AU625856B2 - Second generation monoclonal antibodies having binding specificity to tag-72 and human carcinomas and methods for employing the same - Google Patents

Description

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AU-AI-23085/88 PCr WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau I Dl INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 00692 G01N 33/53 Al (43) International Publication Date: 26 January 1989 (26.01.89) (21) International Application Number: PCT/US88/01941 (74) gt TER in,et 1 H olman Stern, e1 W1ngton, DC 20009 (22) International Filing Date: 7 June 1988 (07.06.88) (US).

(31) Priority Application Number: 073,685 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 15 July 1987 (15.07.87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: US tent), NL (European patent), SE (European patent).

(71) Applicant: THE UNITED STATES OF AMERICA, as Published represented by THE SECRETARY, U.S. DEPART- With international search report.

MENT OF COMMERCE [US/US]; 5285 Port Royal Before the expiration of the time limit for amending the Road, Springfield, VA 22151 claims and to be republished in the event of the receipt of amendments.

(72) Inventors: SCHLOM, Jeffrey 10525 Tyler Terrace, Potomac, MD 20854 COLCHER, David 6 A. 0. J. P 6 APR 1989 Kirkwall Court, Potomac, MD 20854 (US).

AUSTRALIAN

1 3 FEB1989 PATENT OFFICE (54) Title: SECOND GENERATION MONOCLONAL ANTIBODIES HAVING BINDING SPECIFICITY TO TAG- 72 AND HUMAN CARCINOMAS AND METHODS FOR EMPLOYING THE SAME (57) Abstract The present invention relates to second generation monoclonal antibodies having binding specificity to a tumor associated glycoprotein having an approximate molecular weight of 10 6 d ("TAG-72") and human carcinomas and methods for employing the same. Hybridomas producing such antibodies have been prepared.

WO 89/00692 PCT/US88/01941 -1- 1 SECOND GENERATION MONOCLONAL ANTIBODIES HAVING 2 BINDING SPECIFICITY TO TAG-72 AND HUMAN 3 CARCINOMAS AND METHODS FOR EMPLOYING 4 THE SAME FIELD OF THE INVENTION 6 The present invention relates to second generation 7 monoclonal antibodies having binding specificity to a 8 tumor associated glycoprotein having an approximate 9 molecular weight of >10 6 d (hereinafter "TAG-72") and human carcinomas, and methods for employing the same.

11 BACKGROUND OF THE INVENTION 12 Numerous monoclonal antibodies have been developed 13 which have binding specificity for a variety of human 14 carcinomas (see Schlom, et al., "Important Advances in Oncology", Philadelphia, PA, J.B. Lippincott Co., Vol. 1, 16 pp. 170-192 (1984) and Schlom, Cancer Res., 46:3225-3238 17 (1986)). One of these monoclonal antibodies designated 18 B72.3.(see Colcher, et al., Proc. Natl. Acad. Sci. USA, 19 78:3199-3203 (1981) and U.S. Patents Nos. 4,522,918 and 4,612,282), is a murine IgG1, and was developed using a 21 human breast carcinoma extract as the immunogen.

22 Monoclonal antibody B72.3 is produced by hybridoma B72.3 23 (ATCC No. HB-8108) and has been extensively studied.

1 1 i i i WO 89/00692 PCT/US88/01941 -2- I Monoclonal antibody B72.3 has been shown to be distinct 2 from other known monoclonal antibodies on the basis of: 3 its binding specificity to TAG-72 (see Johnson, et 4 al., Cancer Res., 46:857-859 (1986)); its binding specificity to various types of human carcinoma tissues, 6 including breast, ovarian, lung, colorectal, endometrial, 7 and pancreatic carcinoma tissues (see Nuti, et al., Intl.

8 J. Cancer, 29:539-545 (1982); Stramignoni, et al., Intl.

9 J. Cancer, 31:543-552 (1982); Thor, et al., J. Natl.

Cancer Inst., 76:995-1006 (1986); and Thor, et al., 11 Cancer Res., 46:3118-3124 (1986)); its lack of 12 binding specificity to normal adult human tissues (see 13 Nuti, et al., Intl. J. Cancer, 29:539-545 (1982); 14 Stramignoni, et al., Intl. J. Cancer, 31:543-552 (1983); Thor, et al., J. Natl. Cancer Inst., 76:995-1006 (1986); 16 and Thor, et al., Cancer Res., 46:3118-3124 (1986)); (4) 17 its ability to detect TAG-72 in serum (see Paterson, et 18 al., Intl. J. Cancer, 37:659-666 (1986) and Klug, et al.

19 Intl. J. Cancer, 38:661-669 (1986)); its ability to detect carcinoma cells in human effusions and fine needle 21 aspiration biopsies (see Szpak, et al., Acta Cytologica, 22 28:356-367 (1984); Johnston, et al., Cancer Res., 23 1894-1900 (1986); Szpak, et al., Am. J. Path., 122:252- 24 260 (1986); Johnston, et al., Human Path., 17:501-513 (1986); Martin, et al., Am. J. Clin. Path., 86:10-18 26 (1986); Nuti, et al., Intl. J. Cancer, 37:493-498 (1986); 27 and Johnston, et al., Cancer Res., 46:6462-6470 (1986)); 28 and its binding specificity and prolonged binding to 29 human carcinomas both in experimental animal systems (see Kennan, et al., J. Nucl. Med., 25:1197-1203 (1984) and 31 Colcher, et al., Cancer Res., 44:5744-5751 (1984)) and in 32 clinical trials (see Colcher, et al., Cancer Res., 47: 33 1185-1189 (1987) and Esteban, et al., Intl. J. Cancer, 34 39:50-58 (1987)).

I i i SPCT/US 8 80194 1 -PEA/ 24JUL1989 -3- 1 However, monoclonal antibody B72.3 is disadvantageous 2 in that B72.3 does not have binding specificity to 3 every human carcinoma tissue of a particular type, to 4 every ovarian, colon carcinoma tissue, etc. (See Nuti, et 3 al., Intl. J. Cancer, 29:539-545 (1982); Stramignoni, et 6 al., Intl. J. Cancer,31: 543-552 (1983); Thor, et al., J.

7 Natl. Cancer Inst., 76:995-1006(1986); Thor, et al., Cancer 8 Res., 46:3118-3124 (1986); and Hand, et al., Cancer 9 Res.,43: 728-735 (1983)); B72.3 does not have binding specificity to all carcinoma cells within a given human 11 carcinoma mass (see Nuti, et al,, supra.; Stramignoni, et 12 al., supra.; Thor, et al. J. Natl. Cancer Inst., 76:995- 13 1006(1986); Thor, et al., Cancer Res., 46:3118-3124 (1986); 14 and Hand, et al., supra.); B72.3 does not have binding specificity to most human carcinoma cell lines in culture 16 (see Hand, et al., supra.; Hand et al., Cancer Res., 17 45:833-840 (1985); and Friedman et al., Cancer Res.

18 45:5648-5655 (1985)); it is difficult to obtain highly 19 immunoreactive F(ab') 2 F(ab') and F(ab) fragments from B72.3, such fragments being necessary for efficient in vivo 21 immunodiagnostic and therapeutic applications; and 22 since B72.3 is of the IgG 1 isotype, it is difficult to 23 conduct monoclonal antibody effector cell mediated 24 cytotoxicity or complement mediated cytotoxicity studies using B72.3 (IgG 2 a IgG 2 b or IgM isotypes being more 26 efficient for these applications).

27 DEPOSIT OF THE BIOLOGICAL MATERIAL OF THIS INVENTION 28 Viable samples of Hybridoma Cell Lines CC 83; CC 92; 29 CC 11; MATAG 12; CC 30; CC 46; CC 49; and CC 15; have been deposited at the American Type Culture Collection, 12301 31 Parklawn Drive, Rockville, MD. 20852, on June 26, 1987, 32 and respectively have the following ATCC designations HB 33 9453, HB 9454, HB 9455, HB 9456, HB 9457, HB 9458, HB 9459, 34 and HB 9460.

RAZ/ s /US -o "-/NVT 1 WO 89/00692 PCT/US88/01941 4- 1 SUMMARY OF THE INVENTION 2 Accordingly, an object of the present invention is 3 to provide monoclonal antibodies which have binding 4 specificity to a variety of human carcinomas, including human carcinomas of a given type for which B72.3 6 essentially has no binding specificity.

7 Another object of the present invention is to 8 provide monoclonal antibodies, having high binding 9 affinity for TAG-72 and human carcinomas.

A further object of the present invention is to 11 provide monoclonal antibodies from which highly 12 immunoreactive F(ab') 2 F(ab') and F(ab) fragments can be 13 easily obtained for use in in vivo immunodiagnosis and 14 therapy of human carcinomas.

A still further object of the present invention is 16 to provide monoclonal antibodies from which recombinant 17 antibodies can be obtained for use in in vivo 18 immunodiagnosis and therapy of human carcinomas.

19 An additional object of the present invention is to provide monoclonal antibodies of the IgG2a, IgG2b and 21 IgM isotypes which have binding specificity for human 22 carcinomas for use in conducting monoclonal antibody 23 effector cell mediated cytotoxicity or complement 24 mediated cytotoxicity studies.

Still an additional object of the present 26 invention is to provide methods for diagnosing in vitro 27 and in vivo human carcinomas and methods for treating 28 human carcinomas employing these monoclonal antibodies. 29 Other objects and advantages of the present invention will become apparent from the Detailed 31 Description of the Invention presented hereunder.

32 The above and various other objects and advantages 33 of the present invention are achieved by the second

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i WO 89/00692 PCT/US88/01941 5 1 generation monoclonal antibodies of the present invention 2 which have binding affinity to both TAG-72 and to LS-174T 3 antigen(s).

4 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as 6 commonly understood by one of ordinary skill in the art 7 to which this invention belongs. Although any methods 8 and materials similar or equivalent to those described 9 herein can be used in the practice or testing of the present invention, the preferred methods and materials 11 are now described. All publications mentioned hereunder 12 are incorporated herein by reference.

13 As used herein, the expression "second generation 14 monoclonal antibodies" means monoclonal antibodies produced using, as the immunogen, an antigen which has 16 been affinity purified with a first generation monoclonal 17 antibody. As used herein, the expression "first 18 generation monoclonal antibody" means a monoclonal 19 antibody produced using, as the immunogen, a crude cell extract.

21 The term "substantially" as used herein means 22 almost wholly or to a large extent, but not entirely.

23 LS-174T (ATCC No. CRL-188) is a variant of the 24 LS180 (ATCC No. CRL-187) colon adenocarcinoma line. It is more easily subcultivated than the parent line. It 26 is tumorigenic in nude mice. The karyotype is similar 27 to that of LS180 with a missing X chromosome in a 28 majority of the cells. Electron microscopic studies 29 reveal abundant microvilli and intracytoplasmic mucin vacuoles (see Tom, et al., In Vitro, 12:180-191 (1976)).

31 TAG-72 is an antigen found in the LS-174T tumor 32 cell line. Monoclonal antibody B72.3 binds to a high 33 molecular weight tumor associated glycoprotein identified 34 as TAG-72. Data has been presented as described in H I 1 i I^r^ WO 89/00692 PCT/US88/01941 6 1 Johnson, et al., Cancer Res., 46:850-857 (1986), to 2 characterize the TAG-72 molecule as a mucin. This 3 conclusion is based on the following observations: (a) 4 TAG-72 has a high molecular weight x 106) as shown by its exclusion from a Sepharose CL-4B column; the 6 density of TAG-72 determined by equilibrium 7 centrifugation in CsCl was 1.45 gm/ml, indicating a 8 heavily glycosylated glycoprotein; TAG-72 9 demonstrates a change in migration after neuraminidase digestion, indicating that it is a heavily sialylated 11 molecule with an abundance of O-glycosidically linked 12 oligosaccharides characteristic of mucins; blood 13 group antigens commonly found on mucins are found on 14 affinity-purified TAG-72; and chondroitinase ABC digestion had no effect on TAG-72, thus demonstrating 16 that the TAG-72 epitope is not expressed on a chondroitin 17 sulfate proteoglycan.

18 More specifically, the above-described objects of 19 the present invention have been achieved by the second generation monoclonal antibodies of the present 21 invention, immunoreactive fragments or recombinants 22 thereof which have binding specificity to TAG-72 and to 23 human carcinomas, including human carcinomas to which 24 antibody B72.3 has minimal binding specificity and with minimal binding specificity to normal adult human 26 tissues. The term "minimal" means the least possible or 27 substantially inconsequential.

28 To another embodiment, the above-described objects 29 of the present invention have been achieved by a method for diagnosing a human carcinoma or metastases thereof 31 comprising: 32 obtaining a body simple, such as body 33 fluid, tissue or biopsy from a patient; I i l 1 1

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r' i WO 89/00692 PCT/US88/01941 7 1 contacting the body sample material 2 with a second generation monoclonal antibody of the 3 present invention, an immunoreactive fragment or a 4 recombinant thereof; determining the level of binding of 6 second generation monoclonal antibody, immunoreactive 7 fragment or recombinant thereof to the body sample 8 material; and 9 comparing the amount of second generation monoclonal antibody, immunoreactive fragment 11 or recombinant thereof bound to substances present in the 12 body sample to a control sample or to a predetermined 13 base level, so that a binding greater than the control 14 level is indicative of the presence of human carcinomas or metastases thereof.

16 In still another embodiment, the above-described 17 objects of the present invention have been achieved by a 18 method for diagnosing the presence of a human carcinoma 19 or metastases thereof comprising: administering to a patient a second 21 generat.io monoclonal antibody of the present invention, 22 an immunoreactive fragment or recombinant thereof, 23 conjugated to an imaging marker; and 24 exposing the pati-nt to a means for detecting said imaging marker to identify areas of 26 imaging marker corresponding to a human carcinoma or 27 metastatic sites thereof in said patient.

28 In a still further embodiment, the above-described 29 objects of the present invention have been achieved by a method of treating a patient afflicted with a human 31 carcinoma or metastases thereof, comprising administering 32 to a patient afflicted with carcinoma or metastases, a 33 pharmaceutically effective amount of a second generation 34 monoclonal antibody of the present invention, an WO 89/00692 PC'/US88/01941 WO 89/00692 PCT/USS8/01941 21 WO 89/00692 PCT/US88/01941 8 1 immunoreactive fragment or recombinant thereof conjugated 2 to a therapeutic agent.

3 BRIEF DESCRIPTION OF THE DRAWINGS 4 Figure 1 is a schematic diagram of: the differential binding specificities of the CC and MATAG 6 monoclonal antibodies of the present invention to LS-174T 7 colon carcinoma cells (ATCC No. CRL-188) in a competition 8 radioimmunoassay (hereinafter "RIA") with B72.3; the 9 isotypes of the CC and MATAG monoclonal antibodies of the present invention; and the binding specificity of the 11 CC and MATAG monoclonal antibodies of the present 12 invention to various colon carcinomas in a solid phase 13 RIA (hereinafter "SPRIA)".

14 Figure 2 is an analysis of the binding specificity of monoclonal antibody CC41 to LS-174T colon carcinoma 16 cell extract in a competition RIA with B72.3. Figure 2B 17 is a quantitative analysis of the binding specificities 18 of monoclonal antibodies B72.3 and CC41 to LS-174T colon 19 carcinoma cell line extract (LS) and a breast carcinoma biopsy extract (Br. Ca.) in a SPIRA.

21 Figure 2C is an analysis of the binding 22 specificity of monoclonal antibody CC60 to LS-174T colon 23 carcinoma cell extract in a competition RIA with B72.3.

24 Figure 2D is a quantitative analysis of the binding specificities of monoclonal antibodies B72.3 and CC60 to 26 LS-174T colon carcinoma cell line extract (LS) and a 27 breast biopsy extract (Br. Ca.) in a SPRIA.

28 Figure 2E is an analysis of the binding 29 specificity -of monoclonal antibody CC83 to LS-174T colon carcinoma cell extract in a competition RIA with B72.3. i 31 Figure 2F is a quantitative analysis of the binding 32 specificities of monoclonal antibodies B72.3 and CC83 to Ii.

I: S WO 89/00692 PCT/US88/01941 9 1 LS-174T colon carcinoma cell line extract (LS) and a 2 breat carcinoma biopsy extract (Br. Ca.) in a SPRIA.

3 Figure 2G is an analysis of the binding 4 specificity of monoclonal antibody CC49 to LS-174T colon carcinoma cell extract in a competition RIA with B72.3.

6 Figure 2H is a quantitative analysis of the binding 7 specificities of monoclonal antibodies B72.3 and CC49 to 8 LS-174T colon carcinoma cell line extract (LS) and a 9 breast carcinoma biopsy extract (Br. Ca.) in a SPRIA.

Figure 3 is an analysis of a competition RIA with 11 CC49, wherein 125 1-labelled CC49 monoclonal antibody was 12 reacted with LS-174T colon carcinoma cell extract and 13 purified CC30, CC46, CC49, CC83 and B72.3 were used as 14 competing antibodies.

Figure 4A is an analysis of the in vivo targeting 16 of a LS-174T colon carcinoma xenograft with monoclonal 17 antibody CC11.

18 Figure 4B is an analysis of the in vivo targeting 19 of a LS-174T colon carcinoma xenograft with monoclonal antibody CC46.

21 DETAILED DESCRIPTION OF THE INVENTION 22 I. Characteristics of the Monoclonal Antibodies 23 The monoclonal antibodies specifically developed 24 in the present invention, designated CC1 to CC92 (IgG monoclonal antibodies) and MATAG 1 to MATAG 18 (IgM 26 monoclonal antibodies) (see Figure 1) all have binding 27 specificity to TAG-72 and numerous types of .human 28 carcinomas (including breast, ovarian, lung, colorectal, 29 endometrial and pancreatic carcinomas), and are different from B72.3 in that they: 1 k l :i 1 I WO 89/00692 PCT/US88/01941 10 1 have binding specificity to more human 2 carcinomas than B72.3 while still maintaining essentially 3 no specificity to normal adult human tissues; 4 have a higher binding affinity for TAG-72 than B72.3, on the order of greater than 3 x 6 10 9 M, preferably greater than 8 x 10 9 M and consequently 7 bind human carcinomas in vivo at a higher efficiency; 8 exhibit a 50% or more efficiency than 9 B72.3 in targeting human carcinomas in situ more injected dose/gram tumor than B72.3 and preferably 11 greater than 100% more injected dose/gram tumor than 12 B72.3); 13 can be easily fragmented with pepsin to 14 obtain F(ab') 2 F(ab') and F(ab) fragments that are highly immunoreactive; and 16 include monoclonal antibodies of the 17 IgG2a, IgG2b, and IgM isotypes so they can more 18 efficiently be used in monoclonal antibody targeted 19 effector cell mediated cytotoxicity or complement mediated cytotoxicity studies.

21 The development of the CC and MATAG monoclonal 22 antibodies of the present invention also now makes 23 feasible the use of double determinant RIAs (hereinafter 24 "DDRIA"s) for more efficient detection of human carcinoma antigens in body fluids and biopsies of cancer patients.

26 II. Production of the Monoclonal Antibodies 27 The CC and MATAg monoclonal antibodies of the 28 present invention are produced by immunizing mice (or 29 other animals such as rats, rabbits, goats, and humans) with purified TAG-72 obtained from various xenografts, 31 such as LS-174T human colon carcinoma xenografts prepared S I S WO 89/00692 PCT/US88/01941 Aoi i- 1 using LS-174T carcinoma cells (ATCC No. CRL-188) and 2 OVCAR-3 human ovarian cancer xenografts, prepared using 3 OVCAR-3 carcinoma cells (see Hamilton, et al., Cancer 4 Res., 43:5379-5389 (1983)).

TAG-72 is purified from the xenografts by methods 6 well known in the art. More specifically, by the 7 following steps: b:eaking the cells; (2) 8 centrifuging and/or filtering to remove cellular debris; 9 carrying out sizing column chromatography to obtain proteins having a molecular weight of >10 6 d, the 11 molecular weight of TAG-72; and then carrying out 12 B72.3 affinity column chromatography to obtain the 13 desired TAG-72 (see Paterson, et al., Intl. J. Cancer, 14 37:659-666 (1986)).

Immunizing the animals, mice, with the 16 purified TAG-72, isolating the immunized cells, fusing 17 the immunized cells with mouse myeloma cells (or myeloma 18 cells of other species such as rats, rabbits, goats and 19 humans), all of which are well known in the art and readily available, and culturing the resulting fused 21 cells under conditions which allow for growth of 22 hybridomas, are all conducted by methods well known or 23 readily determined in the art (see Herzenberg, et al., 24 "Handbook of Experimental Immunology", Oxford, Blackwell, pp. 25.1-25.7; Colcher, et al., Proc. Natl. Acad. Sci.

26 USA, 78:3199-3203 (1981); and Muraro, et al., Intl. J.

27 Cancer, 39:34-44 (1987)).

28 The resulting hybridomas are then tested to 29 isolate those which produce monoclonal antibodies having binding specificity to TAG-72 and human carcinomas but 31 not to normal adult human tissues. This screening is 32 carried out using a SPRIA as decribed in greater detail 33 in the Examples provided hereinafter. V L• WO 89/00692 PCT/US88/01941 12 1 The binding affinity of monoclonal antibodies for 2 TAG-72 is determined by means well known in the art (see 3 Heyman, et al., J. Immunol. Methods, 68:193-204 (1984)) 4 and as described in ,detail in the Examples provided hereinafter.

6 The isotypes (IgG, IgG IG, IgG2b IgG 3 or IgM) of 7 the monoclonal antibodies are determined by means well 8 known in the art (see Colcher, et al., Cancer Res., 9 41:1451-1459 (1981)) and as described in detail in the Examples provided hereinafter.

11 In the non-limiting Examples provided hereinafter, 12 in excess of four thousand hybridomas were produced by 13 fusing spleen cells of mice immunized with purified 14 TAG-72 which was obtained from a LS-174T human colon carcinoma xenograft, and (ii) the well known and readily 16 available NS-1 mouse myeloma line (ATCC No. TIB-18).

17 From these hybridomas, 44 double cloned hybridomas (29 CC 18 second generation monoclonal antibodies and 15 MATAG 19 second generation monoclonal antibodies) were selected and characterized as described in the Examples provided 21 hereinafter.

22 The CC monoclonal antibodies of the present 23 invention are fragmented to obtain highly immunoreactive 24 F(ab') 2 and F(ab) fragments using the enzyme pepsin by methods well known in the art (see Colcher, et al., 26 Cancer Res., 43:736-742 (1983)) and as described in 27 greater detail in the Examples provided hereinafter.

28 The immunoreactivity of the resulting F(ab') 2 F(ab') and 29 F(ab) fragments are determined in a competition RIA or SPRIA as described above for the complete monoclonal 31 antibody molecule.

32 The second generation antibodies of the present 33 invention are also made into recombinant forms by

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i rl i l ll__~liii~ WO 89/00692 PCT/US88/01941 13 1 techniques of molecular biology well known in the art 2 (see Rice, et al., Proc. Natl. Acad. Sci. USA, 79:7862- 3 7865 (1982); Kurokawa,. et al., Nucleic Acids Res., 11: 4 3077-3085 (1983); Oi, et al., Proc. Natl. Acad. Sci. USA, 80:825-829 (1983); Boxx, et al. Nucleic Acids Res., 12: 6 3791-3806 (1984); Boulianne, et al., Nature (London), 7 312:643-646 (1984); Cabily, et al., Proc. Natl. Acad.

8 Sci. USA, 81:3273-3277 (1984); Kenten, et al. Proc. Natl.

9 Acad. Sci. USA, 81:2955-2959 (1984); Liu, et al., Proc.

Natl. Acad. Sci. USA, 81: 5369-5373 (1984); Morrison, et 11 al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); 12 Neuberger, et al., Nature (London), 312:604-608 (1984); 13 Potter, et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165 14 (1984); Neuberger, et al., Nature (London), 314:268-270 (1985); Jones, et al., Nature (London), 321:522-525 16 (1986); Oi, et al., BioTechniques, 4:214-221 (1986); 17 Jahagan, et al., J. Immunol., 137:1066-1074 (1986); Sun, 18 et al., Hybridoma 5 (Suppl. 1):S-17-S20 (1986); and Sun, 19 et al., Proc. Natl. Acad. Sci. USA, 84:214-218 (1987)) all of which are specifically incorporated herein by 21 reference.

22 More specifically, the second generation 23 monoclonal antibodies of the present invention are 24 altered to a chimeric form by substituting, human constant regions (Fc domains) for mouse constant regions 26 by recombinant DNA techniques known in the art as 27 described in the above cited references. These Fc 28 domains can be of various human isotypes, IgG 1 29 IgG 2 IgG 3 IgG 4 or IgM.

In addition, the second generation monoclonal 31 antibodies of the present invention are. altered to an 32 affinity modified form, avidity modified form, or both, 33 by altering binding sites or altering the hinge region WO 89/00692 PCT/US88/01941 14 1 using recombinant DNA techniques well known in the art as 2 described in the above cited references.

3 The recombinant antibody forms are also fragmented 4 to produce immunoreactive fragments F(ab') 2 or F(ab) in the same manner as described above in which the 6 second generation monoclonal antibodies of the present 7 invention are fragmented.

8 Accordingly, as used herein, the expression 9 "recombinant antibodies" collectively includes chimeric/ recombinant forms of the second generation monoclonal 11 antibody of the present invention wherein the Fc domain 12 is substituted for an Fc domain of another species or 13 isotype, affinity modified forms of the second generation 14 monoclonal antibody of the present invention wherein the binding sites are altered, avidity modified forms of the 16 second generation monoclonal antibody of the present 17 invention wherein the hinge regions are altered, 18 immunoreactiv- fragments thereof and combinations 19 thereof.

The second generation monoclonal antibodies of the 21 present invention are produced in large quantities by 22 injecting a hybridoma producing a second generation 23 monoclonal antibody of the present invention into the 24 peritoneal cavity of pristane-primed mice, and after an appropriate time (about 1-2 weeks), harvesting ascites 26 fluid from the mice, which yields a very high titer of 27 homogenous monoclonal antibody, and isolating the 28 monoclonal antibodies therefrom by methods well known in 29 the art (see Stramignoni, et al., Intl. J. Cancer, 31: 543-552 (1983)). Alternatively, the second generation 31 monoclonal antibodies are produced by culturing a 32 hybridoma producing a second generation monoclonal 33 antibody of the present invention in vitro and isolating 34 secreted monoclonal antibodies from the cell culture __11 i i ii;i-r -IY1PIII-X~ WO 89/00692 PCT/US88/01941 15 1 medium by methods well known in the art (see Colcher, et 2 al., Proc. Natl. Acad. Sci. USA, 78:3199-3203 (1981)).

3 The CC and MATAG monoclonal antibodies of the 4 present invention are thus produced arcording to the above method. The binding specificity and binding 6 affinity of these monoclonal antibodies and a comparison 7 of such with B72.3 are discussed in greater detail in the 8 Examples provided hereinafter.

9 III. Uses of the Monoclonal Antibodies The second generation monoclonal antibodies of the 11 present invention, immunoreactive fragments or 12 recombinants thereof, can be used either alone, in 13 combination with one another, or in combination with 14 other antibodies, such as B72.3 or immunoreactive fragments thereof, in: in vitro diagnostic assays 16 using labelled monoclonal antibodies for the detection of 17 TAG-72 in body fluids of patients; in vivo diagnostic 18 assays (diagnostic imaging) using the second generation 19 monoclonal antibodies of the present invention, immunoreactive fragments or recombinants thereof, 21 conjugated to an imaging marker, for the in situ 22 detection of carcinoma lesions; in vivo cancer 23 treatment using the second generation monoclonal 24 antibodies of the present invention, immunoreactive fragments or recombinants thereof alone or conjugated to 26 a therapeutic agent such as radionuclide, drug, toxin, 27 effector cells, other antibodies or via a complement 28 mechanism; immunohistopathology or 29 immunocytochemistry for the detection or phenotyping of carcinoma cells; and as immunogens to activate the 31 anti-idiotype network for active immunotherapy against 32 carcinomas.

WO 89/00692 PCT/US88/01941 16 1 A. In Vitro Diagnostic Assays 2 In ritro diagnostic assays of human carcinomas or 3 metastases thereof by detecting TAG-72 in body fluids of 4 patients using the second generation monoclonal antibodies of the present invention, immunoreactive 6 fragments or recombinants thereof are described in 7 greater detail below.

8 The body fluid obtained from a patient is 9 contacted with the monoclonal antibody of the present invention, immunoreactive fragment or recombinant 11 thereof. A diagnosis is then made by determining the 12 amount of monoclonal antibody, immunoreactive fragment or 13 recombinant thereof binding to substances (TAG-72) 14 present in the body fluid and comparing the amount of monoclonal antibody, immunoreactive fragments or 16 recombinants thereof bound to the body fluid substances 17 to a predetermined base level as hereinafter defined.

18 The amount of bound monoclonal antibody, immunoreactive 19 fragment or recombinant thereof exceeding the base level indicates the presence of a human carcinoma or metastases 21 thereof.

22 Examples of body fluids which can be used in the 23 in vitro method are any body fluids suspected of 24 containing TAG-72. Preferred examples thereof include blood (serum or plasma), sputum, nipple discharge, cyst 26 fluid, ascites fluids, pleural effusions, seminal plasma, 27 semen, urine and prostatic fluid and/or biopsy specimens.

28 Serum or plasma are the more preferred body fluids 29 employed in the present invention. The body fluids can be obtained by methods readily known to or determined by 31 those skilled in the art.

32 The body fluid is contacted with the second i 33 generation monoclonal antibody of the present invention,

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,J l^_ N( WO 89/00692 PCT/US88/01941 17 1 immunoreactive fragment or recombinant thereof and the 2 amount of monoclonal antibody, immunoreactive fragment or 3 recombinant thereof bound to substances in the body fluid 4 is determined by means of immunochemical assays well known to those skilled in the art, as described, for 6 example, in Klug, et al., Cancer Res., 44:1048-1053 7 (1984); Klug, et al., Intl. J. Cancer, 38:661-669 (1986); 8 Herlyn, et al., J. Clin. Immunol., 2:135-140 (1982); 9 Metzgar, et al., Proc. Natl. Acad. Sci. USA, 81:5242-5246 (1984); Papsidero, et al., Cancer Res., 44:4653-4657 11 (1984); Hayes, et al., J. Clin. Invest., 75:1671-1678 12 (1985); Killian, et al., Cancer Res., 45:886-891 (1985); 13 Hedin, et al., Proc. Natl. Acad. Sci. USA, 80:3470-3474 14 (1983); Pekary, et al., Clin. Chem., 30:1213-1215 (1984); Bast, et al., New England J. Med., 309:883-887 (1983); 16 and Bellet, et al., Proc. Natl. Acad. Sci. USA, 81: 17 3869-3873 (1984), the disclosures of all of which are 18 specifically incorporated herein by reference.

19 An example of one type of immunochemical assay useful in the present invention is a sandwich 21 immunoradiometric assay (hereinafter "IRMA"). In this 22 type of assay, the presence of antigen (TAG-72) is 23 measured directly by reacting it with an excess of 24 labelled monoclonal antibody. In such an assay, before the antigen is reacted with the labelled monoclonal 26 antibody, the antigen in insolubilized on an 27 immunoadsorbent which specifically binds the antigen.

28 The immunoadsorbent is formed by affixing a second 29 generation monoclonal antibody, immunoreactive fragment or recombinant thereof to a substrate such as an 31 immunobead. In sandwich assays for an antigen which is 32 monomeric, two antibodies which recognize' distinct 33 epitopes on the antigen are required, a so-called 34 "double determinant" assay, so that there is no 1

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WO 89/00692 PCT/US88/01941 18 1 competition for binding to the antigen. In sandwich 2 assays, one antibody is bound to the immunoadsorbent and 3 the other antibody is used as the labelled tracer. In 4 assays for dimeric or polymeric antigens, the same antibody can be bound to the immunoadsorbent as the 6 labelled tracer.

7 Sandwich IRMA's may be performed in a forward, 8 reverse or simulantaneous mode.

9 In a forward sandwich assay for TAG-72, a monoclonal antibody is affixed to a solid phase such as 11 an immunobead to form an immunoadsorbent specific for 12 TAG-72. A body liquid sample containing TAG-72 is then 13 incubated with the immunoadsorbent. Incubation is 14 maintained .for a sufficient period of time to allow TAG-72 in the body fluid to bind to the immobilized 16 monoclonal antibody on the immunoadsorbent. After this 17 first incubation, the solid phase immunoadsorbent is 18 separated from the incubation mixture. The 19 immunoadsorbent may be washed to remove unbound interfering substances, such as non-specific binding 21 proteins, which may also be present in the body fluid.

22 The immunoadsorbent containing TAG-72 bound to an 23 immobilized monoclonal antibody is subsequently incubated 24 with a labelled monoclonal antibody, immunoreactive fragment or recombinant thereof. Again, the incubation 26 is carried out for a period of time and under conditions 27 sufficient to ensure binding of the labelled monoclonal 28 antibody, immunoreactive fragment or recombinant thereof 29 to TAG-72. After the second incubation, another wash may be performed to remove unbound labelled monoclonal 31 antibody, immunoreactive fragment or recombinant thereof 32 from the solid phase immunoadsorbent. The labelled 33 monoclonal antibody, immunoreactive fragement or 34 recombinant thereof bound to the solid phase ii 4

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PCT/US88/01941 WO 89/00692 19 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 immunoadsorbent is then measured, and the amount of labelled monoclonal antibody, immunoreactive fragment or recombinant thereof detected serves as a direct measure of the amount of TAG-72 present in the body fluid.

The sandwich IRMA may also be performed in reverse and simultaneous modes. In the reverse mode, an incubation mixture is formed of the body fluid to -be tested and soluble labelled monoclonal antibody, immunorsactive fragment or recombinant thereof directed against TAG-72. The mixture is incubated, then contacted with a solid phase immunoadsorbent also containing a monoclonal antibody, immunoreactive fragment or recombinait thereof directed against TAG-72. After another incubation, the immunoadsorbent is separated from the mixture and the label bound to the immunadsorbent is taken as an indication of the amount of TAG-72 in the body fluid.

In the simultaneous mode, an incubation mixture is formed of the body fluid, the labelled monoclonal antibody, immunoreactive fragment or recombinant thereof and the solid phase immunoadsorbent. After incubation for a sufficient time, the solid phase immunoadsorbent is separated from the mixture and the label associated with the immunoadsorbent is measured to give an indication of the amount of TAG-72 in the body fluid.

For each incubation step in the various assay modes described above, the time and conditions of incubation are selected to ensure maximum binding of TAG-72 to the immobilized monoclonal antibody, immunoreactive fragment or recombinant thereof and to labelled monoclonal antibody, immunoreactive fragment or recombinant thereof, but generally are about 6 to 16 hours at room temperature (220 to 27 0

C).

I

I

r iL I' I I l II I. I WO 89/00692 PCT/US88/01941 20 1 In addition to the IRMA's described above, other 2 immunoassays useful in the present invention include 3 competitive binding assays such as RIAs and fluorescent 4 or enzymelinked immunoassays (hereinafter "ELISA"). On suitable type of RIA is a SPRIA.

6 For a SPRIA, a solid phase immunoadsorbent is 7 prepared as described for the IRMA.

8 The immunoadsorbent is then incubated with the 9 body fluid and a'known amount of labelled TAG-72 for a period of time and under conditions which permit binding 11 of TAG-72 to the immunoadsorbent. The immunoadsorbent is 12 separated from the body fluid and the amount of label 13 associated therewith is assessed. By reference to a pre- 14 established inhibition curve defining the relationship between labelled TAG-72 associated with the 16 immunoadsorbent, the amount of unlabelled human TAG-72 in 17 the body fluid is determined.

18 In the various SPRIA's, the immunoadsorbent is 19 separated from incubation mixtures containing the body fluid, the labelled antibody or both. Separation can be 21 accomplished by any conventional separation technique 22 such as sedimentation or centrifugation. Preferably, 23 though not necessarily, the immunoadsorbent is washed 24 prior to contacting it, when required, with a second incubation medium and prior to measuring the amount of 26 label associated with the immunoadsorbent. The washing 27 removes non-specific interfering substances or excess 28 labelled antibody which may affect the accuracy and 29 sensitivity of the assay.

The particular label employed to label the second 31 generation monoclonal antibodies of the present 32 invention, immunoreactive fragments or recombinants 33 thereof or TAG-72 in the above-described assays is not 34 critical to the present invention and can be a

L

,1

P

8: Li i; I- i WO 89/00692 PCT/US88/01941 21 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 radioisotope such as 32 p, 14 C, 3 H, 1251, 13 11 or 35 S for the IRMA and RIA or a fluorescent molecule such as fluorescein or rhodamine or an enzyme, which, under the presence of an appropriate substrate converts the substrate to a color product for the ELISA. Examples of such enzymes include alkaline phosphatase and horseradish peroxidase.

As the last step in the in vitro diagnostic method according the present invention, the amount of second generation monoclonal antibody, immunoreactive fragment or recombinant thereof, binding to substances (TAG-72) present in the body fluid is compared to a predetermined base level.

The determination of the base level of monoclonal antibody assay binding to be expected is a determination routinely made by those of ordinary skill in the art when defining the parameters necessary for the reading of a diagnostic test of this sort. These determinations may be made without undue experimentation, particularly in light of the teachings set forth herein.

Generally, the "base level" is defined as two standard deviations above the mean of the normal population, or the level below which 99% of the normal population falls.

B. In Vivo Diagnostic Assays In vivo diagnostic assay of human carcinomas or metastases thereof using the second generation monoclonal antibodies of the present invention, immunoreactive fragments or recombinants thereof, are described in more detail below.

A second generation monoclonal antibody of the present invention, immunoreactive fragment or recombinant 'i r

I

i:::a i PCT/US88/01941 WO 89/00692 22 1 thereof, conjugated to an imaging marker is administered 2 to a patient (or subsequently administering the marker or 3 linker conjugate marker after administration of the 4 second generation monoclonal antibody) and then the presence of the imaging marker in the patient is detected 6 by exposing the patient to an appropriate means for 7 detecting the marker.

8 Administration and detection of the antibody- 9 imaging marker conjugate as well as a methods of conjugation of the antibody to the imaging marker are 11 accomplished by methods readily known to or readily 12 determined by those skilled in the art, as described, for 13 example, in Goldenberg, et al., New England J. Med., 298: 14 1384-1388 (1978); Goldenberg, et al., 250:630- 635 (1983); Goldenberg, et al., Gastroenterol., 84:524- 16 532 (1983); Siccardi, et al., Cancer Res., 45:4817-4822 17 (1986); Epenetos, et al., Cancer, 55:984-987 (1985); 18 Philben, et al., Cancer, 57:571-576 (1986); Chiou, et 19 al., Cancer Res., 45:6140-6146 (1985); Hwang, et al., J Natl. Cancer Inst., 76:849-855 (1986); Colcher, et al., 21 Cancer Res., 43:736-742 (1983); Colcher, et al., 22 "Laboratory Research Methods in Biology and Medicine 23 Immunodiagnostics", New York, Alan R. Liss, pp. 215-258 24 (1983); Keenan, et al., J. Nucl. Med., 25:1197-1203 (1984); Colcher, et al., Cancer Res., 43:1185-1189 26 (1987); Esteban, et al., Intl. J. Cancer, 39:50-59 27 (1987); Martin, et al., Curr. Surg., 41:193-194 (1984); 28 Martin, et al., Hybridoma, 5:S97-S108 (1986); and Martin, 29 et al., Am. J. Surg., 150:672-675 (1985); the disclosures of all of which are specifically incorporated herein by 31 reference.

32 The dosage will vary depending upon the age and 33 weight of the patient, but generally a one time dosage of 34 about 0.1 to 20 mg of antibody-marker conjugate per i -f t i MONO& WO 89/00692 PCT/US88/01941 23 1 patient is sufficient. A more preferred dosage is about 2 1.0 to 2.0 mg of antibody-marker conjugate per patient.

3 Examples of imaging markers which can be 4 conjugated to the antibody are well known to those skilled in the art and include substances which can be 6 detected by diagnostic imaging using a gamma scanner or 7 hand held gamma probe or Positron Emission Tomography or 8 the like as described by the references cited above and 9 substances which can be detected by nuclear magnetic resonance imaging using a nuclear magnetic resonance 11 spectrometer or the like as described in the references 12 cited above.

13 Suitable examples 'of substances which can be 14 detected using a gamma scanner or the like include 1 25 1, 1311, 1 23 I, 111 In, and 9 9 mTc. 111 Tn and 99 mTc are 16 preferred due to their low energy and suitability for 17 long range detection.

18 An example of a substance which can be detected 19 using a nuclear magnetic resonance spectrometer or the like is the nuclear magnetic spin-resonance isotope 21 gadolinium (Gd).

22 C. In Vivo Treatment 23 In vivo treatment of human carcinomas or 24 metastases thereof using second generation monoclonal antibodies of the present invention, immunoreactive 26 fragments or recombinants thereof is described in greater 27 detail below.

28 A pharmaceutically effective amount of a second 29 generation monoclonal antibody of the present invention, immunoreactive fragment or recombinant thereof 31 unconjugated or conjugated to a therapeutic agent is 32 administered to a patient.

I:!

i-:

I

j i! .i

B

Kei. WO 89/00692 PCT/US88/01941 24 1 Methods of preparing and administering the 2 monoclonal antibody-therapeutic agent conjugate as well 3 as suitable dosages will depend on the age and weight of 4 the patient and the therapeutic agent employed and are well known to or readily determined by those skilled in 6 the art. Representative protocols are described in the 7 references cited below.

8 Examples of the monoclonal antibody-therapeutic 9 agent conjugates which can be used in therapy include antibodies coupled to radionuclides, such as 1 31 T, 90 y, 11 105 Rh, 47 Sc, 67 Cu, 212 Bi, and 211 At, as described, for 12 example in Goldenberg, et al., Cancer Res., 41:4354-436C 13 (1981); Carrasquillo, et al., Cancer Treat. Rep., 14 68:317-328 (1984); Z~icberg, et al., J. Natl. Cancer Inst., 72:697-704 (1984); Jones, et al., Int. J. Cancer, 16 35:715-720 (1985); Lange, et al., Surgery, 98:143-150 17 (1985); Kaltovich, et al., J. Nucl. Med., 27:897 (1986); 18 Order, et al., Intl. J. Radiother. Oncl. Biol. Phys., 19 8:259-261 (1982); Courtenay-Luck, et al., Lancet, 1:1441-1443 (1983); and Ettinger, et al., Cancer Treat.

21 Rep., 66:289-297 (1982), the disclosure of all of which 22 are specifically incorporated herein by reference; 23 antibodies coupled to other drugs or biological response 24 modifiers such as methotrexate, adriamycin, and interferon as described, for example in Chabner, et al., 26 "Cancer, Principles and Practice of Oncology", 27 Philadelphia, PA, J.B. Lippincott Co., Vol. 1, pp.

28 290-328 (1985); Oldham, et al., "Cancer, Principles and 29 Practice of Oncology', Philadelphia, PA, J.B. Lippincott Co., Vol. 2, pp. 2223-2245 (1985); Deguchi, et al., 31 Cancer Res., 46:3751-3755 (1986); Deguchi, et al. Fed.

32 Proc., 44:1684 (1985); Embleton, et al., Br. J. Cancer, 33 49:559-565 (1984); and Pimm, et al., Cancer Immunol.

34 Immunother., 12:125-134 (1982), the disclosure wow f ii i.

WO 89/00692 PCT/US88/01941 25 1 of all of which are specifically incorporated herein by 2 reference; antibodies coupled to toxins, as described, 3 .for example, in Uhr, et al., "Monoclonal Antibodies and 4 Cancer", Academic Press, Inc., pp. 85-98 (1983); Vitetta, et al., "Biotechnology and Bio. Frontiers", Ed. P.H.

6 Abelson, pp. 73-85 (1984); and Vitetta, et al., Sci., 7 219:644-6540 (1983), the disclosures of all of which are 8 specifically incorporated herein by reference; 9 heterobifunctional antibodies for example, antibodies coupled or combined with another antibody so that the 11 complex binds both to the carcinoma and effector cells, 12 killer cells, such as T cells, as described, for 13 example, in Perez, et al., J. Exper. Med., 163:166-178 14 (1986); and Lau, et al., Proc. Natl. Acad. Sci. USA, 82:8648-8652 (1985); the disclosures of both of which are 16 specifically incorporated herein by reference; and 17 native, non-conjugated or non-complexed, antibody, 18 as described in, for example, Herlyn, et al., Proc. Natl.

19 Acad. Sci. USA, 79:4761-4765 (1982); Schulz, et al., Proc. Natl. Acad. Sci. USA, 80:5407-5411 (1983); Capone, 21 et al., Proc. Natl. Acad. Sci. USA, 80:7328-7332 (1983); 22 Sears, et al., Cancer Res., 45:5910-5913 (1985); Nepom, 23 et al., Proc. Natl. Acad. Sci. USA, 81:2864-2867 (1984); 24 Koprowski, et al., Proc. Natl. Acad. Sci. USA, 81:216-219 (1984); and Houghton, et al., Proc. Natl. Acad. Sci. USA, 26 82:1242-1246 (1985), all of which are specifically 27 incorporated herein by reference.

28 In this method, the monoclonal antibody- 29 therapeutic agent conjugate can be delivered to the carcinoma site thereby directly exposing the carcinoma 31 tissue to the therapeutic agent.

i WO 89/00692 PCT/US88/01941 26 1 D. Immunohistochemistry and Immunocytochemistry 2 Assays 3 Immunohistochemistry (hereinafter "IHC") and 4 immunocytochemistry (hereinafter "ICC") assays for the diagnosis of human carcinomas or metastases thereof or to 6 make differential diagnoses using the second generation 7 monoclonal antibodies of the present invention, are 8 carried out as described in detail below.

9 A second generation monoclonal antibody of the present invention, is added to a slide containing a 5 p 11 section of a biopsy specimen (for IHC) or cells (for ICC) 12 from body fluid (such as pleural effusion, ascites, 13 sputum, or vaginal fluid). A series of linkers 14 biotinylated horse anti-mouse IgG followed by avidin DH:biotinylated horseradish peroxidase complex) and dyes 16 diaminobenzidine) are then added to the slides to 17 detect binding of the second generation monoclonal 18 antibody, immunoreactive fragment or recombinant thereof 19 to carcinoma cells in the biopsy or body fluid by a color reaction, carcinoma cells will look reddish-brown 21 while normal and benign cells will look blue (the 22 background stain). Alternate linkers, dyes and 23 subsequent color reactions, may of course be applied, as 24 incorporated by reference herein (see Sternberger, "Immunocytochemistry", New York, John Wiley Sons, 26 Second Edition, pp. 82-169 (1979)). By this method: 27 carcinoma cells can be detected in biopsy specimens 28 and body fluids as an adjunct to making a diagnosis of 29 cancer, and a differential diagnosis can be made; .for example, TAG-72 has been shown to be present in 31 adenocarcinoma of the lung and adenosquamous carcinoma of 32 the lung but not in small cell carcinoma. Thus, .33 detection of binding of the second generation monoclonal .1 WO 89/00692 PCT/US88/01941 27 1 antibody of the present invention, immunoreactive 2 fragment or recombinant thereof to a lung biopsy would 3 rule out small cell lung cancer. Furthermore, since 4 TAG-72 has been shown not to be expressed in malignant mesothelioma, the second generation monoclonal antibody 6 of the present invention, therefore can be used to 7 differentiate adenocarcinoma of the lung from malignant 8 mesothelioma.

9 The use of IHC and ICC assays for the diagnosis of cancer or to make differential diagnoses are accomplished 11 by methods known or readily determined by those skilled 12 in the art, as described, for example, in Nuti, et al., 13 Intl. J. Cancer, 29:539-545 (1982); Stramignoni, et al., 14 Intl. J. Cancer, 31:543-552 (1983); Szpak, et al., Acta Cytologica, 28:356-367 (1984); Johnston, et al., Cancer 16 Res., 45:1894-1900 (1985); Szpak, et al., Am. J. Path., 17 122:252-260 (1986); Martin, et al., Am. J. Clin. Path., 18 86:10-18 (1986); Nuti, et al., Intl. J. Cancer, 37:493- 19 498 (1986); Johnston, et al., Cancer Res., 46:850-857 (1986); Thor, et al., Cancer Res., 46:3118-3124 (1986); 21 Ohuchi, et al., Intl. J. Cancer, 38:643-650 (1986); 22 Johnston, et al., Cancer Res., 45:6462-6470 (1986); and 23 Thor, et al., Cancer Res., 47:505-512 (1987), the 24 disclosures of all of which are specifically incorporated herein by reference.

26 The amount of second generation monoclonal 27 antibody of the present invention, used per slide and the 28 incubation time and temperature may vary, but generally, 29 the IHC and ICC assays are conducted at about 4 0 C for about 18 hours using about 40 ug per ml of monoclonal 31 antibody.

r- 1. i WO 89/00692 PCT/US88/01941 28 1 E. Activating the Anti-Idiotype Network 2 Activating the anti-idiotype network for cancer 3 therapy using the second generation monoclonal antibodies 4 of the present invention, immunoreactive fragments or recombinants thereof is carried out as described in 6 detail below.

7 A second generation monoclonal antibody of -he 8 present invention, immunoreactive fragment or recombinant 9 thereof (designated Ab i) is administered to a patient at multiple intervals. The immune system of the patient 11 will respond by the generation of antibodies (designated 12 Ab 2) which having binding specificity to the binding 13 site of Ab 1. These anti-idiotype antibodies (Ab 2's) 14 will then elicit the formation of antibodies (designated Ab 3) which have binding specificity to the binding site 16 of Ab 2. The Ab 2 antibodies will be an internal image 17 of the original TAG-72, 4and thus the Ab 3 antibodies will 18 have binding specificity and potentially destroy a 19 carcinoma producing TAG-72.

The use of monoclonal antibodies to activate the 21 idiotypic network and the procedures used to accomplish 22 this are readily known or readily determined by those 23 skilled in the art, as described, for example, in 24 Nisonoff, et al., Clin. Immunol. and Path., 21:397-406 (1981), Forstrom, et al., Nature, 303:627-629 (1983); 26 Kauffman, et al., J. Immunol., 131:2539-2541 (1983); 27 Reagen, et al., J. Virol., 48:660-666 (1983); Koprowski, 28 et al., Proc. Natl. Acad. Sci. USA, 81:216-219 (1984), 29 Herlyn, et al., J. Immunol., 143:1300-1304 (1985); Koprowski,e t al., J. Immunol. Metho., 85:27-38 (1985), 31 Koprowski, et al., Science, 232:100-102 (1985); Greene, 32 et al., J. Immunol., 137:2930-2936 (1986), Kohler, et 33 al., J. Immunol., 137:1743-1749 (1986), Notkins, et al., Ii t\ WO 89/00692 PCT/US88/01941 29 1 2 3 4 6 7 8 9 J. Exp. Med., 163:1355-1360 (1986), the disclosures of all of which are specifically incorporated herein by reference.

The activation of the anti-idiotypic network can be used to stimulate a patient's immune system so that the patient can mount an active immune response against carcinomas producing TAG-72.

The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention.

Example 1 Preparation of Monoclonal Antibodies A. Preparation of Immunogen LS-174T colon carcinoma cells (ATCC No. CRL-188) were grown in Eagle's minimum essential medium with non-essential amino acids supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin and 100 pg/ml streptomycin. The LS-174T cells were tested for the presence of Mycoplasma species and were found to be negative.

Four-week old female athymic mice were inoculated subcutaneously with 1 x 106 LS-174T cells in 0.1 ml of culture medium. Carcinoma xenografts were harvested when they reached approximately 1.0 cm in diameter (15-20 days after cell implantation), quick frozen in liquid nitrogen and stored at -70 0 C. Large carcinoma xenografts were not used due -to necrosis.

Thereafter, aprpoximately 3 grams of frozen LS-174T human carcinoma xenograft was homogenized with an Omni Mixer for 45 sec in buffer comprising 20 mM Tris (pH 7.2) and 150 mM NaCI (hereinafter The I.r; WO 89/00692 PCT/US88/01941 1 homogenized xenograft was then filtered through glass 2 wool and loaded onto a Sepharose CL-4B column sizing 3 column (Pharmacia, Upsala, Sweden) x 25 cm) which 4 was previously equilibrated in TBS. The column was elusted using TBS (pH 7.2).

6 7.0 ml fractions were collected and examined in a 7 direct binding assay 1/10 volume dilutions. More 8 specifically, 50 pi of the dilutions were added to wells 9 of a 96-well polyvinyl chloride microtiter plate (Dynatech Laboratories, Inc., Alexandriva VA). To 11 minimize nonspecific protein adsorption, the microtiter 12 wells were treated with 100 pl of 5.0% bovine serum 13 albumin (hereinafter "BSA") in phosphate buffered saline, 14 comprising 8.0 mM Na 2

HPO

4 2.5 mM KC1, 140 mM NaC1, M' MgC12, 1.0 mM CaC12, (pH 7.2) (hereinafter "PBS") and 16 incubated for 1 hour at 37 0 C. Next, the BSA was removed 17 and 125 I-B72.3, prepared as described in Colcher, et al., 18 Cancer Res., 44:5744-5751 (1984) at 50,000 cpm/25 pl per 19 well, was added to each well. Following an overnight incubation at 4 0 C, unbound 125 I-B72.3 was removed by '21 washing with 1.0% BSA in PBS. The bound 125 I-B72.3 22 was detected by cutting individual wells from the plate 23 and measuring the radioactivity in a gamma counter 24 (RIAgamma, LKB, Bromma, Sweden).

Thereafter, the peak fractions were pooled (130 26 mls of material), and loaded onto a B72.3 affinity column 27 which was washed with TBS. The B72.3 affinity column was 28 prepared as described in Johnson, et al., Cancer Res., 29 46:850-857 (1986) and comprised 100 ml of 1,1'-carbonyldiimidazole activated affinity matrix Reacta-Gel 31 (Pierce, Rockford, TL) coupled with 200 mg of B72.3. The 32 column was washed with TBS and the bound protein was *I t- L Riri

'-A

PCT/US88/01941 WO 89/00692 31 1 2 3 4 6 7 8 9 eluted with 3.0 M Nal in TBS. The column was finally washed with TBS.

5.0 ml fractions were collected and examined in a second direct binding assay carried out as described above. The peak fractions were pooled (92 mls of protein) dialyzed against 4.0 liters of 20 mM Tris (pH 7.2) at 4 0 C overnight. The purified TAG-72 thus obtained was concentrated in Aquacide II, sodium salt of carboxymethyl cellulose (Calbiochem, San Diego, CA) and used as the immunogen.

B. Immunizations 1. CC Group For the group designated CC hereinafter, three four-week old BALB/c mice were immunized by intraperitoneal inoculation of 10 pg of TAG-72 purified as described above which had been pre-mixed with an equal volume of complete Freund's adjuvant. After 80 days, the mice received booster doses intraperitoneally of g of TAG-72 purified as described above which had been pre-mixed with an equal volume of incomplete Freund's adjuvant. Seven days later the mice received 10 yg of TAG-72 in saline, by intravenous inoculation. Spleens were harvested three days later for cell infusion.

2. MATAG Group For the group designated MATAG hereinafter, two four-week old BALB/c mice were immunized by intraperitoneal incoulation of 50 4g of TAG-72 purified as described above which had been pre-mixed with an equal volume of complete Freund's adjuvant. After seven days, the mice received booster doses intraperitoneally of 50 pg of TAG-72 purified as described above which had been pre-mixed with an equal volume of incomplete Freund's ai i t 8 ii ii

E

i_-C i rlU 1 WO 89/00692 PCIT/US88/01941 32 1 adjuvant. Seven days later the mice received 10 jg of 2 TAG-72 in saline, by intravenous inoculation. Spleens 3 were harvested three days later for cell fusion.

4 C. Preparation of Hybridomas Somatic cell hybrids (hybridomas) were prepared 6 using a modification of the method of Herzenberg, et al., 7 "Handbook of Experimental Immunology", Oxford, Blackwell, 8 pp. 25.1-25.7 (1978). More specifically, single cell 9 suspensions of spleen cells from the immunized mice were made by passing the spleen tissue of the mice through a 11 No. 3 mesh stainless steel screen Fenenco Co., Inc., 12 Norcester, MA). The spleen cells and NS-1 mouse myeloma 13 cells (ATCC No. TIB-18) were washed in RPMI-1640 medium, 14 containing 2.0 mM glutamine, 1.0 mM sodium pyruvate, units/ml penicillin, 50 pg/ml streptomycin and 0.25 pg/ml 16 Fungizone, an antimycotic mixture (Grand Island 17 Biological Company, Grand Island, NY). Then, the spleen 18 cells and NS-1 mouse myeloma cells were mixed at a 4:1 19 ratio, and fused with 50% polyethylene glycol (M.W.

1500) (BDH Chemical Ltd., Poole, England). After fusion, 21 individual wells of 96-well microtiter plates (Costar, 22 Cambridge, MA) were seeded with 1 x 106 total cells (0.1 23 ml) of .the cell suspension. Fused cells were then 24 selected for growth with HAT media.

Cloning of hybridoma cell lines was performed by 26 limiting dilution. Specifically, twenty-four wells of a 27 96-well microtiter plate (Costar, Cambridge, MA) were 28 seeded with one of the following concentrations of 29 hybridoma cells: 10 cells/well, 5 cells/well, cell/well, or 0.5 cell/well. Mouse thymocytes, dervied 31 from the thymus glands of four-week old BALB/c mice, were 32 added to each well as feeder cells at a concentration of 33 106 cells/well. Wells were seeded at the b 1 q i WO 89/00692 PCT/US88/01941 33 1 concentration that eventually resulted in the growth of 2 single cell cultures.

3 A total of 2,567 initial hybridoma cultures were 4 obtained for the CC group and a total of 2,000 initial hybridoma cultures were obtained for the MATAG group.

6 All hybridoma cell lines selected for further screening 7 were cloned twice.

8 D. Solid Phase Radioimmunoassays 9 1. CC Group The CC group was assayed in a SPRIA using 11 the cell extracts from a metastatic breast carcinoma and 12 normal spleen and liver.

13 More specifically, 50 pl of the cell 14 extracts (5 pg) were added to each well of a Cooke round bottom polyvinyl chloride microtiter (Dynatech 16 Laboratories, Alexandria, VA) plate and allowed to dry.

17 To minimize non-specific protein adsorption, microtiter 18 wells were treated with 100 1 of 5.0% BSA in PBS 19 and incubated with the sample covered for 1 hour. This and all subsequent incubations were at 37 0 C. The BSA was 21 then removed and the wells were washed one time with 22 BSA in PBS. Next, 50 pl of hybridoma supernatant 23 was added per well. After a 1 hour incubation, the 24 unbound immunoglobulin was removed by washing the plates three times with 1.0% BSA in PBS at 100 26 pl/well/wash.

27 To determine antibody binding, the wells 28 were then incubated with 25 pl of 125 I-goat-anti- mouse 29 IgG (y chain specific) (Kirkegaard Perry, Gaithersburg, MD) at 75,000 cpm/25 pl per well for 1 hour at 37 0

C.

71 WO 89/00692 PCT/US88/01941 34 1 The supernatant was aspirated and the plates were washed 2 four times with 1.0% BSA in PBS at 100 3 pl/well/wash.

4 The plates were then subjected to autoradiography using Kodak XAR film and Dupont 6 Lightning-Plus intensifying screens. The films were 7 developed after 16 hours at -70 0 C. The bound cpm were 8 also detected by cutting the individual wells from the 9 plate and measuring the cpm in a gamma counter.

The results yielded 433 CC cultures which 11 had binding specificity in the SPRIA, to the carcinoma 12 extract but not to the normal extracts.

13 All of these 433 CC cultures were then 14 assayed, in a SPRIA as described above, using the cell extracts shown in Table I below.

16 TABLE I 17 Primary colon carcinoma 18 Metastatic breast carcinoma 19 Normal kidney Normal liver 21 Normal colon 22 Normal stomach 23 Normal bone marrow 24 Normal lung Normal thyroid 26 Polymorphonuclear leukocyte 27 Red blood cell 28 The results yielded 99 CC cultures which had 29 binding specificity, in the SPRIA, to the carcinoma extracts but not to the normal extracts listed in Table I 31 above.

32 Next, all of the 99 cultures were cloned 33 into 9,504 wells and each well was checked for growth of 34 a single colony. Those with a single colony were i i" -n WO 89/00692 PCT/US88101941 35 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 34 selected for further assay. Those that were selected were assayed, in a SPRIA as described above, using extracts of a human breast carcinoma and primary colon carcinoma as well as normal liver. The colonies that had binding specificity, in the SPRIA, to the carcinoma extracts but not to normal liver extract were recloned and again assayed for binding specificity, in the SPRIA, to the colon carcinoma extract but not to the normal liver extract. This resulted in the generation of 29 CC monoclonal antibodies which had binding specificity to the colon carcinoma but not the normal liver extracts (see Figure 1).

All of the 29 CC monoclonal antibodies shown in Figure 1 exhibit binding specificity to extracts of colon adenocarcinoma, but lack binding specificity to extracts of the following normal and/or benign tissues: colon (minimal binding specificity to superficial goblet cells), ovary, stomach (minimal binding specificity to goblet cells of intestinal metaplasia), endocervix (minimal binding specificity to glandular epithelium), brain, kidney, spleen, lung (minimal binding specificity to epithelium), skin (minimal binding specificity to sebaceous glandular epithelium), liver, prostate, uterus (binding specificity to secretory phase endometrium only), adrenal, pancreas, heart, lymph node, bone marrow, breast and small intestine (minimal binding specificity to superficial mucosal cells).

Of the 29 CC monoclonal antibodies so produced, the hybridomas producing preferred monoclonal antibodies have been deposited at the American Type Culture Collection under CC 49 (ATCC No. HB-9459); CC 83 (ATCC No. HB-9453); CC 46 (ATCC No. HB-9458); CC 92 (ATCC No. HB-9454); CC 30 (ATCC No, HB-9457); CC 11 (ATCC No.

HB-9455); and CC 15 (ATCC No. HB-9460).

I. 1 WO 89/00692 PCT/US88/01941 36 1 2. MATAG Group 2 The MATAG group was assayed in a SPRIA 3 essentially as described above for the CC group using a 4 1/80 dilution per well of TAG-72 in PBS except that to detect binding of antibody, 50 pl of rabbit-anti-mouse 6 IgM (Cooper Biomedical, Malvern, PA) was added to each 7 well. The plates were incubated for 1 hour at 37 0

C,

8 after which time 125 1-labelled Protein A (SPA) 9 (Pharmacia, Upsala, Sweden) at 50,000 cpm/25 pi was added per well and again allowed to incubate at 37 0 C for 1 11 hour. The unbound SPA was removed by extensive washing 12 with 1.0% BSA in PBS.

13 Of the 2000 MATAG' cultures assayed using 14 TAG-72 and PBS, 110 were found to have binding specificity to TAG-72. Further cloning and assaying in a 16 SPRIA as described above, using TAG-72 yielded 34 17 cultures which had binding specificity with colon cancer 18 extract and TAG-72 but not a normal liver extract. These 19 were cloned into 3,264 wells and approximately 20 wells of each of the original 34 cultures were assayed, in a 21 SPRIA as described above, using TAG-72 and PBS. This 22 yielded 23 cultures which had binding specificity to 23 TAG-72. The 23 cultures were subsequently grown up and 24 further assayed, in a SPRIA as described above, for lack of binding specificity to normal spleen and normal liver 26 and binding specificity to a metastatic breast carcinoma 27 extract, as well as being assayed, in a SPRIA as 28 described above, using TAG-72 and PBS. The results 29 yielded 15 cultures which exhibited binding specificity to the carcinoma extract and TAG-72 but not to the normal 31 extracts. These cultures were then recloned and 32 reassayed, in a SPRIA as described above, to produce 33 MATAG monoclonal antibodies (see Figure 1).

r :.iiih F WO 89/00692 PCr/US88/01941 37 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 All of the MATAG monoclonal antibodies shown in Figure 1 exhibit binding specificity to extracts of ovarian carcinoma, colon adenocarcinoma, infiltrating ductal carcinoma of the breast, non-small cell lung carcinoma, but lack binding specificity to extracts of the following normal and/or benign tissues: colon (minimal binding specificity to mucosal goblet cells), ovary, benign effusions (minimal binding specificity to lymphocytes and mesothelial cells), lung (minimal binding specificity to brochial epithelium), spleen, liver, breast, kidney, bone marrow, stomach (minimal binding specificity to superficial epithelium), skin, nerve, parathyroid, heart, pancreas, lymph node, adrenal, thyroid, small intestive (minimal binding specificity to superficial mucosa), brain, gall bladder, cervix, uterus (binding specificity to secretory phase of endometrium only), endocervix (minimal binding specificity to endocervical glandular epithelium), bladder, appendix, fallopian tube, muscle, salivary gland, thymus, testis, and esophagus.

Of the 15 MATAG monoclonal antibodies so produced, the hybridoma producing MATAG 12 is preferred and has been deposited at the American Type Culture Collection under MATAG 12 (ATCC No. HB-9456).

Example 2 26 Isotyping Assay 27 1. CC Group 28 For the CC group, 50 pl of polyclonal anti-mouse 29 IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was absorbed onto a 96-well polyvinyl chloride i r :1 14 is WO 89/00692 PCT/US88/01941 38 1 (Dynatech Laboratories, Alexandria, VA\ microtiter 2 plate. The IgG was diluted with PBS. The plates were 3 incubated overnight at 37 0 C. The following day, 100 pl 4 of 5.0% BSA in PBS was added to each well and allowed to incubate for 1 hour to minimize non-specific 6 absorption. The wells were then washed with 1.0% (w/v) 7 BSA in PBS. 5 pl of undiluted CC culture supernatant was 8 added to each of two wells. The plates were again 9 incubated for 1 hour at 37 0 C after which time they were washed 3 times with 1.0% BSA in PBS. Rabbit- 11 anti-mouse IgGl, IgG2b, IgG 3 IgM, IgA (Cooper 12 Biomedical, Malvern, PA) DC-12 (NIH, NCI, LTIB) and 13 control 1.0% BSA in PBS were added at 50 pl per 14 well. Following a 1 hour incubation, the plates were washed 3 times as described above. Then 50,000 cpm of 16 125 I-labelled Protein A (SPA) were added to each well, 17 incubated for 1 hour, washed 4 times with 1.0% BSA 18 in PBS and the cpm per well was counted in a gamma 19 counter. The results are shown in Figure 1.

2. MATAG Group 21 For the MATAG group, isotypes were determined by 22 parallel assays essentially as described above for the CC 23 group. However, for detection, one assay used 24 125 I-labelled goat-anti-mouse IgG (Kirkegaard Perry, Gaithersburg, MD) and the other assay used 125 1-labelled 26 goat-anti-mouse IgM (Kirkegaard Perry, Gaithersburg, 27 MD) in place of 1 25I-labelled Protein A (SPA).

28 The MATAG group was further characterized by High 29 Performance Liquid Chromatography (herreinafter "HPLC") analysis for their pentameric structure. HPLC analysis 31 was performed using a Zorbax GF-450 column, 0.94 x 25 cm if_ ,i- WO 89/00692 PCT/US88/01941 39 (Dupont, Wilmington, DE), equilibrated in 0.2 M sodium phosphate (pH 100 il MATAG culture supernatant was loaded on the column and the column was run at a flow rate of 0.5 ml/min, 0.5 ml fractions were'collected at 1 min intervals. The fractions were analyzed for isotypes as described above. The results are shown in Figure 1.

7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 Example 3 Competition RIA Competition RIAs were perfomred to determine whether B72.3 and the CC monoclonal antibodies of the present invention recognize di1t rent antigenic determinants. More specifically, B72.3 and the CC monoclonal antibodies were assayed for their ability to compete for the binding of 125 i-iabelled B72.3 to an ex-tract of LS-174T colon carcinoma cells in the following manner.

5.0 Pg of LS-174T colon carcinoma cell extract was absorbed in each well of a polyvinyl chloride microtiter plate (Dynatech Laboratories, Alexandria, VA) and varying amounts of competing CC monoclonal antibody (from Vg/pl to 0.004 pg/pl) was added to saturate the binding sites. After incubation for 6 hours at 40C, 50,000 cpm/25 Ul of 125 1-B72.3, was added to each well and incubated for 12 hours at 4 0 C. Bound 125 1-B72.3 was determined by cutting individual wells and measuring cpm in the wells in a gamma counter. The cpm in the wells pre-incubated with saturating amounts of B72.3 as a competitor was considered 100% competition. The results are shown in Figure 2A, 2C, 2E, and 2G. In Figure 2A, CC 41 was used as the competing antibody. In Figure 2C, CC 60 was used as the competing antibody. In Figure 2E, ii WO 89/00692 PCT/US88/01941 40 1 CC 83 was used as the competing antibody. In Figure 2G, 2 CC 49 was used as the competing antibody.

3 As shown in Figures 2A and 2C, CC 41 and CC 60 did 4 not compete at all with B72.3. This demonstrates that CC 41 and CC 60 having specificity for a different epitope 6 on TAG-72 than B72.3. As shown in Figures 2E and 2G, CC 7 83 and CC 49 partially compete with B72.3. This 8 demonstrates that the epitopes recognized by CC 83 and CC 9 49 share partial (but not complete) homology with the B72.3 epitope on the TAG-72 molecule, or that the CC 83 11 and CC 49 epitopes are distinct from but near the B72.3 12 epitope, resulting in steric hinderance.

13 The' after, competition RIAs were performed to 14 determine whether CC 49 recognizes the same or different antigenic determinants than B72.3, CC 30, CC 46, and CC 16 83. More specifically, these monoclonal antibodies were 17 assayed for their ability-to compete for the binding of 18 125 1-lebelled CC 49 to an extract of LS-174T colon 19 carcinoma cells as described above. The results obtained are shown in Figure 3. Figure 3 demonstrates that (1) 21 the epitopes on TAG-72 recognized by monoclonal 22 antibodies CC 46 and B72.3 share little or no homology 23 with the epitope recognized by monoclonal antibody CC 49; 24 the epitope recognized by CC 83 shares considerable homology with that recognized by CC 49 but is not 26 identical as revealed by the displacement of the CC 83 27 curve; and the epitope recognized by monoclonal 28 antibody CC 30, shares partial homology to that 29 recognized by CC 49, or is distinct from that of CC 49 but is in proximal location resulting ih steric 31 hinderance.

i 7 89/00692 PCT/US88/01941 WO 89/00692 41 1 Example 4 2 Binding Affinity 3 The binding affinities (affinity constants) of the 4 second generation monoclonal antibodies of the present invention to TAG-72 were determined by a SPRIA using a 6 modification of the procedure of Heyman, et al., J.

7 Immunol. Methods, 68:193-204 (1984). More specifically, 8 30 pl of purified TAG-72 diluted in PBS at a 9 concentration of 280 units/ml (units determined as described in Paterson, et al., Intl. J. Cancer, 37:659- 11 666 (1986)) were dried in 96 well polyvinyl chloride 12 miicrotiter plates (Dynatech Laboratories, Alexandria, 13 VA). Any remaining non-specific active groups were 14 blocked with 5.0% BSA in PBS. Then, 20 ul of 1:1.5 serial dilutions of the purified monoclonal antibody 16 (purified as described in Colcher, et al., Cancer Res., 17 44:5744-5751 (1984)), shown in Table 2 below, starting at 18 1.0 g/ml were added to the wells. After incubating 19 overnight at 4 0 C, the plates were washed three times with 1.0% BSA in PBS. Next, 1 25 1-labelled 21 goat-anti-mouse IgG (Kirkegaard Perry, Gaithersburg, 22 MD) at 75,000 cpm/25 pl per well was added and left to 23 react for 1 hour at 37 0 C. After washing three times with 24 1.0% BSA in PBS, the cpm in the individual wells were counted as described above.

26 In order to convert the cpm values to 27 concentration of bound monoclonal antibody, the remaining 28 free monoclonal antibodies in the supernatant, which had 29 been incubated with TAG-72 but not bound thereto, were incubated on another 96 well polyvinyl chloride 31 microtiter plate which had been precoated with 4.0 g/ml 32 of sheep anti-mouse IgG (Jackson Immunoresearch 33 Laboratories, Inc., West Grove, PA) and detected with

I..

I 'i i- C;I, WO 89/00692 PCT/US88/01941 42 125 I-labelled goat anti-mouse IgG (Kirkegaard Perry, Gaithersburg MD). In this manner, the concentration at which there was no free monoclonal antibodies remaining in the supernatant was determined for each monoclonal antibody. From these data, computer curves were generated to determine the binding affinity constant of each monoclonal antibody. The results are shown in Table 2 below.

TABLE 2 Binding Affinity Constants Measured Using TAG-72 Purified Antibody B72.3 CC 46 CC 30 CC 15 CC 29 CC 92 CC 49 CC 83 Affinity Constant x 10 9

M)

2.54 3.64 8.15 9.13 9.49 14.26 20.58 27.72 Table 2 demonstrates that the second generation monoclonal antibodies CC 46, CC 30, CC 15, CC 29, CC 92, CC 49 and CC 83 all have higher binding affinity constants than.the first generation monoclonal antibody B72.3.

The CC group was assayed in a SPRIA using the cell extracts from the LS-174T cell line and a metastatic breast carcinoma. 50 pl of the cell extract (5 g) was a.

I

WO 89/00692 PCT/US88/01941 43 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 -19 21 22 23 24 26 27 28 29 31 32 33 added to each well of a Cooke round bottom polyvinyl chloride microtiter plate (Dynatech Laboratories, Alexandria, VA) and allowed to dry. To minimize non-specific protein absorption, microtiter wells were treated with 100 Ul of 5.0% BSA in PBS and incubated covered for 1 hour. This and all subsequent incubations were at 37 0 C. The BSA was then removed and the wells were washed one time with 1.0% BSA in PBS. Next, 50 pi of hybridoma supernatant and dilutions of the supernatant fluid was added per well.

After a 1 hour incubation, the unbound immunoglobin was removed by washing the plates three times with 1.0% (v/v) BSA in PBS at 100 pl/well/wash.

To determine antibody binding, the wells were then incubated with 25 pl of 125 1-goat-anti-mouse IgG (gamma chain specific) (Kirkegaard Perry, Gaithersburg, MD) at 75,000 cpm/25 pl per well for 1 hour at 37 0 C. The supernatant was aspirated and the plates were washed four times with 1.0% BSA in PBS at 100 pl/well/wash.

The bound cpm were detected b r cutting the individual wells from the plate and measuring the CPM in a gamma counter.

As shown in Figure 2B, CC 41 reacts with the LS extract but B72.3 does not. Note, Figure 2B and Table 2 demonstrates that CC 41 has a higher binding affinity (slope of the curve) to the Br. Ca. than B72.3. Figure 2D demonstrates that although CC 60 does not have binding specificity to the LS extract like B72.3, CC 60 has a higher binding affinity (slope of the curve) to the Br.

Ca. than B72.3. Figure 2F demonstrates that CC 83 and B72.3 have similar binding properities to the Br. Ca.

extract but that CC 83 has high binding affinity to the LS extract while B72.3 does not. Figure 2H demonstrates V

'K

1_1 WO 89/00692 PCT/US88/01941 44 1 that CC 49 has high binding affinity to both the LS and 2 Br. Ca. extracts while B72.3 has essentially no binding 3 affinity to the LS extract.

4 Example Western Blotting 6 40 pg of LS-174T cell extracts or an extract of a 7 human breast carcinoma diluted in SDS-PAGE sample buffer 8 comprising 0.125 M Tris-HCl (pH.6.8) 4.0% SDS, 9 glycerol 'and 10% 2-mercaptoethanol, loaded onto a 3 to 12% linear gradient SDS-PAGE. After 11 electrophoresis for 8 hours at 5 milliamps/gel at 9 0

C,

12 the gels were treated with transfer buffer comprising 13 mM Tris-HCl (pH 192 mM glycine, 20% methanol 14 with 4 M urea and 0.5% Triton-X-100 for 1 hour at room temperature. The gel 'was then equilibrated with 16 transfer buffer and the proteins were transferred to 17 nitrocellulose paper (0.45 pm pore size) at 4 0 C for 16 18 hours at 30 V. Then, the nitrocellulose paper was 19 incubated with 5.0% BSA with 0.05% in PBS for 3 hours at room temperature and washed with 21 0.05% Tween-20 in PBS. Next, 10 ml of hybridoma 22 tissue culture supernatant of all the CC and MATAG 23 monoclonal antibodies were added, and incubation 24 continued for 2 hours at room temperature with gentle agitation. After washing with PBS containing 0.05% (v/v) 26 Tween-20, the nitrocellulose paper was incubated for 1 27 hour at room temperature with 12 5 I-labelled 28 goat-anti-mouse IgG (Kirkegaard Perry, Gaithersburg, 29 MD). The nitrocellulose paper was then extensively washed overnight and exposed to Kodak XAR-5 X-ray film 31 with a DuPont Lightning Plus intensifying screen at Y i. i. i WO 89/00692 PCT/US88/01941 45 1 -700C for 2 hours. For all experiments, NS-1 tissure 2 culture supernatant was used as a negative control.

3 The Western blotting analysis demonstrated the 4 reactivity of the CC and MATAG antibodies to a diffuse band beginning at the interface of the stacking gel with 6 the 5-12% resolving gel that penetrated the resolving gel 7 approximately 1 cm. This diffuse band is consistent with 8 the high molecular weight TAG-72 mucin-like molecule.

9 The high molecular weight band was observed with all the CC and MATAG antibodies tested and detected in both the 11 LS-174T cell line extract and the human breast carcinoma 12 metastases extract.

13 Example 6 14 Immunoperoxidase Studies 5.0 p sections of formalin-fixed or frozen 16 sections of tissue on slides were used. Fixed tissues 17 were deparaffinized in xylene and hydrated in graded 18 H20/ethanol rinses. A 15 minute incubation with 0.3% 19 H202 in methanol was used to block any endogenous peroxidase activity. After rinsing in PBS without Ca 2 21 and Mg 2 the slides were incubated with a 1:10 (v/v) 22 dilution of normal goat serum for the MATAG designated 23 antibodies for 15 minutes. This incubation and all 24 subsequent incubations were carried out at room temperature with the exception of the primary MATAG 26 antibody which was a 16 hour incubation at 4 0 C. The 27 normal blocking serum was removed and undiluted tissue 28 culture supernatant of the monoclonal antibody was placed 29 on the tissue sections and the slides were incubated overnight. The supernatant IgM was i Ik L _I 1 r WO 89/00692 PCT/US88/01941 -46 1 removed and the slides were rinsed for 15 minutes in PBS 2 without Ca 2 and Mg+2. For the MATAG designated 3 antibodies at 1:167 dilution of biotinylated goat 4 anti-murine IgM (Vector Laboratories, Inc.), was added to each of the tissue sections and allowed to incubated for 6 30 minutes. The slides were again rinsed in PBS without 7 Ca 2 and Mg 2 and then incubated for 30 minutes with ABC 8 (Vector Laboratories, Inc.) peroxidase at room 9 temperature. After another PBS rinse, 0.06% 3,3' diaminobenzidine (Sigma Chemical Co., St. Louis, MO) with 11 0.01% H 2 0 2 was added for 5 minutes. The sections 12 were rinsed briefly in water, counterstained with 13 hematoxylin, dehydrated in graded ethanol/H 2 0 rinses, 14 cleared (eliminating residual H 2 0) in xylene, mounted with Permount (histologic mounting medium, Fisher 16 Scientific Co.) under a coverslip, and examined with a 17 light microscope. Each section was evaluated for the 18 presence of reddish-brown diaminobenzidine precipitate 19 indicative of monoclonal antibody binding. The approximate percentage of positive carcinoma cells was 21 assigned according to the number of carcinoma cells 22 positive with each monoclonal antibody divided by the 23 total number of carcinoma cells times 100. The results 24 are shown in Table 3 below.

WO 89/00692 PCT/US88/01941 47 1 TABLE 3 2 Binding Specificity of B72.3 vs. MATAG-12 in 3 an Immunoperoxidase Assay of Tissue Sections 4 Percent MAb Reactive Carcinoma Cells B72.3 MATAG-12 6 Ovarian Cancer 1 6 7 Ovarian Cancer 2 5 8 Ovarian Cancer 3 10 9 Colorectal Cancer 1 10 Colorectal Cancer 2 40 11 As shown in Table 3, the percent carcinoma cells 12 reactive with B"2.3 is considerably lower than that for 13 MATAG-12. This demonstrates that MATAG-12 has a higher 14 binding specificity for the above carcinomas and thus is more useful in immunohistochemical or immunocytochemical 16 assays, as well as in in vivo diagnosis and therapy of 17 cancer.

18 Example 7 19 In Vivo Carcinoma Testing The monoclonal antibodies shown in Table 4 below 21 were labelled with Na 1 25 I using lodogen (Pierce Chemical, 22 Rockford, IL). More specifically, 40 pg of monoclonal 23 antibody shown in Table 4 below were adjusted 0.1 ml 0.1 24 M sodium phosphate buffer (pH 7.2) and then added to a 12 cm x 75 cm glass tube coated with 20 pg of Iodogen 26 followed by addition of 0.5 mCi of Na 1 25I (New England 27 Nuclear, Boston, MA). After a 2 min L- i WO 89/00692 PCT/US88/01941 48 1 incubation at room temperature, the protein was removed 2 from the insoluble lodogen, and the unincorporated 1251 3 was separated from the antibody by gel filtration through 4 a 10 ml column Sephadex G-25 with a buffer comprising mM sodium phosphate, pH 7.2. The labelled monoclonal 6 antibody in the void was pooled and dialy. d against 7 mM sodium phosphate buffer (pH 7.2) containing 5.0 mM 8 Nal. The iodination protocol yielded labelled IgG 9 monoclonal antibody with a specific activity of 5.0 to UCi/pg (approximately 8.0 to 25 x 106 cpm/pg).

11 Female athymic mice (nu/nu) on a BALB/c background 12 were obtained from Charles River, Inc., o:r the Frederick 13 Cancer Research Facility at approximately 4 weeks of 14 age. One week later, mice were inoculated subcutaneously (0.1 ml/mouse) with the LS-174T human colon carcinoma 16 cells (1 x 106 cells/animal).

17 Athymic mice bearing carcinomas 0.3 to 1.5 cm in 18 diameter, approximately 2 to 3 weeks after inoculation of 19 the cells were given injections intraperitoneally of pCi (0.1 pg) in PBS of the monoclonal antibodies shown in 21 Table 4 below, which had been iodinated as described 22 above. Groups of five mice were sacrificed at varying 23 times by exsanguination, the carcinoma and normal tissues 24 were excised and weighed, and the cpm were measured in a gamma counter. The cpm/mg of each tissues was then 26 determined and compared to that found in the carcinoma.

27 The results are shown in Table 4 and Figures 4A and 4B.

I WO 89/00692 PCT/US88/01941 49 1 TABLE 4 2 Percent Injected Dose Per Gram of 3 125 I-Labelled Antibody* 4 Tissue B72.3 CC 11 CC 46 CC 30 CC 92 CC 83 CC 49 Carcinoma 6.6 26.6 13.2 23.1 12.4 22.9 23.4 6 Liver 0.8 1.2 0.5 0.8 0.8 0.7 1.2 7 Spleen 0.5 1.1 0.5 1.0 1.0 0.7 1.2 8 Kidney 0.6 1.1 0.4 1.0 1.0 0.7 0.4 9 Lung 1.4 2.4 1.1 2.1 2.0 1.8 0.6 Blood 2.9 6.2 2.1 4.1 3.8 4.6 1.1 11 *At 168 hours post monoclonal antibody administration.

12 As shown in Table 4, the percent of injected dose 13 to tumor for B72.3 is considerably lower than that for 14 the CC antibodies of the present invention. Even though monoclonal antibody CC 46 has only a slightly higher 16 affinity constant than B72.3, Table 4 shows that CC 46 is 17 clearly more efficient in targeting the human tumor in 18 situ than is B72.3. This demonstrates that the second 19 generation monoclonal antibodies of the present invention are more efficient for in vivo carcinoma targeting than 21 monoclonal antibody B72.3 and thus are more useful in in 22 vivo diagnosis and therapy of cancer. Figures 4A and 4B 23 show the different binding kinetics and carcinoma/normal 24 tissue ratios at various time points for CC 11 and CC 46, respectively. Figures 4A and 4B demonstrate that these 26 monoclonal antibodies have the ability to bind the 27 carcinomas efficiently and stay bound to the carcinomas 28 over a prolonged time at least 7 days).

I t WO 89/00692 PCT/US88/01941 50 1 Example 8 2 Fragmentation of Monoclonal Antibodies 3 Biodistribution studies both in animal models and 4 in clinical trials have demonstrated that intact IgG may not be the best form of the antibody molecule to obtain 6 optimal tumor localization with minimal background in 7 normal organs. As a result, studies were undertaken to 8 fragment the second generation monoclonal antibodies of 9 the present invention and B72.3 with pepsin as described in Colcher, et al., Cancer Res., 43:736-742 (1983). The 11 resulting fragments were radiolabelled with 125, as 12 described above and tested for binding specificity in a 13 SPRIA as described above, using a LS-174T colon carcinoma 14 cell extract. The results are shown in Table TABLE 16 Binding Specificity of Immunoreactive 17 F(ab')2 Fragments 18 Binding Specificity to LS-174T 19 F(ab')2 Fragment colon carcinoma cell extract B72.3 <2% 21 CC 49 22 CC 46 23 As shown in Table 5, F(ab') 2 fragments of CC 49 24 were able to bind greater than 50% of the input counts in a SPRIA using limiting amounts of antigen and CC 46 26 fragments bound over 70% of the input activity while 27 fragments obtained from B72.3 essentially lack all 28 immunoreactivity, maintained less than 2% binding 29 specificity.

j ]I I -"t WO 89/00692 PCT/US88/01941 51 1 A pharmaceutical composition comprising the second 2 generation antibodies of the present invention in a 3 pharmaceutically acceptable, non-toxic, sterile carrier 4 such as physiological saline, non-toxic buffers and the like, now also becomes possible. The amount of said 6 antibodies in the pharmaceutical composition should be 7 sufficient to achieve effective binding with the antigens 8 against which said antibodies have.specific affinity or 9 neutralization reactivity. The pharamaceutical composition may be administered in a single or multiple 11 dosage with other adjuvants or additives, if necessary, 12 in any suitable manner to the host in need of said 13 antibodies.

14 While this invention has been described in detail and with reference to specific embodiments thereof, it 16 will be apparent to one skilled in the art that various 17 changes and modifications could be made therein without 18 departing from the spirit and scope thereof.

I

Claims (12)

1. 4. (amended) The second generation monoclonal 16 antibody of Claim 1, wherein said antibody exhibits 0-30% 17 competition with B72.3 for binding to LS-174 cell line 18 antigens. 19 5. (amended) The second generation monoclonal antibody of Claim 1, wherein said antibody has an isotype 21 selected from the group consisting of IgG 2 a, IgG 2 IgG 3 and 22 IgM. 23 6. The second generation antibody of Claim 1, wherein 24 said antibody is conjugated to a label, a tumor detecting marker or to a therapeutic agent. 26 7. The second generation antibody of Claim 6, wherein 27 said label is selected from the group consisting of a 28 radioisotope, a fluorescent molecule and an enzyme. IPEAIUS -3 T s- LO i ii -ir ~1 i:lr;r; c i WO 89/00692 PCT/US88/01941 53 1 8. The second generation antibody of Claim 7, 2 wherein said radioisotope is selected from the group 3 consisting of 3 2 p, 14 C, 3 H, 1 25 I, and 35 S. 4 9. The second generation antibody of Claim 7, wherein said fluorescent molecule is selected from the 6 group consisting of fluorescin and rhodamine. 7 10. The second generation antibody of Claim 7, 8 wherein said enzyme is selected from the group consisting 9 of alkaline phosphatase and horseradish peroxidase.
2. 11. The second generation antibody of Claim 6, 11 wherein said tumor detecting marker is selected from the 12 group consisting of 1311, 1231, l 1 1In, 67a, 99 mTc and 13 Gd. 14 12. The second generation antibody of Claim 6, wherein said therapeutic agent is selected from the group 16 consisting of a radionuclide, drug, toxin and second 17 antibody. 18 13. The second generation antibody of Claim 12, 19 wherein said radionuclide is selected from the group consisting of 1311, 90 y, 105 Rh, 47 Sc, 67 Cu, 212 Bi, and 21 211 At. 22 14.. The second generation antibody of Claim 12, 23 wherein said drug is selected from the group consisting 24 of methotrexate and adriamycin.
3. 15. The second generation antibody of ClaimAr, wherein said second antibody has specific bindina 2 7U A affinity to killer T-cells. V' 0 q/ 0 I WO 89/00692 PCT/US88/01941 54 1 16. The second generation antibody of Claim 1, 2 obtained from a hybridoma selected from the group 3 consisting of the hybridomas having the identifying 4 characteristics of ATCC No. HB-9459, ATCC No. HB-9453, ATCC No. HB-9458, ATCC No. HB-9454, ATCC No. *-B-9457, 6 ATCC No. HB-9455, ATCC No. HB-9460, and ATCC No. HB-9456. 7 17. A method for detecting a human carcinoma or 8 metastases thereof comprising: 9 obtaining a sample of body fluid or biopsy from a patient; 11 contacting the body fluid or biopsy 12 with the second generation monoclonal antibody, 13 immunoreactive fragment or recombinant thereof of Claim 14 1; determining the amount of binding of 16 second generation monoclonal antibody, immunoreactive 17 fragment -r recombinant thereof to the body fluid or 18 biopsy material; and 19 comparing the amount of binding in step to a control sample or to a predetermined base level; 21 a binding greater than the base level being indicative of 22 the presence of carcinomas or metastases thereof. 23 18. The method of Claim 17, wherein said body 24 fluid is selected from the group consisting of blood, plasma, serum, nipple discharge, cyst fluid, ascites 26 fluids, pleural effusions, seminal plasma, semen, urine 27 and prostatic fluid. 28 19. The method of Claim 17, wherein the amount 29 of monoclonal antibody binding to material present in the body fluid or biopsy is determined by means of a 31 radioimmunoassay. L 1 1 1_11* 1 WO 89/00692 PCT/US88/01941 55 1 20. The method of Claim 17, wherei the amount 2 of monoclonal antibody binding to substances present in 3 the body fluid or biopsy is determined by means of an 4 enzyme immunoassay.
4. 21. The method of Claim 17, wherein said 6 antibody has a binding affinity of greater than 3 x 109 7 M. 8 22. The method of Claim .17, wherein said -For yv L-S- k74 a*i'. 9 antibody exhibits 0-30% competition with B72.3\antibody.
5. 23. The method of Claim 17, wherein said 11 antibody is of an isotype selected from the group 12 consisting of IgG 2 a, IgG2b, IgG 3 and IgM. 13 24. The method of Claim 17, wherein said 14 antibody is obtained from a hybridoma selected from the group consisting of hybridomas having the identifying 16 characteristics of ATCC No. HB-9459, ATCC No. HB-9453, 17 ATCC No. HB-9458, ATCC No. HB-9454, ATCC No. HB-9457, 18 ATCC No. HB-9455, ATCC No. HB-9460, and ATCC No. HB-9456. 19 25. A method for localizing carcinoma or metastases thereof comprising: 21 administering to a patient a second 22 generation monoclonal antibody, immunoreactive fragment 23 or recombinant thereof of Claim 1, conjugated to an 24 imaging or detecting marker; and exposing a patient to means for 26 detecting said tumor detecting marker, an area of 27 localization of the tumor detecting marker being 28 indicative of the site of the carcinoma or metastasis in R;i said patient. i- ~'NT WO 89/00692 PC/US88/01941 56 1 26. The method of Claim 25, wherein said tumor 2 detecting marker is selected from the group consisting of 3 1251, 1311, 123I, 111 1n, 113 1n, 67 Ga, 68 Ga, 99 mTc and Gd. 4 27. The method of Claim 25, wherein said antibody has a binding affinity of greater than 3 x 109 6 M. 7 28. The method of Claim 25, whereins aid 8 antibody exhibits 50% more efficiency than B72.3 in 9 targeting human carcinoma in situ.
6. 29. The method of Claim 25, wherein said 11 antibody exhibits 0-30% competition with B72.3 antibody. 12 30. The method of Claim 25, wherein said 13 antibody is of an isotype selected from the group 14 consisting of IgG2a IgG2b, IgG 3 and IgM.
7. 31. The method of Claim 25, wherein said 16 antibody is obtained from a hybridoma selected from the 17 group consisting of hybridomas having the identifying 18 characteristics of ATCC No. HB-9459, ATCC No. HB-9453, 19 ATCC No. HB-9458, ATCC No. HB-9454, ATCC No. HB-9457, ATCC No. HB-9455, ATCC No. HB-9460, and ATCC No. HB-9456. 21 32. A method. for treating carcinoma or 22 metastases thereof, comprising administering to a patient 23 afflicted with carcinoma or metastases thereof, an 24 effective amount of a second generation monoclonal antibody, immunoreactive fragment or recombinant thereof 26 of Claim 1 to destroy or inhibit growth and proliferation 27 of said carcinoma or metastases thereof. 1 iN uti m WO 89/00692 PCT/US88/01941 57 1 33. The method of Claim 32, wherein said 2 antibody is conjugated to a therapeutic agent. 3 34. The method as claimed in Claim 33, wherein 4 said therapeutic agent is selected from the group consisting of a radionuclide, a drug, a toxin, a 6 biological response modifier, and a second antibody. 7 35. The method of Claim 34, wherein said 8 radionuclide is selected from the group consisting of 9 1311, 90 y, 105 Rh, 47 Sc, 67 Cu, 212 and 211 At.
8. 36. The method of Claim 34, wherein said drug is 11 selected from the group consisting of methotrexate and 12 adriamycin. 13 37. The method of Claim 34, wherein said second 14 antibody has specific binding affinity to killer T-cells.
9. 38. The method of Claim 32, wherein said 16 antibody has a binding affinity of greater than 3 x 109 17 M. 18 39. The method of Claim 32, wherein said 19 antibody exhibits 50% or more efficiency than B72.3 in targeting human carcinoma in situ. 21 40. The method of Claim 32, wherein said fv \oi-Vtna to S- \74 o s 22 antibody exhibits 0-30% competition with B72.3\antibody. 23 41. The method as claimed in Claim 32,. wherein 4 said antibody is of an isotype selected from the group SRA2 consisting of IgG2a, IgG2b, IgGi and IgM. 1 i ^W .r -I S PCT/US 88/01941 IPEAIUC. 24JULIS -58- 1 42. (amended) The method of Claim 32, wherein said 2 antibody is obtained from a hybridoma selected from the 3 group consisting of hybridomas having all of the identi- 4 fying characteristics of ATCC No. HB-9459, ATCC No. 9453, ATCC No. HB 9458, ATCC No. HB-9454, ATCC No. HB-9457, 6 ATCC No. HB-9455, ATCC No. HB-9460, and ATCC No. HB-9456. 7 43. (amended) A hybridoma that produces a second 8 generation monoclonal antibody having specific binding 9 affinity to both TAG-72 and LS-174T antigens without substantial binding affinity to normal adult tissues. 11 44. (amended) The hybridoma of Claim 43, which is 12 selected from the group consisting of hybridomas having all 13 of the identifying characteristics of 'ATCC No. HB-9459, 14 ATCC No. 9453, ATCC No. HB 9458, ATCC No. HB-9454, ATCC No. HB-9457, ATCC No. HB-9455, ATCC No. HB-9460, and ATCC 16 No. HB-9456. 17 45. (amended) A pharmaceutical composition comprising 18 the antibody, immunoreactive fragment or recombinant 19 thereof, of Claim 1 in an amount that is sufficient to bind antigens for which said antibody, immunoreactive fragment 21 or recombinant thereof has binding affinity and a 22 pharmaceutically acceptable, non-toxic, sterile carrier. 23 46. (amended) A composition of matter comprising a 24 second generation monoclonal antibody, an immunoreactive fragment or a recombinant thereof of claim 1. 26 47. (amended) A method of producing anti-idiotype 27 antibodies by administration.to an animal of an amount of 28 the composition of claim 46 that is effective for inducing 29 production of said anti-idiotype antibodies. SUBSTITUTE SHEET \s jIPEA/US NT 0 59
10. 48. A method of eliciting antibodies against a tumor of a cancer patient by administration of a composition of claim 4k
11. 49. The second generation monoclonal antibody of claim 1, wherein said binding affinity for TAG-72 cell line antigen -9 is in a range from about 3.64 x 10 M to about 27.72 x -9 lM. The second generation monoclonal antibody of claim 1 produced by a process comprising utilizing affinity chromatography purified TAG-72.
12. 51. The second generation monoclonal antibody of claim wherein the monoclonal antibody B72.3 is employed in affinity chromatography. $i o