFA
SP2/0 100 iv 8400 PE 23/50 PBS
~35 rbl/d~ 1 ~.c tO0 'E 1~11 CtA
2g Ip50 ~tg 'E ~7n ~A
3~i Ip 2500 'E 1711 rFA
2~ Ip 500d 'E 2U50 IFA
SP~/O 32 l~ 8150 PE 10~1 PBS

~137~I/dn ~ ~.c 100 PE t7n CFA
2g IP 50 ~g PE 1711 iFA
3~ Ip 2500 PE 1711 iFA
i~ SoO0 PE 28/50 FA
SP210 39 iv 8000 PE 1011 SAL
ig3~ l/dn 1 ~.~ 100 PE 17n C~A
41 ip 100 PE 1711 C~A
2g ip 500 PE 311 IFA
41 ip 2500 PE 1~11 rFA
37 Ip 5000 PE 28/50 ItA ~
/ny 60 iv 8250 PE 3711 PBS i--~40rbl/dn 1 Ip 100 PE 17~l CFA
41 ~p ~oo PE 17~1 crA O
29 ip 500 PE 311 IFA I C~;~
41 ip 2500 PE 1711 iFA ~,, 37 ip 5000 PE 2B/50 IFA J -52 iv 3000 PE 3711/40 PBS
SP2/o 16 iv 3000 PE 3711/40 PBS
~45Balb/c 1 ip 5000 PE 28/60 CFA
28 ip 5000 PE 28/60 CFA
44 ip 3333 PE 311 WGA

TABLE XI
~usion Proces~

SP210 16 ip 6333 PE 3711 WGA
P67 CAF1 1 sc 5000 PE3711 C~A
31 ip 5000 PE2~12 iFA
21 Ip 5000 PE3711 IFA
ip 5000 PE3711 Pi3S
19 ip 5000 PE3711 P~S
1 B Ip 5000 PE3711 Pi3S
SP2/0 39 iv 10000 PE 26 AP Pi3S
B74 CAF1 1 sc 5000 PE 37~1 CtA
31 ip 5000 PE 2312 iFA
21 ip 5000 PE 3711 iFA
ip 5000 PE 3711 Pi3S
19 ip 5000 PE 3711 Pi3S
16 ip 5000 PE 3711 P~3S
39 Ip 5000 PE 3711 PgS
14 ip 5000 PE 37t1 Pi3S
31 ip 5000 PE 37~1 Pi3S
14 Ip 10000 PE 37~1 Pi3S
SP2/0 10 sc 10000 PE 3711 Pi3S

Claims (47)

1. A method of screening a plurality of antibody-producing clones to identify clones producing antibodies which specifically bind a particular carbohydrate determinant comprising the steps of:
(a) incubating the antibody produced by each such clone with a mucinous body fluid including at least one first glycoprotein known to bear said carbohydrate determinant, and selecting clones whose antibodies bind to the first glycoprotein; and (b) incubating the antibodies produced by clones selected in step (a) with the mucinous body fluid which has been treated with an enzyme that makes a predetermined modification to carbohydrate chains, the so-treated mucinous body fluid including at least one second glycoprotein in which the carbohydrate structure corresponding to said determinant differs therefrom in a predetermined manner, the difference resulting from the enzyme having modified the carbohydrate structure corresponding to the determinant of the first glycoprotein, and selecting clones whose antibodies essentially do not bind to said the second glycoprotein.

2. The method of claim 1 in which at least one of the first and second glycoproteins is provided as a component of a mucinous cystic, peritoneal or thoracic body fluid.

3. The method of claim 2 wherein the body fluid is obtained from at least one patient suffering from a cancerous condition.

4. The method of claim 2 in which the at least one glycoprotein that is provided as a component of a mucinous cystic, peritoneal or thoracic body fluid is provided as a mucin-enriched fraction of the body fluid which was at least partially purified to enrich its mucin content.

5. The method of claim 4 in which the purification is by carbohydrate affinity chromatography.

6. The method of claim 4 in which the purification is by size, and the mucin-enriched fraction is higher in molecular weight than the remaining material.

7. The method of claim 6 in which the mucin-enriched fraction comprises molecules having a molecular weight of at least about 200,000 daltons.

8. The method of claim 7 in which the mucin-enriched fraction comprises molecules having a molecular weight of at least about 2,000,000 daltons.

9. The method of claim 1 in which the second glycoprotein is obtained by treating the first glycoprotein with a glycosidase which selectively removes a predetermined sugar of said first glycoprotein.

10. The method of claim 9 in which the glycosidase is a sialidase and the removed sugar is sialic acid or a derivative thereof.

11. The method of claim 1 in which the second glycoprotein is obtained by treating the first glycoprotein with a glycosyltransferase which selectively adds a predetermined sugar to one or more carbohydrate chains of said first glycoprotein.

12. The method of claim 1 in which the second glycoprotein is obtained by treating the first glycoprotein with an isomerase which selectively modifies a predetermined sugar of the first glycoprotein.

13. The method of claim 9 in which fucose is removed.

14. The method of claim 9 in which galactose is removed.

15. The method of claim 1, further comprising the step of selecting clones whose antibodies essentially do not bind to a third glycoprotein known not to bear said carbohydrate determinant.

16. The method of claim 1, further comprising identifying clones whose antibodies are competitively inhibited from binding to said first glycoprotein by a hapten bearing said carbohydrate determinant.

17. A hybridoma producing antibodies against a particular carbohydrate structure, said hybridoma being obtained by screening a plurality of antibody-producing hybridomas according to the method of claim 1 and selecting a hybridoma producing antibodies against said carbohydrate structure which bind to said first glycoprotein and essentially do not bind to said second glycoprotein under the proviso that the monoclonal antibody is not HB 8059.

18. A monoclonal antibody produced by a hybridoma according to claim 17 or by a reclone thereof, and antigen-binding fragments and conjugates thereof.

19. A monoclonal antibody of claim 18 wherein said first glycoprotein is a human glycoprotein obtained from at least one patient suffering from a cancerous condition.

20. A monoclonal antibody of claim 18 in which said second glycoprotein is obtained by treating the first glycoprotein with a glycosidase which selectively removes a predetermined sugar of said first glycoprotein.

21. A monoclonal antibody of claim 20 in which fucose is removed.

22. A monoclonal antibody of claim 20 in which galactose is removed.

23. A monoclonal antibody of claim 20 in which the glycosidase is a sialidase and the removed sugar is sialic acid or a derivative thereof.

24. A monoclonal antibody of claim 18 in which the second glycoprotein has an additional sugar residue.

25. A monoclonal antibody of claim 24 in which the second glycoprotein is obtained by treating the first glycoprotein with a glycosyltransferase which selectively adds a predetermined sugar to one or more carbohydrate chains of said first glycoprotein.

26. A monoclonal antibody of claim 18 in which the second glycoprotein is obtained by isomerizing a sugar residue of said first glycoprotein.

27. A monoclonal antibody of claim 26 in which the second glycoprotein is obtain by treating the first glycoprotein with an isomerase which selectively modifies a predetermined sugar of the first glycoprotein.

28. A monoclonal antibody of claim 18 in which the antibody binds to a sialosyl Lewis a hapten but essentially does not bind to an unsialosylated Lewis a hapten.

29. The hybridoma clone B25.10 and any reclone thereof.

30. A monoclonal antibody produced by the hybridoma of claim 29, and antigen-binding fragments and conjugates thereof.

31. A device for screening a plurality of hybridomas according to the method of claim 8, which comprises: a support means to which the first glycoprotein and the second glycoprotein are attached at distinct and known locations, whereby it may be determined whether the antibodies secreted by the hybridomas are bound by the first glycoprotein and essentially not bound by the second glycoprotein, said support means further providing means for maintaining the antibodies produced by said hybridomas in contact with said first and second glycoproteins.

32. The device of claim 31, wherein a first and second series of depressions are formed in the support means, and the first glycoprotein is attached to the support means within the first series of depressions, and the second glycoprotein is attached to the support means within the second series of depressions, said first and second series of depressions being provided to receive said antibody-producing hybridomas or their antibodies.

33. The device of claim 31, wherein said glycoproteins are mucins.

34. The device of claim 33, wherein the second glycoprotein is the asialo form of the first glycoprotein.

35. The device of claim 34, wherein the first glycoprotein bears sialosyl Lewis-a.

36. In an immunoassay for a tumor-associated glycoantigen, wherein the glycoantigen is bound by a monoclonal antibody, the improvement which comprises selecting the antibody according to the method of claim 1.

37. The method of claim 36 wherein the antibody binds to a sialylated Lewis-a carbohydrate epitope on said antigen.

38. In an immunological method wherein a glycoantigen is bound by a monoclonal antibody, the improvement which comprises using an antibody such that the percentage of antigen bound by the antibody within a pH range of 4-9 is always at least about 66% of the percentage of the antigen bound at the pH within the range of 4-9 at which the binding is maximized.

39. The method of claim 38 wherein the antibody binds to a sialylated Lewis-a carbohydrate epitope on said antigen.

40. The method of claim 38 wherein the assay is a competitive immunoassay for the detection or quantification of a glycoantigen in an aqueous solution.

41. The method of claim 1 in which the antibody-producing cell is obtained by immunization of an animal with a mucinous body fluid or a component thereof and removal of antibody-producing cells from the immunized animal.

42. A method of screening antibody-producing cells which comprises incubating the candidate antibody produced by said cells with an antigen and with a known antibody produced by cells selected according to the method of claim 1, and determining whether the candidate antibody's binding to said antigen is competitively inhibited by the known antibody.

43. A method according to claim 2 in which the at least one glycoprotein is provided by a mixture of mucinous body fluids from a plurality of patients.

44. A method of claim 39 wherein the average percentage of the antigen bound by the antibody at pH 7.1 is at least 75% of the percentage of the antigen bound by the antibody at pH 5.

45. A monoclonal antibody which binds to the same carbohydrate determinant as is bound by the antibody produced by hybridoma B25.10, and antigen-binding fragments and conjugates thereof, with the proviso that the monoclonal antibody is not the antibody produced by HB8059.

46. In an immunological method wherein a glycoantigen is bound by a monoclonal antibody, use of an antibody of claim 18 or claim 45 such that the percentage of antigen bound by the antibody within a pH range of 4-9 is always at least about 66% of the percentage of the antigen bound at the pH within the range of 4-9 at which the binding is maximized.

47. A method of claim 46 wherein the average percentage of the antigen bound by a said antibody at pH 7.1 is at least 75% of the percentage of the antigen bound by the antibody at pH 5.