TW523521B - High affinity humanized anti-TAG-72 monoclonal antibodies - Google Patents

Abstract

Novel humanized monoclonal antibodies, humanized antibody fragments and derivatives thereof which specifically bind TAG-72 are provided as well as methods for their manufacture. These humanized antibodies are useful in the treatment of cancers which express TAG-72 as well as for diagnostic purposes, e.g., for in vivo imaging of tumors or cancer cells which express TAG-72.

Description

523521 A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (1) Field of the invention The present invention relates to anthropomorphic monoclonal antibodies and fragments or derivatives thereof that can specifically bind to tumor-related glycoproteins ( tu__as_iated glye_tein) Tumor-related glycoproteins-a pancarcinoma antigen expressed by various human tumor cells. In particular, the present invention relates to anthropomorphic monoclonal antibodies, anthropomorphic antibody fragments, and derivatives derived from murine monoclonal antibodies CC49 or other amidine antibodies that specifically bind TAG-72. The present invention further includes a method by which Lin Lin produces such anthropomorphic individual record for TAG-72, a pharmaceutical and diagnostic group containing anthropomorphic antibody, and its use in cancer. Methods. BACKGROUND OF THE INVENTION The determination of antibodies expressed by tumor cells and the preparation of monoclonal antibodies that specifically bind to such antigens are known in the art. Antitumor monoclonal antibodies have potential applications as therapeutic and diagnostic agents. Such monoclonal antibodies have potential applications as diagnostic tests because they can specifically bind to tumor antigens and therefore detect the presence of tumor cells or tumor antigens in the assay. For example, an antibody that can bind to a tumor antigen is available-this single antibody in a labeled form is used for tumor cell or tumor imaging in vitro and in vivo, as is well known in the industry. Also, a monoclonal antibody that can be bound to a tumor is known to be used as a therapeutic agent. The antibody itself is used as a therapeutic agent, or the monoclonal antibody is directly or indirectly attached to an active moiety (for example, a tritium Intercellular hormones are known. ^ Importantly, if a monoclonal antibody is attached to an effective part, the body part is used as the target part, that is, it guides the desired part. The role of sub-segment: -Generally therapeutically active—to the desired target (for example, to show that it can be a single strain _______4 ϋ: Zhang scale application (CNS) Please read the back}

Page order • f 523521 A7 ________ B7 V. Description of the invention (2) Tumor of the antigen bound to the body. In contrast, when the monoclonal antibody itself is used as a therapeutic agent, the antibody system acts as a target part-that is, it specifically binds to a cell that displays the antibody-and a cell with a therapeutic activity ( Generally, it has two functions: the role of decomposing tumor cells. Such interactor functions include, for example, in particular antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) —by means of a portion of the antibody molecule, generally referring to the literature The pe part described above. A specific tumor antigen system against which various monoclonal antibodies have been developed is tumor-associated glycoprotein TAG-72cTAG-72 is displayed on the surface of various human tumor cells, such as the LSI74T tumor cell line (American Type Tissue Collection (ATCC) No. CL188, a variant of the cell line LS180 (ATCC No. CL187)), a colon adenocarcinoma strain. Various research groups have reported the production of monoclonal antibodies against TAG-72. See, eg, Muraro d α / ·, Cawcer 7? Team, 4588-4596 (1988); Johnson to α / ·, Cancer Res., 46: 850-857 (1986); Molinolo et alf Cancer Res., 50: 1291-1298 (1990); Thor et al} Cancer Res., 46: 3118-3127 (1986); EP 394277 of Schlom et "/ (assigned to National Cancer Institute); and Jeffrey Schlom ei a / ... Patent No. 5,512,443. Specific antibodies against TAG-72 that are publicly available by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs include CC49 (ATCC No. HB 9459), CC83 (ATCC No. HB 9453), and CC46 (ATCC No. HB 9458), CC92 (ATCC No. HB 9454), CC30 (ATCC No. HB 9457), CC11 (ATCC No. HB 9455), and CC15 (ATCC No. HB 9460). As an example, CC49 is an IgGi heterogeneous class This monoclonal antibody system is a second-generation monoclonal antibody, which uses the first-generation anti-5 paper standard applicable to the Chinese National Standard (CNS) A4 Zhuge (2l〇 X297 mm) 523521 Δ7 Α7 • 丨 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (3) TAG-72 purified from B72.3 It was produced by immunizing mice. Colcher is α /., Prac. Li / · 5W · 21: 319 ^ 3203 (1981). CC49 specifically binds to TAG-72 'and has a more southern antigen than B72.3 -Binding affinity. Muraro βί α /., •, 4588-4596 (1988). This monoclonal antibody has been reported to be effective against human colon cancer tumor xenografts and to reduce this xenotransplant efficiently. Growth. Molinolo eh / ”Omcer 7? Catch, welcome: 1291-1298 (1990); ColchereM /” J · Wi / · Ka ·, pull: 1191-1197 (1990). And, the radioactively marked CC49 was also Reports have shown excellent tumor localization in several ongoing clinical trials. However, murine antibodies have applicability as human therapeutic agents, and they have disadvantages in some respects. In particular, because murine resistance systems are of heterogeneous origin, they may be immune in humans. This may lead to a neutralizing antibody response (human anti-murine antibody (HAMA) response), which is particularly problematic if the antibody is intended to be administered repeatedly, for example, for chronic diseases or recurrent Treatment of disease conditions. And, because these antibody molecules contain murine fixed blocks, they may not exhibit the functions of human acting elements. In an effort to eliminate or reduce such problems, chimeric antibodies have been disclosed. Post-binding antibodies contain two different antibodies, typically two different kinds. Generally, such antibodies include human fixed regions and another type of variable regions (typically murine variable regions). For example, some mouse / human chimeric antibodies have been reported to have maternal mouse antibody binding properties and effector functions associated with human fixed regions. See, for example, U.S. Patent No. 4,816,567 to Cabilly et al .; U.S. Patent No. 4,978,745 to Shoemaker et al .; U.S. Patent No. 4,975,369 to Beavers et al. And U.S. Patent No. 4,816,397 to Boss et al. Generally speaking, these chimeric resistance systems are extracted from DNA derived from preexisting murine fusion tumors. 6 paper sizes are applicable to the Chinese National Standard (CNS) A4 Zhuge (210 X 297 mm) ~- 523521 A7 B7 V. Description of the invention (4) An all-genotype gene bank is constructed by the preparation. Nishimura et al., Cancer Res "47: 999 (1987). Then screen the variable regions of the heavy and light chains that show a recombinant version of the forward-reading antibody fragment. Alternatively, prepare RNA extracted from the fusion tumor RNA cDNA gene library and screening, or the variable region is obtained by polymerase chain reaction. The selected variable region group is then linked to a fixed region gene containing a suitable human heavy or light chain The expression vector of the colony cassette. The chimeric gene is then expressed in a selected cell line, usually a murine myeloma rod. Such chimeric antibodies have been used in human therapy. And, A chimeric murine anti-human antibody derived from CC49 and CC83 has been reported that specifically binds to TAG-72. In this respect, see, for example, EPO 0,365,997 (for Dow Chemical) (Owned by the company). One such chimeric CC49 resistance system was deposited as ATCC No. HB 9884 (Budapest). Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs In addition, Morrison ei α / · reported a variety of anti-tumor Preparation of chimeric monoclonal antibodies, Published in Ijnportant Advances in Oncology, Recombinant Chimeric Monoclonal Antibodies, pp. 3-18 (SA Rosenberg, ed., 1990) (JB Lippincott, Philadelphia, PA). At present, anabolic cMAb-17-lA has metastatic rectum. The results of clinical trials in cancer patients show that this antibody has a 6-fold longer circulation time and significantly reduces its immunity compared to its parental murine monoclonal antibody. LoBuglio et aL, Proc. Natl. Acad. Sci. USA, 86: 4220-4224 (1989); Meredith et alf J. Nucl Med., 32: 1162-1168 (1991). However, when such chimeric monoclonal antibodies typically display less immunity When they are sexually active, they still have implicit immunity in the human body, because they contain murine variable sequences, which may cause antibody responses. Therefore, it is possible that these chimeric antibodies may cause primary antibodies when administered to patients- Anti-idiotypic response. Saleh uses α /., 7 paper sizes to apply Chinese National Standard (CNS) Α4 Zhuge (210 X 297 mm) 523521 A7B7 Consumption cooperation printed by employees of the Central Standards Bureau of the Ministry of Economic Affairs Fives (5) Cancer Immunol. Immunother ,, 32: 185-190 (1990). For example, when cB72.3 (r4) is administered to a patient with colorectal cancer, 62% of such patients will Generate a human anti-murine antibody (HAMA) response that includes a primary anti-V-domain response. This is disadvantageous because a HAMA response is due to an increased antibody clearance in blood · clearance (Saleh ei a / ·, Cancer 32: 180-190 (1990)) and also due to a tendency to allergic reactions (LoBuglio W a /. , 5: 5117-5123 (1986)) and repeated administration of anti-tumor antibodies will tend to fail. Many genetic variants that may have clinical utility have been developed from Mab CC49. These include cCC49, a CH2 region-incomplete cCC49 (Slavin-Chiorini et al., Int. J. Cancer, 53: 97-103 (1993)), and a single-chain Fv (SFV) (Milenic a /., Cwcer Res .j 5χ: 6365-6371 (1991); Sawyer et aLt Protein Eng., 7: 1401-1406 (1994)). These molecules can cause a relatively reduced HAmA response in patients because they have faster plasma and systemic clearance rates in mice and monster monkeys than intact IgG. Slavin_Chiorini et al. (1993) (id.); Milenic et al. (1991) (id.). Moreover, novel single-chain immunoglobulin (scig) molecules derived from cCC49 have been reported and are encoded by single-gene construction. One such molecule, SCIgACH1 is composed of a human glFc region CC49 sFv (Shu is manufactured by a / ·, Proc .. Welcome: 7915: 7999 (1993)), while the other SCIg-IL-2 has a human interleukin. Molecules 2 (IL-2) is genetically engineered to the carboxy terminus of the region of SCIgACHi (Kashmiri / Voc. XFJJni / · Cnig ·,] _: 183-187 (1994)). Both SCIgs are comparable to cCC49 in both antigen binding and antibody cell lytic activity. The biological activity of IL-2 is also stored in sCIg-IL-2. In order to reduce the immunogenicity of chimeric antibodies and murine antibodies, it is also known that "humanizing" antibodies are produced. Ideally, "humanizing" can obtain a non-immunity in the human body __ __ 8 This paper size applies Chinese National Standard (CNS) A4 Zhuge (210X 297 mm) (Please read the notes on the back first

This page) Order 523521 A7 B7 Printed by the Consumers' Cooperative of the Central Government Bureau of Commerce of the Ministry of Economic Affairs V. The antibody of the invention description (6), which can substantially completely retain the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its binding site has been faithfully overlaid in an anthropomorphic "antibody. This may be done by transplanting the #human antibody binding site to a human architecture This is achieved by (a) transplanting only non-human CDRs to human skeletal regions and fixed regions, retaining or not retaining critical skeletal residues (Jones as α /., 321: 522 (1986) and Verhoeyen ei a / ·, ISWwce, 239: 1539 (1988); or (b) transplant the entire non-human variable block (to retain ligand-binding properties) but also to a human-like surface by intentional substitution of exposed residues "Mask" them (to reduce antigenicity) (Padlan, Mo / π. / Mmw⑽ / ·, 28: 489 (1991)). The point is that the anthropomorphic effect of CDR-transplantation involves only the transplantation of CDRs into the human skeleton and On a fixed area. In theory, this should essentially eliminate immunity (except if a heterotype or autogenous difference exists). Jones W a /., 321: 522-525 (1986); Verhoeyen et al, Science 239: 1534-1536 (1988), Riechmann et al ”Nature, 332: 323-327 (1988) While this technique is effective in some cases, CDR-grafting sometimes fails to achieve the desired results. Moreover, it has been reported that certain backbone residues of the maternal antibody may also need to be retained to retain antigen-binding activity Riechmann, al, Nature, 332: 323-327 (1988); Queen et al, Proc. Natl. Acad. Scl USA, 10023-10029; Tempest et alf Biol Technology, 9: 266-271 (1991); Co et alf Nature, 351: 501-502 (1991). As mentioned above, in order to retain the antigen-binding properties of the maternal antibody, the structure of its binding site must be faithfully reproduced in the anthropomorphic molecule. X-ray crystallographic pattern Studies have shown that antibody binding sites are basically established by CDR residues, although some adjacent backbone residues have also been found to be involved in antigen binding. Amit et al., 233: 747-753 (1986) Colman et alt Nature, 326: 358-363 (1987); Sheriff 9 This standard is applicable to China National Standard (CNS) A4 Zhuge (210X297 mm) '— Island 23521'; 爿. 化 j A7 ♦ B7 Ministry of Economic Affairs Printed by the Consumer Standards Cooperative of the Central Bureau of Standards V. Invention Description (7) et al, Proc. Natl Acad. ScL USA, M: 8075-8079 (1987); Padian et alf Proc. Natl Acad. Sci. USA, 86: 593δ-5942 (1989), Fischmann et al, J. Biol · Chem ·, 266: 12915-12920 (1991); Tulip et aL, J. Molec. Biol., Yi 2: 122-148 (1992). It was also found that the structure of the CDR loop is significantly affected by the surrounding backbone structure. Chothia ei α / ·, / · Mo / ec · 5zW ·, 196: 901-917 (1987); Cholhia et aL, Nature ^ 342: 877-883 (1 989); Tramomonteno et al., J. Molec · Biol ·, 215: 175-182 (1990). In addition to the role of this backbone residue on CDRS, small but significant differences in the relative arrangement of variable light chains (VD and variable heavy block (VH)) have been noted (Colman et al. Nature, 326: 358-363 (1987)) and those different surfaces are due to variability in residues involving contact between blocks (Padian a /., Mo / ee. / Mm 31: 169-217 ( (1994)) 〇 Also, structural studies of the role of internal residue mutations involving changes in the side chain volume have shown that the resulting regional destructuring system can be extended to the side chain positions at the distal end of the molecule Positional tolerance. This means that during anthropomorphic action, internal residues in the variable block and at the junctions between these blocks, or at least the internal volume can also be preserved; one of which An anthropomorphic scheme in which an internal residue is replaced by a different physical property (such as size, charged or hydrophilic, etc.) can cause a significant change in the structure of the antigen-binding site. An identification needs to be preserved The method of skeleton residues is computerized. On the other hand, key backbone residues can be identified by comparing known binding site structures of antibodies (Padlan, Mo / ec · / mmwn ·, 31 (3): 169-217 (199 sentences). It can affect antigen binding The residues can be divided into several groups. The first group contains residues that are close to the surface of the binding site and therefore can directly contact the antigen. These residues contain 10 amino-terminus. Zhuge (210X 297mm) (Please read the notes on the back first

Ben Gong) Order J ·. 2 5 3 2? 5V- Printed by the J. Industry and Consumer Cooperative Bureau of the Central Bureau of Standards of the Ministry of Economic Affairs A7 • B7 V. Description of the Invention (8) Residues and those adjacent to CDRs. The second group contains residues that can alter the structure or CDRs by contacting the CDRs or relative strands. The third group contains amino acids with buried side chains that can affect the structural integrity of the variable block. Residues in these groups are usually found in the same position according to the adopted numbering system (ibid ·). See Kabat ei aLy Sequences of Proteins of Immunological Interest, NIH Pub. No. 91-3242 (5th ed., 1991) (U.S. Dept. Health & Human Services, Bethesda, MD) and Genbank. However, despite their possible low immunity in the human body, anthropomorphic resistance systems are desirable, and their production is unexpected. For example, a sequence modification of an antibody can result in a substantial or even total loss of antigen-binding affinity, or a loss of binding specificity. On the other hand, "humanoid antibodies" may still show immunity in humans, regardless of sequence modification. Therefore, the industry still has a significant need for novel anthropomorphic antibodies against target antigens. More specifically, there is a need in the industry for anthropomorphic antibodies specifically directed against tAG-72, as they can be used as immunotherapeutic and immunodiagnostic reagents. OBJECTS OF THE INVENTION Accordingly, it is an object of the present invention to provide anthropomorphic antibodies specific for human TAG_72. One of the objectives of the present invention is to provide anthropomorphic antibodies derived from mouse-antibody antibodies, and in particular derived from CC49. Cc49 is a specific mouse that can be bound to TAG_72. antibody. Another aspect of the present invention provides a pharmaceutical composition comprising a personified antibody specific to TAG_72. A more specific object of the present invention is to provide

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(This page), ν'χί —τ · 523521 Α7Β7 Printed by the Consumer Cooperative Bureau of the Ministry of Economic Affairs of the Central Bureau of Commerce of the People's Republic of China. 5. Description of the invention (9) Pharmaceutical pharmacist with anthropomorphic antibody derived from CC49, bottle 7 composition The CC49 is a murine antibody that can specifically bind to TAG-72. r Another specific object of the present invention is to provide a method for treating cancers exhibiting TAG-72, especially human colorectal cancer, using a humanized antibody against tag_72. Another object of the present invention is to provide an immunodiagnostic composition for detecting cancer cells, which comprises an anthropomorphic antibody that can specifically and specifically bind to TAG.72, and preferably is derived from CC49. The system is identified or unidentified. Another object of the present invention is to provide a method for immunological diagnosis of cancer, which uses a composition comprising a humanized antibody specifically binding to TAG-72, which is in a labeled or unlabeled form. Another object of the present invention is to provide a nucleic acid sequence capable of encoding a humanized antibody or fragment thereof to TAG_72. A more specific object of the present invention is to provide a nucleic acid sequence encoding a humanized antibody derived from CC49, which is a murine antibody that can specifically bind to TAG-72. Another object of the present invention is to provide a carrier that can express a humanized antibody to tag_72, in particular a humanized antibody derived from CC49, a murine antibody that can specifically bind to TAG-72. Brief description of Formula M. Figure 1 shows the arrangement of murine CC49 VH, the newm framework region composed of FR starting material, and the anthropomorphic NEWM-based v (HuVH) amine disclosed in Example 1. Amino acid sequence. CDRs are tied in a box. Murine residues retained in FRS are distinguished by the symbol (t). Murine FR residues remaining in other aspects of HuVH are indicated by the letter symbols (A), (S), and (K). Figure 2 shows the mouse CC49 VK, the REI framework region encoded by the FR starting material, and the anthropomorphic REI-based amine group disclosed in Example 1 (please read the first * 1 This page) The size of the paper used in the edition is in accordance with the Chinese National Standard (CNS) Α4 Zhuge (21〇 > < 297 mm) 523521 A7 B7 Employees ’cooperation cooperation with the Central Procurement Bureau of the Ministry of Economic Affairs. Description (10) The acid sequence. CDRs are tied in a box. Figure 3 shows the CC49 variable heavy chain, HuCC49 disclosed in Implementation 1, and NEWM. Figure 4 shows the CC49 variable light chain, HuCC49 disclosed in Implementation 1, and REI. Figure 5 contains a schematic representation of the anthropomorphic VH and VK vectors. Figure 6 is an ELISA showing the binding of the CC49 antibodies HuVHA / MuVK and HuVHA / HuVK to TAG-72. Figure 7 is an ELISA showing the binding of CC49 antibodies MuVH / MuVK and HuVH / HuVK to TAG-72. Figure 8 is an ELISA showing the binding of CC49 antibodies MuVH / MuVK and HuVHA / HuVK to TAG-72. Figure 9 is an ELISA showing the binding of CC49 antibodies MuVH / MuVK and HuVHA / HuVK to TAG-72. Figure 10 is an ELISA showing the binding of the CC49 antibodies HuVH / HuVK and HuVHK / HuVK to TAG-72. Panel VII is ELISa showing the binding of CC49 antibodies HuVHS / HuVK and HuVH / HuVK to TAG-72. Figure 12 is a catchar (j analysis) of one of the humanized (HuVH / HuVK) and chimeric (MuVH / MuVK) CC49 monoclonal antibodies. Figure U shows the initial humanized NEWM-based Single-strand dnA sequence of VHs, HuVH, and HuVHA templates. Figure 14 shows the double-strand DNA sequence used to generate additional anthropomorphic vHs, HuVHS, and HuVHK templates. ____ 13 This paper is applicable to countries of the country 檩 隼 ( CNS) A4 · (210x297 public attack) (Please read the winter precautions on the back first '

(This page) Order 5 丨 23 521 A7 • B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (11) Explaining the details of the invention ir Before explaining the invention, it is used for the specific exclusive use of this disclosure. The definitions of terms are explained as follows: Antibodies refer to single-chain, double-chain and multi-chain proteins and glycoproteins belonging to multiple strains, single strains, chimeric and heteroimmunoglobulins (single antibody 俾 is better) Also included are synthetic and genetically engineered variants of these immunoglobulins. "Antibody fragments" include Fab, Fab ', F (ab') 2, and Fv fragments, as well as any portion of an antibody that is specific for one or more epitopes of a desired target. Anthropomorphic antibody refers to an antibody derived from a non-human antibody (typically murine) that retains or substantially retains the antigen-binding properties of its parental antibody but has lower immunity in the human body. This can be achieved by a variety of methods, including (a) transplanting only non-human CDRSs onto human skeletal regions and fixed regions, retaining or not retaining critical skeletal residues, or (b) transplanting entire non-human variability Blocks are replaced with surface-like residues by human-like parts, obscuring, them. Such methods used in the practice of the present invention include those disclosed in Jones et al., Morrison et al., Proc. TWm .. dead, iSW, ^ /, 81: 6851-6855 (1984); Morrison and Oi , Adv. Immunol, 44: 65-92 (1988); Verhueyer, et al., Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec Immun., 31 (3): 169-217 (1994). Ajfuding, or CDR—The proper term CDR as used herein refers to the amino acid sequence binding affinity and specificity of the Fv region that collectively determine the natural immunoglobulin binding position, such as by Kabatatetal (1991 ). 14 This paper size applies to Chinese National Standards (CNs) μ Zhuge (210X297 mm) (Please read the & intention on the back j

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1T 523521, printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (12) _ ~-Yu Niu—The proper term Fr used in this article refers to the amino acid sequence separated by CDRs . These parts of the antibody are used to support the proper orientation of the CDRs to maintain an antigen binding. I_-antibodies good fortune function. In the present invention, the mouse fixed area is replaced by a human fixed area. The fixed region of a chimeric antibody or anthropomorphic antibody is derived from a human immunoglobulin. The fixed region of the double bond can be selected from any of five categories: α, 5, e, r, or ^. Furthermore, the heavy bonds of various subclasses (such as the IgG subclass of the heavy chain) are responsible for different acting subfunctions and therefore by selecting the desired fixed region of the heavy bond, the desire to have the desired acting subfunction can be generated. The preferred immobilized regions are ri (IgG1), w (igG3) ^ 4 (IgG4). Even more preferred is a fixed region of the rl (IgGl) class. The fixed area of the soft keys can be of the public or lambda type, preferably the moxibustion type. This is an antibody containing two different antibodies, which are typically of different species. The most typical chimeric antibodies include human and murine antibody fragments, which are generally human fixed regions and murine variable regions. Mammals are animals that nourish their young children with milk secreted by the mammary glands. The better are warm mammals and the better are humans. Avoid the calculation of the ability of the target protein or therapeutic part to elicit an immune response (humoral or cellular) when administered to a recipient. The focus of the present invention is on the immunity of predominantly humanized antibodies or fragments thereof. Star waste, a low-immunity anthropomorphic body—This refers to a personified antibody that exhibits reduced immunity relative to maternal antibodies. Anthropomorphic antibodies essentially retain the binding properties of maternal antibodies—this refers to an anthropomorphic antibody that retains the mother that specifically binds it to produce such anthropomorphic antibodies. 15 This paper is in accordance with Chinese national standards (CNS ) 8 Zhuge (210x297 mm) (Please read the • Notes on the back page first) This order 2 5 3 2 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 ____B7__ V. Description of the Invention (13) Source Antibody Institute Ability to recognize antigens Preferably, the personification antibody will exhibit the same or substantially the same antigen-binding affinity and avidity as its parent antibody (eg CC49). Preferably, the affinity of the antibody is at least about 10% of the affinity of the maternal antibody. More preferably, the affinity is at least about 25%, that is, at least two-fold lower than the affinity of the maternal antibody. . Optimally, the affinity is at least about 50% of the maternal antibody. Methods for analyzing antigen-binding affinity are well known in the industry and include semi-maximum binding analysis, competitive analysis, and Scatchard analysis. Suitable antigen binding assays are discussed in this application. In its broadest embodiment, the present invention relates to a humanized antibody that specifically binds TAG-72, which is a whole cancer antigen expressed by various human cancers. Preferably, such anthropomorphic antibodies will be derived from antibodies with good binding affinity for TAG-72, for example: B72.3 (Thor ei a / ·, 7? Team, ^: 3118-3127 ( 1986); Johnson ei a / "team, ^: 850-857 (1986)), deposited as ATCC No. HB8108; CC49 (ATCC No. HB 9459); CC83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9452); CC92 (ATCC No. HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. HB 9455) and CC15 (ATCC No. HB 9460); or their chimeric forms (see, For example, EPO 0,365,997 by The Dow Chemical Company of Mezes et al.) Best of all, this type of anthropomorphic resistance system is CC49, which has been reported to effectively target human colorectal cancer xenograft and can also be efficiently xenogeneic. Growth of transplants. Molinolo et al, Cancer Res., 50: 1291-1298 (1990); Colcher et alf J. TWzi / · Cancer JwW ·, so: 1191-1197 (1990). As mentioned above, the anthropomorphic antibody ratio Murine and / or chimeric antibodies may provide many advantages, such as reduced immunity in humans. This advantage is because when such anthropomorphic 16 *--1 1 · (Please read first Precautions face of this ^^ I)

、 1T This paper size applies the Chinese National Standard (CNS) A4 Zhuge (210 × 297 mm) 523521 r! • · S A7 V. Description of the invention (14-antibodies can be reduced and possibly eliminated-HA when administered in vivo) The initiation of a response, such as in the treatment of cancer or the diagnosis of cancer, for example in tumor imaging. However, as noted above, anthropomorphic effects may have adverse effects on antigen binding in some cases. Preferably, the humanized antibody capable of specifically binding tag_72 of the present invention has a binding affinity for TAG-72 of at least its parental murine antibody (for example, B72.3, CC49, CC46, CC30, ccu, CC15, CC83 or its parent-derived antibody) has a TAG-72 antigen binding affinity of about 10%, and more preferably at least about 25%, and the best is at least about 50%. The best, the Anthropomorphic antibodies have a binding affinity for TAG-72 of at least about 10% of the TAG-72 antigen binding affinity of CC49 or a chimeric CC49 antibody, and more preferably at least about 25%, and most preferably It is at least about 50/0. Preferably, the present invention Anthropomorphic antibodies will bind to the same epitope as CC49. Such antibodies can be identified based on their ability to compete with CC49 for binding to TAG_72 or to TAG72-expressing cancer cells. In general, the subject anthropomorphic antibody The manufacturing process is performed by obtaining a nucleic acid encoding a variable heavy chain and a variable light chain of an antibody (preferably CC49) capable of binding to TAG-72, and identifying the sequences in the variable heavy chain and variable light chain. CDRs, and transplant this CDR nucleic acid sequence onto a human backbone nucleic acid sequence. Preferably, the selected human backbone system is expected to be suitable for living administration, that is, it will not exhibit immunity. This can be determined For example, by previous experiments using such antibodies in vivo and by studying the similarity of amino acid sequences. In the latter manner, the amine group of the backbone of the antibody to be anthropomorphic (such as CC49) The acid sequence should be compared with known human backbone regions, and the human backbone to be used for CDR transplantation should contain 17 copies of its parental antibody (such as a murine antibody bound to TAG-72). Paper size Chinese National Standard (CNS> A4 size (training > < 297 mm) please first read the © back to item ,,

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. The invention description (15) is selected based on the same size and sequence. Many human skeletal regions have been isolated and their sequences have been reported in the literature. See 'e.g. Kabat et al., (Id.). This further reinforces the hope that the resulting CDR-grafted, anthropomorphic, antibodies, including maternal (e.g., murine) antibodies, transplanted onto selected human skeletal regions will substantially preserve the antigen-binding structure and therefore The possibility of binding affinity of the maternal antibody is preserved. As a result of this study, FRs of REI and VJEWM antibodies have been identified with amino acid sequences that may allow CC49 CDRs to retain a significant degree of antigen-binding affinity. As stated, the selected human skeletal region is preferably suitable for in vivo administration, i.e. non-immunogenic. Based on their amino acid sequences, rEI and NEWM human skeletal regions are expected to be substantially non-immune. Methods for breeding nucleic acid sequences encoding immunoglobulins are well known in the industry. Such methods generally involve the use of appropriate primers to amplify the immunoglobulin sequence to be selected by polymerase chain reaction (pCR). Primers suitable for the amplification of immunoglobulin nucleic acid sequences and specific murine variable heavy and variable light chain sequences have been reported in the literature. After this immunoglobulin-coding sequence has been selected, it can be sequenced by methods well known in the industry. This is useful for identifying the VH- and VL-coding sequences (and more specifically, the parts encoding CDRS and FRs). This can be done using known methods. Once the CDRs and FRs of the cloned antibody sequence to be personified are identified, the amino acid sequences encoding the CDRs can be identified (based on the nucleic acid sequence and the genetic code, and compared with the previous antibody sequence And inferred by comparison) and the corresponding nucleic acid sequence is transplanted onto the selected human FR. This can be accomplished through the use of appropriate primers and linkers. Methods for selecting suitable primers and linkers to provide the desired binding of nucleic acid sequences are within the scope of those skilled in the art. After the CDRs have been transferred to the selected human FRs, the resulting, anthropomorphic, 18 paper sizes are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 523521 A7 • B7 Central Bureau of Standards, Ministry of Economic Affairs Printed by the Employee Consumption Cooperative. V. Description of the Invention (16) Variable heavy chain and variable light chain sequences can be expressed to produce a personified & or personified antibody that can bind tag-72. Typically, the anthropomorphic variable heavy chain and variable light bond sequences will be expressed as-a fusion protein with a human @definite block sequence, and thus a complete antibody that can bind tag-72 will be obtained. However, this is not necessary because the variable heavy bond and light chain sequences can also be exhibited in the absence of a fixed sequence to produce a personified Fv that can bind TAG-72. However, the fusion of human fixed sequences to the anthropomorphic variable region may be desirable, because the resulting anthropomorphic antibody that can bind TAG-72 will have human effector functions, such as complement-dependent cytotoxicity ( CDC) and antibody-dependent cellular cytotoxicity (ADCC) activity. Such activities can be found in chimeric antibodies, including CC49. The anthropomorphic anti-TAG_72 antibody of the present invention can also support such effector functional activity with the added advantage of greatly reducing the risk of a HAMA response. Methods for synthesizing dNA encoding a protein of a known sequence are well known in the industry. Using such methods, DNA sequences encoding the anthropomorphic VL and vH sequences are synthesized 'and then expressed in a vector system suitable for the expression of recombinant antibodies. This can be achieved in any vector system that provides humanized vL and vH sequences that are to be expressed as fusion proteins with human fixed block sequences and are used to generate functional (antibody-binding) antibodies. Vectors and host cell lines suitable for the expression of recombinant antibodies and especially humanized antibodies are well known in the industry ^ The following reference materials show methods and vectors suitable for the expression of recombinant immunoglobulins, which can be used in the practice of the present invention Utilized: Weidleetal ·, 仏, 釭: 21-29 (1987); Dorai et al. 5 J. Immunol ^ 13 (12): 4232-4241 (1987); De Waele et al., Eur. J. Biochem ., 176: 287-295 (1988); Colcher et al., Cancer Res., 49: 1738-1745 (1989); Wood et al., J. Immunol, 145 (a): 3011-3016 (1990); Bulens _ 19 (Please read the notes on the back first

This book I) is a revised version, which is considered to be suitable for financial use_ 家 县 (CNS) A tank grid (21 () > < 297 public directors) 523521 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 17) et al., Eur. J. Biochem., 195: 235-242 (1991); Beggington et al., Biol Technology, 10: 169 (1992); King ret al., Biochem. J., 281: 317 -323 (1992); Page et al., Biol. Technology ^ 9:64 (1991); King et al. 5 Biochem. J "290: 723-729 (1993); Chaudary et al.? Nature, 339: 394 -397 (1989); Jones et al., Nature ^ 321: 522-525 (1966); Morrison and Oi, jdv 44: 65-92 (1988), Benhar et al., Proc. Natl. Acad. Sci. USA, 91: 12051-12055 (1994); Singer et al.? J. Immunol, 150: 2844-2857 (1993); Cooto et al. 5 Hybridoma, 13 (3): 215-219 (1994); Queen et al.5 Proc. Natl Acad. Sci · USA, 86: 10029-10033 (1989); Caron et al., Cancer Rest <) 32: 6761-6767 (1992); Cotoma et al., J. Immunol Meth. 9 152: 89-109 (1992). Also, vectors suitable for the expression of recombinant antibodies are commercially available. Host cells known to be capable of expressing functional immunoglobulins Examples include: mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, myeloma cells such as NSO and SP2 / 0 cells; such as E. coli C & ycAeWc / n'a co / ί) bacterial cells; yeasts such as Saccharomyces cerevisiae; and other host cells. Among them, the CH0 cell line has been used by many researchers to effectively express and secrete immunoglobulins. Ability. NS0 cells are a preferred type of host cell that can be used in the present invention. Primarily, the recombinant expression of a personified antibody is obtained by one of two general methods. In the first method, the host cell line is transfected with a single vector that provides the expression of two variable sequences, the heavy chain and the light chain, and is optionally fused to a selected fixed region. In the second method, the host cell line is transfected with two vectors, each of which encodes a different variable chain (ie, a variable heavy or variable light chain); each variable chain · —The carrier can optionally be fused to the selected fixed area —-_ 20 This paper size is applicable to the Chinese national standard (centimeter millimeter) Please read the note of the back ft order ft order • f 523521 A7 B7 5. Description of the invention (18) Performance of variable chain. The human fixed block sequence has been known in the industry and has been reported in the literature. Preferred human-variable fixed sequences include A and fixed light chain sequences. Preferred human heavy bond solid sequences include human gamma 人类, human ^ 2, human γ3, humans, and mutations that provide alterations or functions, such as enhanced half-life in vivo or reduced Fc receptor binding. After expression, the antigen-binding affinity of the resulting anthropomorphic antibody can be analyzed by known methods', such as a Scatchard analysis. In a particularly preferred embodiment, the anthropomorphic antibody has an antigen-binding affinity of at least 25% of its parental antibody (eg, CC 4 9), that is, two-fold lower than that of natural or chimeric CC49 The minimum value. Most preferably, the affinity of the humanized antibody is at least about 50% of its parental antibody (e.g. CC49).

In some cases, by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, 'the transfer of CDRs (from an antibody that binds to TAG-72) to a selected human skeleton region can provide the desired A humanized antibody with affinity for TAG-72. However, there may be a need to further modify specific residues of the selected human backbone to obtain enhanced antigen binding. This is possible because some backbone residues are believed to be necessary or influential for antigen binding. Preferably, those structures in the backbone residues of the parent antibody (e.g., murine) that maintain or affect the binding position will be retained. These residues can be identified from the X-ray crystallographic pattern of the parent antibody or Fab fragment, thereby identifying the third-order structure of the antigen-binding site. And, backbone residues involved in antigen binding may be identified based on previously reported humanized murine antibody sequences. Therefore, retaining such backbone residues or other murine M residues from the maternal murine antibody is likely to make the binding of TAG · 72 most likely. Preferably, 'such methodologies can give the resulting anthropomorphic antibody—species, similar to anthropomorphic,' characteristics', so that the interior of the antigen · binding and contact residues can be reduced by 21 paper sizes. Applicable to China National Standard (CNS) A4 specification (210X297 mm) | 23521

:: 5 --- ..:-¾. • 丨 A7 • B7

Immunity. Furthermore, the invention encompasses variants and equivalents that include conservative substitution mutations, that is, one or more amino acids are replaced by similar amino acids. For example, conservation substitution refers to the replacement of one amino acid by another in the same general classification, such as' One acidic amino acid is replaced by another acidic amino acid, or one basic amino acid is replaced by another Basic amino acid substitutions, etc. It is well known in the art how the desired conserved amino acid substitutions are known. The invention further relates to nucleic acid sequences from which such humanized antibodies can be expressed, and to expression vectors that can be used to produce such humanized antibodies in transfected host cells. In a preferred embodiment, such humanized antibodies and corresponding nucleic acid sequences are derived from CC49. Preferably, the anthropomorphic heavy chain has the amino acid sequence described in Figure 1 or 3 and the anthropomorphic light chain has the amino acid sequence described in Figure 2 or 14. However, as stated, the present invention further includes other modifications of these personified heavy and light chain sequences, for example, further comprising one or more conserved amino acid substituted sequences or retaining one or more affecting (Enhancing) sequences of additional murine backbone residues bound by the antibody which are different from or those that have been shown in these figures. Because it specifically binds TAG-72, an antigen expressed on many different cancer cell types (ie, colorectal, breast, ovarian, and prostate cancers), and because they are expected to be apparently Non-immunity, so the anthropomorphic antibody of the present invention should be far applicable to the treatment or prevention of cancer with TAG-72 performance characteristics, and diagnostic reagents, such as for tumor imaging or radioimmunoguided research. System (Radioimmunoguided Surgery® System) (RIGS㊣). See Hinkle et al

Antibody, Immunoconjugates and Radiopharmaceuticals ^ 4 (3): 339-358 22 This paper size is applicable to Chinese National Standard (CNS) A4 Zhuge (210X 297 mm) ~ '~~ (Please read the notice on the back first_

Order Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 523521 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 --------______ B7 V. Invention Description (20) ~~ '~-(1991). People familiar with this technology (by routine experimentation) determine the amount of antibody that is effective and non-toxic to achieve cancer treatment. However, in general, the effective dose is in the range of about 0.05 to 100 mg per kilogram of body weight per day. The antibody of the present invention can be administered to a mammal in an amount sufficient to produce a therapeutic, preventive or diagnostic effect according to the aforementioned treatment method. Such antibodies of the present invention may be administered to mammals in the form of a -convenient agent #, which is a dosage form based on known techniques by combining the antibody of the present invention with a generally pharmaceutically acceptable carrier, diluent, and / or K-forms are prepared to form a suspension, injectable solution or other formulation. Those skilled in the art will understand that the nature of the form and pharmaceutically acceptable carrier or diluent is determined by the number of active ingredients to be combined, the route of administration, and other known variables. A pharmaceutically acceptable formulation may include, for example, a suitable solvent, a preservative such as benzyl alcohol, if desired, and a buffering agent. Useful solvents may include, for example, water, aqueous alcohols, glycols, and phosphonic acids and carbonates. Such aqueous liquids contain no more than 50% organic solvents by volume. Suspension-type silk recipes may include-Wei Wei material medium as a carrier, for example, water-based polyethylene, birrolidone, inert oils such as vegetable oil or highly refined mineral oil, or water-based Cellulose ether. A diluent such as gelatin or alginate may also be present, one or more natural or synthetic surfactants or antifoaming agents may be used, and one or more suspending agents such as sorbitol and another sugar may be used among them. Such formulations may include one or more adjuvants. The antibody (or fragment thereof) of the present invention can be administered orally, parenterally, by inhalation, or topically. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, anal, vaginal or intraperitoneal administration. The parenteral subcutaneous, intravenous and intramuscular forms are generally preferred. Daily parenteral and oral preparations __11 23 This paper size applies to China) M Zhuge (2 丨 QX29? Meal) " —- (Please read the precautions on the back first

(This page) Order! 3521 A7 B7 V. Description of the invention (21) Amount of intake for preventive or therapeutic use The humanized antibody of the present invention is generally in the range of about 0.005 to 100, but the better is daily About 0.5 to 10 milligrams per kilogram of body weight. The antibodies of the present invention can also be administered by inhalation. By "inhalation" is meant intranasal and intraoral inhalation. Suitable dosage forms for such administration, such as a spray formulation or a fixed dose inhaler, can be prepared by ordinary techniques. To The preferred dose of one of the compounds of the present invention to be used is generally in the range of about 0J to 1,000, and more preferably about 10 to 100 mg per kilogram of body weight. The antibody of the present invention may also be topically Administration. By local administration means non-systemic administration. This includes topically administering an anthropomorphic antibody (or anthropomorphic antibody fragment) of the present invention to the epidermis or oral cavity, and dropping the antibody into the ear, or Intranasal, and any place that does not specifically enter the bloodstream. By systemic administration means oral, intravenous, intraperitoneal, subcutaneous and intramuscular administration. Amount of antibodies required for therapeutic, prophylactic or diagnostic purposes Of course, it varies with the selection of antibodies, the nature and severity of the conditions being treated, and the treatment of L, and is ultimately a doctor's choice. One suitable topical dose of antibodies of the present invention In general lines per kilogram of body weight per day to about Shu 1〇 () mg. (Please read the Notes on the back page) -M5 clothing

1T •• ^. &Quot; f Consumption cooperation printed by employees of the Prospective Bureau in the Ministry of Economic Affairs. Formulation It is possible to administer -antibody or its fragment separately, and it is preferably presented in a pharmaceutical formula. For topical administration, the active ingredient may include caffeine to cryptic W / W, such as 1% to 2% of the weight of the formula. Although it may contain up to muscle w / w, the preferred is not More than 5% w / w, more preferred is 01% to i% of the formula. The topical formula of Shumingming contains _ active ingredients and-an acceptable carrier and optionally any other therapeutic ingredients. Acceptable §23521

Printed by the Consumer Affairs Cooperation Bureau of the Central Bureau of Commerce of the Ministry of Economic Affairs of the People's Republic of China 5. "Invention Note (22)" means compatible with other ingredients of the formula and harmless to the recipient. Formulas suitable for topical administration include liquid or semi-liquid Formulations suitable for penetrating the skin to the place to be treated, such as ointments, lotions, creams, plasters or pastes, and drops suitable for administration to the eyes, ears or nose. The drops of the invention may Contains a sterile aqueous or oily solution or suspension, and can be prepared by dissolving the active ingredient in a suitable liquid solution of a bactericidal and / or fungicide and / or any suitable preservative, and It preferably contains a surfactant. The resulting solution can then be certified and sterilized by filtration and transferred to a container using an aseptic technique. Examples of bactericidal and fungicides suitable for inclusion in the drops are Nitric acid or hydrazine mercury (0.002%), benzikonium chloride (0.01%) and chlorhexidine acetate (0.01%). Solvents suitable for an oily solution Including glycerin, dilute Alcohols and propylene glycols. The emulsions of the present invention include those suitable for application to the skin or eyes. An ophthalmic emulsion may contain a sterile aqueous solution optionally containing a bactericide, and may be prepared by a method similar to that used for drops. The method is prepared. A lotion or plaster for application to the skin may also contain an agent that promotes drying and cooling of the skin, such as an alcohol or acetone, and / or a humectant such as glycerin or a compound such as castor oil or groundnut oil Oils and fats. The creams, plasters or ointments of the present invention are semi-solid formulations of active ingredients for external use. They can be mixed in an aqueous or aqueous solution by subdividing the active ingredients separately or in powder form or in a solution or suspension. It is made in a non-aqueous fluid, which can be aided by a suitable machine and can contain an oily or non-oily base. The base can include hydrocarbons such as hard, soft or liquid paraffin, glycerin, beeswax, metal soap A vegetable secretion; an oil of natural origin such as almond, corn, groundnut, castor or olive oil; hair fat or a derivative thereof, or a product such as hard Acid or liquid fatty acid fatty acid and one such as propylene glycol 25 (please read the unintentional matter on the back page first) Order 'This paper is applicable to Chinese National Standards-2-V-1i Calendar ^ ZE 2 5 32 Central Bureau of Standards, Ministry of Economic Affairs Printed by the consumer consumer cooperative A7 ----------- B7 V. Description of the invention (23) " ——- ~~-or giant gum. The formula can contain any suitable surfactant, such as An anionic, cationic, or nonionic surfactant, such as sorbitol vinegar or its polyoxyethylene derivative. It may also contain suspending agents such as natural gums, cellulose derivatives, or inorganics such as laurel silica Substances, and other ingredients such as lanolin. The set of the present invention contains the necessary solutions; East (optionally followed by _ further dilution) or suspended in-(preferably buffered) Reconstituted frozen or freeze-dried anthropomorphic antibodies or anthropomorphic antibody fragments in a liquid carrier. The kit also includes buffers and / or additional solutions (riding or chilled form) _ or buffers and / or powder formulations to be reconstituted by the property—for easy mixing with anthropomorphic antibodies or anthropomorphic antibody fragments To produce a formulation suitable for administration. Therefore, preferably, the kit comprising the anthropomorphic antibody or anthropomorphic antibody fragment is cold bundle, cold Kang dried, pre-diluted or pre-mixed to a specific concentration, the degree of heat makes a predetermined amount of heat The addition of water, solution or solution will result in a sufficient concentration and pH that can be effectively used in the treatment and diagnosis of cancer in vivo or in vitro. Preferably, this set will also include instructions for reconstructing and using the anthropomorphic antibody or anthropomorphic antibody fragment composition to treat or test cancer. The kit also contains two or more components of the active composition for reconstruction. For example, a second component portion—in addition to a personified antibody or anthropomorphic antibody fragment—may be a dual-function chelator, a dual-function chelate, or a therapeutic agent such as a radioactive special nucleus, when compared to anthropomorphic When the antibodies or humanized antibody fragments are mixed, they form a conjugation system. The aforementioned buffers, additives and other components can be sold separately or co-sold with the kit. Those skilled in the art will understand that the optimum amount and the individual dose interval of the anthropomorphic antibody or anthropomorphic antibody fragment of the present invention will be determined by the nature and scope of the condition to be treated, the form, route and location of administration, and the particular animal being treated It can be determined and can be used by traditional techniques. 26 This paper size is applicable to the Chinese National Standard (CNS) Α4 Zhuge (210X297 mm) (please read the precautions on the back page). Too much order. System 3 2 ^ ^ ---- I-Is. -------- ------- ---------- .1 A7 ---------- ---- B7 V. Invention Description (24) The technique determines the most suitable one. What is known to the artist is the most suitable course of treatment, that is, the number of doses of one antibody or fragment of the present invention given per day on a specific number of days can be used by the skilled artist to use general treatment decision tests Calculate. The humanized antibody of the present invention can also be administered together with other anticancer agents, such as antibodies or drugs thereof. And the humanized antibody or humanized antibody fragment of the present invention can be directly or indirectly adhered to a functional subunit having therapeutic activity. Suitable effector moieties include, for example, interleukins (IL-2, TNF, interferon, colony stimulating factors, ^^, etc.), cytotoxins (Pseudomonas exotoxin, Kuna toxin, Acacia Toxins, etc.), especially such as 9GY, 131I, 99mTc, 111In, 125; ), Immunomodulators, therapeutic enzymes (such as galactosidase), anti-proliferative agents, etc. The adhesion system of antibodies to desired effectors is known. See, for example, US Patent No. 5,435,99 to Cheng et al. No. 0. Furthermore, the bifunctional linker system for promoting this adhesion is known and generally available. Moreover, the chelator (chelator and chelate) for the adhesion of a special nucleus of radiation is Known and available. On the other hand, the anthropomorphic antibody or fragment specific for TAG-72 in this case can be used as an immunodiagnostic reagent in vivo and in vitro. A particularly preferred use is for performance Cancer of TAG-72 In vivo images of damaged cells. Antibodies in this case are preferred because they do not cause significant HAMA or allergic reactions. Therefore, they can be used repeatedly to monitor a patient's disease state. As described above, the present invention Another preferred application of anthropomorphic antibodies or fragments thereof is for use in the Radioimmunoguided System®. This technique is also known as the RIGS® system, which involves the identification of a single antibody or fragment thereof Surgery 27 copies are applicable to Chinese national standards ((: milk) 8 4 Zhuge (210/297 mm) ~~ " (Please read the precautions on the back page first))

Order 523521 η Α7 Β7 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs. 5. Description of Invention (25) for intravenous administration. After the tumor has been taken up and the radioactivity in the blood has been cleared, the patient is taken to the operating room where surgical exploration can be effectively performed with the aid of a one-year grip / active probe (eg, Neopro (g) 1000). This can help the surgeon avoid tumor metastasis and improve the complexity of the resection. The RIGS_ system is advantageous because it can make the detection of tumors need not be measurable by visual inspection or palpation. See et al., Such as •, welcoming 139M394 (1986). This technique is discussed in detail in Hinkle et al., Antibody, Immunoconjugates and Radiopharmaceuticals, 4: (3) 339-358 (1991) (?! References discussing this technique in several articles). This reference also reveals the use of this technique with the CC49 monoclonal antibody itself. This technique is particularly useful for cancers of the large intestine, breast, pancreas and ovaries. The anthropomorphic antibody or anthropomorphic antibody fragment of the present invention can be identified by radiation, such as iodine and nuclei, for example. In addition, luIn and 99mTc are also suitable radiation signs. The humanized antibodies of the invention can be used alone or in combination with other antibodies. Moreover, the humanized antibody of the present invention can be made into the form of a diagnostically effective composition. In general, this requires a combination of diagnostically acceptable carriers and additives and identification for detection purposes. Suitable labels include diagnostic radionuclides, enzymes, etc. Methods for using antibodies for tumor imaging are known in the industry. Without further effort, it is believed that those skilled in the art can utilize the present invention to the widest scope using the foregoing description. The present invention will be further described by using the following examples, which are purely for illustrative purposes of the present invention, and therefore are to be regarded as illustrative examples only and in no way limit the scope of the present invention. Example 28 This paper size is applicable to China National Music i CNS) A4 specification (Please read the precautions on the back first

(This page), May 2 5 3 2 5 Printed by the Ministry of Economic Affairs of the Central Bureau of Commerce for consumer cooperation

A7 _______- B7_ V. Description of the invention (26) Materials and methods Borrowing of the PNA template, all the recombination work is performed on the human sequence in the plastid M13 vector. The source for the initiation of the anthropomorphic CC49 VH2NEWM backbone region is an M13 architecture with a DNA between the positions of M13 5flmH I and calendar w Π_ __ DNA with the nucleotide sequence shown in Figure 13 Fragment. The jujube source used to generate the initiating anthropomorphic CC49 VL REI framework region is a M13 architecture with a position between M13 such as mH I and the calendar "IIΠ position-with an Rm amine group encoding Figure 2 DNA fragments of acid sequences. When the overlapping extension method is used to cause mutations in a DNA sequence, double-stranded M13 DNA is used. Conversely, when the extension conjugation method is used instead, the nucleotides are designed De-adhesion to one strand of the two strands of DNA. In the latter method, M13 DNA is first treated to replace all thymine nucleotides in the DNA with uridine to produce urinary DNA. This can be achieved by M13 plastids 01 ^ 8 were combined with the following components and transfected into competent cells lacking dUTPase and uracil glycolytic enzymes, which are generally RZ1032 cells (although CJ236 cells obtained from Hercules, CA Bi0-Rad are also Appropriate). 1 μL of M13 plastid DNA 4 ml of LB medium 40 pL competent cells RZ1032. This group was cultured at 37. (: was shaken for 5 hours, and the resulting single-stranded plastid DNA (ssDNA) was isolated Come out and dissolve in 50 吣 Tris_EDTA buffer. Then borrow Mixing uracil glycosidic enzyme treatment of the DNA. 1 μί of uridine by the SSDNA 29

(Please read the & precautions on the back

I: this page), ιτ 2 5 3 2

7 B Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (27) 1 pL 10x glycolytic enzyme buffer 1U Uracil glycolytic enzyme (Gfbco BRL, Gathersburg, MD) 40 μI > 25mM MgCl2 Then this The mixture was incubated at 37 ° C for one hour, and then 6.6 pL 25mM MgCl2 and 9.9 pL 1M NaOH were added. The mixture was then incubated at 37 ° C for another 5 minutes, and then 16.5 pL of 0.6M HC1 was added to neutralize the mixture. The DNA was then precipitated with ethanol and dissolved in water. M13 Oligonucleotide Primers The following oligonucleotide primers were used throughout the preparation of the anthropomorphic CC49 VHs and VLs illustrated below. 10. 5, -CTAAAACGACGGCCAGT-3 ,; 11.5f-AACAGCTATGACCATG-3f; 385.5-GCGGGCCTCTTCGCTATTACG03; and 391.5, -CTCTCTCAGGGCCAGGCGGTGA-3, these primers are complementary to the presence of plastid M13 (NEWM or REI ) The region outside the two target skeleton sequences, and it is the segment from M13 to Li · ηί / ΠΙ position. In order to compare the antibody-binding properties of the antibodies produced in the following examples, the murine variable region has a chimeric heavy chain (that is, a heavy chain has a murine CC49 VH region and a human IgG1 fixed region) and / or An antibody to a chimeric light chain (ie, a light chain with a murine CC49 VL region and a human A-fixation region) was expressed. The source of these chimeric chains was the ATCC-host cell line HB9884 (Budapest), which showed a chimeric CC49 antibody with two chains. The heavy chain of this antibody was named "MuVH" and its light chain was named "MuVL". Oligonucleotide and Phosphonium-Based Interaction Scheme 30 This paper size applies to China National Standard (CNS) A4 (210 '乂 297 mm)

523521 Α7 Β7 V. Description of the invention (28) The mutant nucleotides for non-overlapping extension are added according to the following method. In a final volume of 25 pL, the chin components are bound together: 10 pmol of various oligonucleotides, 5 pL of a 5x polynucleotide kinetic enzyme buffer, and 5U of T4 polynucleotide kinetic enzyme (GibcoBRL) The phosphorylation reaction begins with the addition of an enzyme and is allowed to proceed at 37 ° C for one hour. Adhesion scheme for non-overlapping extension-ligation The adhesion step for non-overlapping extension-ligation involves a single adhesion, in which all oligonucleotides with mutations and a primer oligonucleotide are adhered to a single DNA template of the strand, in which all thymidine bases have been replaced by urine nucleoside bases. The nucleotides for mutation are first phosphorylated in accordance with the above-mentioned oligonucleotide plus phosphorylation method. In a final volume of 20 pL, the following components are combined ... 1 pmol of each of the phospho-oxygenated nucleotides with a mutation of 1 pmol of primer oligonucleotide 4pL5x binding buffer 0.2 pmol The ssU-DNA template was printed by consumer cooperation of the Central Standards Bureau of the Ministry of Economic Affairs, and then the mixture was heated to 90 ° C for 30 seconds, then quickly cooled to 70 °, and finally cooled slowly to 37 ° C. The extension junction for extension-junction is completed after the primer and the oxidized mutant oligo & 30 μί 5 HaissU-DNA template adhesion step is completed. In the final volume, the following components are combined: '20 pL of glued SSU-DNA (that is, the contents of the above-mentioned glue 0 illegal) 31 The paper size applies the Chinese National Standard (CNS) Α4 Zhuge (210 × 297 mm) ) 523521

I; J A7 I B7 5. Invention descriptions such as)

2 pL 5x binding buffer 2 pL 0.1 M dithiothreitol f 0.3 μm 0.1 M ATP 1 pL 6.25 mM dATP, dTTP, dGTP, dCTP dNTP mixture 2.5U T7 DNA polymerase (USB , Now Amersham Life Sciences, Cleveland, OH) 0.5U T4 DNA ligase (Gibco BRL) water was added to 30 μϋ / and this mixture was incubated for one hour at room temperature. The standard PCR method uses the following method, or amplifies non-overlapping extension-joined DNA sequences and performs extension of each overlapping DNA sequence. In a final volume of 50 porosies, the following components are combined: 2 pL template DNA (adhered Ssu-DNA or unadhered ssDNA) 5 pL 10x Vent buffer (NEB, also known as New England Biolabs, Beverly, MA ) Or printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, lx Thermalase buffer solution (IBI, CT of New Haven) 2 pL 6.25 mM and other moles of dATP, dTTP, dGTP, dCTP dNTP mixture 25 pmol Nucleotide primer 25 pmol, a mutated nucleotide (for overlapping extension) or a second nucleotide primer 1U Vent DNA Polymerase (NEB) or Thermalase DNA Polymerase _______ 32 CNS)格 (^ 7 ^ 7 2 5 3 2 υϋ 'A7 ___. B7 V. Description of the invention L). _ The reaction starts with the addition of DNA polymerase, and it takes about 15 cycles after the game: (1) 94 (Please read first Intentions on the back

〇C30 sec "(2) 50 ° C 30 sec" and (3) 30-60 seconds at 75 ° C (Vent DNA Polymerase) or 72 ° C (Thermalase). The reaction was completed at a fixed temperature of 75 ° C (Vent DNA Polymerase) or 72 ° C (Thermalase) for 5 minutes. PCR overlap-extension amplification scheme After a pair of PCR reactions are performed, one for each of two (partially complementary) overlapping DNA fragments, the two resulting fragments are ligated according to the following PCR method. In a final volume of 50 pL, the following components are combined: each of the lpL overlapping DNA (from the overlapping pCr extension reaction described above) 5 μ! ≫ 10x Vent buffer (NEB) or Thermalase buffer (IBI),? τ 2 pL 6.25 mM equal mols of dATP, dTTP, dGTP, dCTP dNTP mixture 25 pmol for overlapping nucleotide primers 1U Vent DNA Polymerase (NEB) or Thermalase DNA Polymerization in PCR extension Enzyme (IBI) reaction starts with the addition of DNA polymerase and is then processed in about 15 cycles: (1) 94 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

° C30 sec ”(2) 50 ° C30 sec” and (3) 30_60 seconds at 75 ° C (Vent DNA Polymerase) or 72 ° C (Thermalase). The reaction was completed at a fixed temperature of 75 ° C (Vent DNA polymerase) or 72 ° C (Thermalase) for 5 minutes. The humanized CC49 variable region DNA sequence obtained from M13 was transferred to a PSV vector and subsequent antibody expression. The humanized antibody was expressed in a pSV vector grown in NSO cells. The anthropomorphic variable region construction produced in plastid M13 is constructed with ίου III and both are obtained from BRL) With a final volume of 100 pL and Tris-EDTA _____ 33 This paper size applies Chinese national standards (CNS) A4 specification (210X297 mm) 2 5 3 2 5 Consumers' cooperation of the China Central Standards Bureau of the Ministry of Economic Affairs, printed by A7 __—_. B7_ V. Description of the invention ") The digestion solution was digested at 37 ° C for 1 hour. The obtained DNA fragment was then run on a low melting point glycosaminoglycan colloid. The band containing the DNA designed to be humanized was cut out, and an ELUTIP 'd was used, and the column was set at 20 pL. The ONA was purified with Tris-EDTA buffer. Then 10 pL of the purified DNA preparation was subjected to one of 1 pL > ^ 111 and 5⑽ // I-digested pSV preparation, 3 μΐ 5x ligase buffer Solution and T4 DNA ligase (BRL) binding to make the design inserted into a pSV plastid. The anthropomorphic CC49 VH design was inserted into a pSVgpt vector with a human IgGl heavy chain fixed region; this was used The pSVgpt vector is "aLYS-30" shown in Figure 5. The anthropomorphic CC49 VH construct was inserted into the pSVhyg vector with a fixed block of human moxibustion light chain; the used vector was naLys-l 7 " shown in Figure 5. Each anthropomorphic variable region construct is inserted adjacent to each fixed region, that is, instead of the HuVHLYS or HuVLLys fragment described in Figure 5. The obtained vector was transfected into NSO cells as follows. Then about 3 pg of VH vector or about 6 pg of VL vector prepared by PSV-insertion method was linearized by digestion with 10U Pvwl (Gibco BRL). The digested DNA was then immersed in ethanol and dissolved in 50 μ ： of water. NSO cells were collected by centrifugation and resuspended in 0.5 mL Dulbecco's Modified Eagie's Medium (DMEM) and transferred to a Gene Pulser tube (Bio-Rad). DNA obtained from both a VH and a VL construct was slightly mixed with the cells by aspiration, and the vial was left on ice for 5 minutes, then 'the vial was inserted between the Bio-Rad Gene Pulser electrodes and applied A single pulse of 170V at 960 pF. The contents of the tube were then transferred to a flask containing 20 mL of DMEM, and the cells were allowed to stand at 37 ° C for 1-2 days. The cells were collected again by centrifugation and resuspended in 36 mL of screening DMEM. Each 1.5 mL portion of this re-suspension was placed in each tank of a 24-well plate and incubated at 37 ° C for 4 days. At that time, each tank was _______34_____ This paper size applies to Chinese National Standard (CNS) A4 specifications ( 210 X 297 mm) (Please read the notes on the back first

f this page)

1T 2 5 3 2 5 Printed A7 ____ '—_. B7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs V5. Description of the invention ") The culture medium in") was replaced with 1.5 mL of fresh screening DMEM. At 37 ° C again After 6 days of incubation, the viable cell population can be seen with the naked eye, and the antibody production of the supernatant of each tank is analyzed. The ELISA analysis method uses the enzyme-linked immunosorbent assay (ELISA) method described below to test antibodies Concentration and antibody binding properties., · · »·

Measurement of IgG concentration The concentration of IgG secreted from the transfected cells was measured using an enzyme-linked immunosorbent support assay (ELISA) method as described below. Polyethylene (PVC) microtiter plates (Dynatech Laboratories, Chantilly, VA, catalog # 〇〇i-〇 イ 〇-2101)

Goat anti-human igG (10 mg / mL, GAHIG, Southern) diluted in Milli-Q® water

Biotechnology Associates, Inc "Birmingham, AL, Catalog # 2010-01) Cover with 50 mL / tank in a pan. This titration is air-dried overnight at ambient temperature or 3 hours at 37 ° C. Before use, add 0.2 mL / well of 1% (W / V) bovine serum albumin (Sigma, St. Louis, MO catalogue) in scaly buffered saline (Sigma, catalog # 1000-3) (PBS / BSA) # A7888) Block non-specific binding. All incubations are performed in a humid container. The titration plate is incubated at 37 ° C for 1-2 hours, and the blocking solution is removed before the sample is added. Make triplicate samples in PBS / BSA solution or a two-fold serial dilution of standard IgG set at 500 ng / mL (50 μΐ / well). The titration plate is incubated at 37 ° C for 3 hours or at 4 ° C. ° C overnight. The titration plate was washed three times at 0.025% Tween-20 (v / v, Sigma) using an automatic titration plate washer. A goat anti-human IgG (Horseradish Peroxidase) conjugated to Horseradish Peroxidase ( Southern Biotechnology 35 This paper size is applicable to the National Standard (CNS) A4 specification (210X 297 mm) — " '(Please read the back first ti Matters on this page) Order 523521 A7 .B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention ") Associates Inc ·) was added at a dilution of 1: 1000 at 50 μΐ / slot and added at 37 ° C Train for 15 hours. Wash three times at 0.025% Tween-20 (v / v, Sigma) using an automatic titration plate washer and add 50 μΐ / well OPD substrate buffer. After developing for 4 minutes, stop at 12.5% ΗΘΟ4 and read the absorption at 492 nm. The IgG concentration in the test sample can be estimated by comparing with the average optical density of a standard curve made from standard IgG. 1 ♦ •. * Determination of Relative Affinity of Anthropomorphic Antibodies in an ELISA Using a partially purified immobilized on a vinyl chloride (PVC) microtiter plate (Dynatech Laboratories, Chantilly, VA, catalog 001-010-2101) TAG-72 antigen was tested for antibody binding properties. The PVC discs were covered with TAG-72 (Dow Chemical, lot # 040191) diluted 1: 300 with Milli-Q water at 50 μΐ / slot. The titration was air-dried overnight at ambient temperature or 3 hours at 37 ° C. Prior to use, 0.2 mL / well of 1% (W / V) was added to bovine serum albumin (Sigma, St. Petersburg, Ph.D.) in phosphate buffered saline (Sigma, catalog # 1000-3) (PBS / BSA). Louis, MO Directory # A7888) blocked non-specific binding. The titration plate was incubated at 37 ° C for 1-2 hours, and the blocking solution was removed before the sample was added. All incubations were performed in a humid container. A two-fold serial dilution of the test sample in PBS / BSA solution was added in duplicate to the wells (50 μΐ / well) of the TAG-covered titration plate. The titration plate is incubated overnight at 4 C or 1-2 hours at 37 ° C. The titration plate was washed 3_ times at 0.025% Tween-20 (v / v, Sigma) using an automatic titration plate washer. A goat anti-human IgG (Southem Biotechnology Associates Inc.) conjugated to horseradish peroxidase was added at a 1: 100 dilution at 50 μΐ / well and incubated at 37 ° C for 1.5 hours. Use an automatic titration dish washer to clean 36 at 0.025% Tween-20 (v / v, Sigma) (please read the precautions on the back page first)

L, y 'mouth

T This paper size applies to China National Standards (CNS> A4 (210X 297 mm)) ^ Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 3 2 A7 ______; _JB7__ 5. Description of the invention ") ~ ^-Wash twice Add 50 μΐ / well of 0pd substrate buffer. Color developed for 4 minutes, stopped at 12.5% HAO4 at 12.5 mt and read the absorbance at 492 hm. Determination of affinity constant bound to TAG-72 A two-fold dilution of purified Hu-CC49 was prepared in PBS / BSA in the range of 1.0 mg / ml_0.03 μ§ / ιη1, and the sample (20 μl / slot) was added to TAG-coated, PVC prepared and blocked as described above. The titration plate is incubated overnight at 4 ° C. After this incubation, the sample is transferred from the titration plate to the corresponding on a GAmG-coated titration plate. In the tank, the original TAG titration plate was washed 3-20 times with 0.025% Tween-20 (v / v, Sigma, catalog # P1 379) using an automatic titration plate washer. Once identified, goat anti-human IgG probes (ICN Biomedicals, Inc., catalog # 68088) were diluted to 75,000 cpm / 25y in PBS / BSA and added (25 μl / well) to all wells. A TAG titration plate was incubated for 1 hour at 37 ° C. After 1 hour of incubation at 37 ° C, the titration plate was washed as described above and a 125H labeled GAHIG probe was added. The titration plate was at 3rt Incubate for i hours. Then the two titer plates (TAG and GAHIG groups) containing the probes were washed with a microtiter plate washer to remove unbound probes. A titration plate cutter (D. Lee, Sunnyvale, CA, Model HWC-4) was used to separate each cell from the frame of the titration plate. A T-meter was used to quantify the radioactivity in each cell. The data obtained was based on Scatchard. · Ivnw "51: 600- 672 (1946)). Example 1 Preparation of CDR-transferred (original anthropomorphic) antibodies from mouse CC49 ____— 37 This paper is sized to the Chinese National Standard (CNS) A4 (210X 297 mm) (please read the back first) Precautions ^ Pf this page)

-523521 A7 ________. A7 ________. B7 V. Invention Description | 5) In this example we are talking about using the VH and VL skeletons of human Mabs REI and NEWM respectively. Anthropomorphic CC49 Mabs (CC49 HuVH / HuVK) architecture. The CDRs of murine CC49 were transplanted onto the human skeleton according to known methods discussed. In particular, the human skeletal line is selected from antibodies that were expected to be suitable based on previous studies, that is, it does not negatively affect antibody binding and does not have significant immunity in humans. Human skeletons selected for the variable heavy chain and the variable light chain are NEWM and REI, respectively. In the initial generation of anthropomorphic VH, specific murine skeleton residues were also retained, which, as previously studied, preserved the antigen-binding properties. In detail, Y27, T30, A72, F95 and T97 of the mouse heavy chain are all retained. At the same time, another aspect of the anthropomorphic VH was made, which also retained the murine backbone residue A24. The generation of these NEWM-transferred anthropomorphic CC49 VHs was completed according to the above-mentioned adhesion and extension-joint method, which was performed using a single strand of M13 DNA template, which is in its calendar III and mH I position Between them is a DNA fragment having the nucleotide sequence shown in Figure 13. In this method, primer 11 is used in combination with a set of mutation oligonucleotides. These mutations were designed and synthesized with the following sequence of oligonucleic acid: la. 5, -GCTGTCTC ACCC AGTGMTTGCATGGTCAGTGAAG GTGTAGCCAGACACGGTGCAGGTCA-3CC; and lb · 5, -GCTGTCTCACCCAGTGAATTGCATGGTCAGTCCCCCTCGCTCTCCCTCTCCCTCCTCTCTCTCTCCTCTCTCTCCT-3CT; -3 ,; and 38 (Please read the intentions on the back first

Book I) The size of the paper is applicable to the Chinese National Standard (CNS) A4 (210X297 mm) 523521 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention ")

3 · 5’-GGAC0CTTGGCCCCAGTAGGCCATATTC AGGGATCT TGTACAGAAATAGACCGCG6TGTC-3 ,. Codons designed for the preservation of murine FR amino acids are shown in bold type. After extension-ligation, amplification PCR was performed using Vent DNA polymerase using standard PCR methods. The use of two mutated oligonucleotides 1 resulted in the formation of two initial anthropomorphic VHs. These are named " CC49 NMVH '', also known as nHuVH "(for the construction incorporating oligonucleotide i) and" HuVHA "(for the construction incorporating oligonucleotide lb). In the production of the anthropomorphic VL in the initial state, no special murine skeleton residues were retained. The production of the anthropomorphic CC49 VL via REI-transplantation was completed according to the above-mentioned adhesion and extension-joint method, which uses an SSM13 template with an ssU-DNA fragment between its III and I positions, the ssU-DNA fragment The REIVK sequence shown in Figure 2 can be encoded. In this method, a primer 385 is used in combination with a set of mutation oligonucleotides. These mutations were designed and synthesized with the following oligonucleotide sequences: 21. 5′-GTTCTTCTGATTACCACTGTATAAAAGACTTTGAC TGGAC-3 ?; 22.5′-CAGATTCCCTAGCGGATGGCCAGTAG-3 ,;

23. 5! -TTCTAGTCACGTGTGATTATTCAGCTTGGTCCCTTG

GCCGAACGTGAGGGGATAGGAATAGTATTGCTGGCAGTAGTA G-3 ,; and 24.5, -GCTCTGGGTCATCTGGATGTCGG-3f. After extension-ligation ', amplification PCR is performed using the above-mentioned Thermalase standard PCR method. The resulting anthropomorphic VL is called " CC49REVK, and is also referred to as "HuVK". Construction of two heavy chains in the M13 vector, HuVH and HuVHA, and 39 (please read the intention page on the back)

1T 2 5 3 2 5 A7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. B7____ 5. Description of the invention ") The light chain constructs HuVH, which grows and is expressed in TG1 cells. These constructed polypeptide expression products are Sequenced, and these constructs #amino acid sequences are shown in Figures 1 and 2. These DNA constructs are then inserted into the pSV expression vector as described above. These constructs then each other or encode a chimeric MuVH or MuVL The combination of ATCC (Budapest) HB9884 DNA sequence was inserted into NSO cells. In particular, the following four combinations of heavy and light chain constructs were transfected into NSO cells as described above:

HuVHA and MuVK, HuVHA and HuVK, MuVH and MuVK, and HuVH and HuVK. These combinations were expressed, and then the above-mentioned ELISA analysis was used to test the antigen-binding properties of the obtained antibodies. The results of these analyses are shown in Figures 6-9. The data in Figure 6 show that when combined with HuVHA, the function of the HuVK anthropomorphic light chain is the same as that of the MuVK chimeric light chain. Figures 7 and 8 indicate that the entire anthropomorphic HuVH / HuVK antibody binds TAG-72 approximately two-fold lower than the chimeric MuVH / MuVK antibody. In addition, the ninth graph shows that the A24 mutation did not enhance antigen binding; and when measured by this ELISA analysis, the A24 mutation resulted in an approximately 2-fold reduction in affinity. Further development of the anthropomorphic CC49 Because the function of the HuVK anthropomorphic light chain is apparently the same as that of the MuVK chimeric light chain 'further work is to make another mutational pattern of the HuVH anthropomorphic heavy chain. The first variant of HuVH was made by replacing the lysine residue at position 76 of CC49NMVH shown in Figure 1 with serine Fr residue serine. This is achieved by the above-mentioned Vent DNA polymerase PCR overlapping extension method, which uses two primer-and-mutation oligonucleotide pairs, one oligonucleotides 10 and 4, and oligonucleotides U and 5. ___40 This paper size is applicable to China National Standard Leather (CNS) (210X297mm) '~-(Please read the following page: Notices on this page) • Packing · P521 Printed by the Central Consumers Bureau of the Ministry of Economic Affairs Consumer Cooperatives V. Invention Description ") Each pair is matched with one strand of a dsDNA template and is used one position between the positions Π and I of the plastid M13 and has the guaranty shown in Figure 14 (the above one is related to the ii & 5 one For the nucleic acid sequences used together). Nucleotides 4 and 5 were designed and synthesized with the following sequences: 4.5'-AGACACCAGCAGCAACCAGTTCAG-3, and 5. GCTGAACTGGTTGCTGCTGGTGTC-3 ,. The serine residue code is shown in bold type. The resulting anthropomorphic CC49 VH was designated as 'HuVHS,'. The second variant of this HuVH was isolated by murine FR residues. Amino acid was used to replace the threonine residue shown at position 74 of CC49NMVH in Figure 1. This was achieved by the above-mentioned Vent DNA polymerase PCR overlapping extension method using two primer-and-mutation One pair of oligonucleotides 10 and 6, and oligonucleotides 11 and 7 were used. Each pair is matched with one strand of a dsDNA template and is used one position between the nc / III and mH I positions of the plastid M13 and has the position shown in Figure 14 (the above line and 11 & 7 that A pair of nucleic acid sequences used together. Oligonucleotides 6 and 7 were designed and synthesized with the following sequences: 6. 5f-CTGGCAGACAAGAGCAAGMCCAG-3, 7. 5'-TGGTTCTTGCTCTTGTCTGCCAGG-3 ,. The code for the lysine residue is shown in bold type. The resulting anthropomorphic CC49 VH is designated as " HuVHK, .. The two heavy chain constructs, HuVHS and HuVHK, located in the M13 vector are grown And expressed in TG1 cells. The polypeptide expression products of these constructs are sequenced, and the amino acid sequences of these constructs are shown in Figure 1. These DNA constructs are then inserted into the pSV expression vector as described above. . Then 41 paper sizes apply Chinese National Standards (CNS) M specifications (210X297 mm) (please read the column on the back page first), τ

523521 V. Description of the invention) The combination of these constructs and HuVK was inserted into NS0 cells. These combinations were expressed, and then the above-mentioned ELISA analysis was used to test the antigen-binding properties of the obtained antibodies. The results of these analyses are shown in Figures PD and U. These figures show that neither the κ? 4 or S76 mutations can lead to enhanced antigen binding; in fact, this ⑽ mutation results in approximately 2-fold reduced affinity when measured by ELISA. Example 2 Measurement of the affinity constant of the anthropomorphic CC49 antibody obtained in Example 1 The final anthropomorphic CC49 antibody of Example 1, CC49 HuVH / HuVk㈣ is shown in the variable heavy chain and variable The light chain sequence) affinity is often measured according to the aforementioned ELISA analysis method. The ATCC (Budapest) hb988 chimeric CC49 antibody was used as an internal control group. This eusa analysis was performed by Li Yue purified humanized silk-combined ⑽9 monoclonal antibody and recorded by Wei Xing. The accuracy was calculated and the variability in analysis-and-analysis was calculated. The age-oriented results obtained from this analysis are shown on page 12 @ 巾. A summary of the results obtained in these analyses is shown in Table 1. (Please read the caution page on the back first)

, -Ir Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs

I quasi-standard home country I country Middle use I suitable sauce 7 29 523521 A7 .B7 7; I. Description of the invention) Table 丨 1 Excerpt from analysis of affinity constant of anthropomorphic & chimeric CC49

Chimeric CC491 CC49 HuVH / HuVK Affinity constant (Ka) ± number of standard deviation analyses 7.62 χ109Μ · 3.94χ109Μ-7 4.27 xlO ^ " 1 2.57 x109m-1 8 (Please read the notice on the back of the Ministry of Economy first. Printed by the Bureau of Standards Consumer Cooperatives1. The published affinity constant (Ka) is 16.18 x H ^ M'Schlom et al., Cancer Fine 48: 4588-4596 (1988)). These results show that the anthropomorphic anti-TAG-72 antibody HixVH / HuVK (having the variable heavy chain and variable light chain sequences shown in Figures 3 and 4 respectively) has an approximate chimeric CC49 antibody (MuVH / MuVK). Therefore, it is expected that this humanized antibody will effectively target TAG-72-expressing tumors in vivo. Moreover, based on its sequence, this antibody should exhibit very low or no immunity in humans, and exhibit excellent plasma clearance, metabolic properties, and relative to murine CC49 and its chimeric appearance. Effective tumorigenicity. The murine plasmacytoma cell line producing this anthropomorphic CC49 antibody has been deposited in the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852 on October 6th, i996), and this cell line was awarded Number ATCC CRL-12209. This deposit was made in accordance with the Budapest Treaty. -Once here-please issue a patent or hereby grant any patent priority under u.s.c · ⑽%, this deposited cell is available without restriction.

(This page)% 523521! A7 4-- · Β7.: ¾ -------------- I V. Explanation of the invention

It can be understood from the foregoing that the anthropomorphic antibody disclosed in Example w exhibits anti-i-binding properties, that is, TAG_72affinity # is comparable to the maternal monoclonal antibody ncc49 (murine antibody), and the antibody derived from nCC49 Chimeric antibodies, ie, cCC49. Furthermore, the results described in the foregoing I ^ j These antibodies have properties that make them suitable for in vivo diagnosis or treatment, such as' improved Jinqing clearance, metabolic properties, and low or no immunity in humans. These properties are of high importance because these properties will allow this personified antibody to be administered repeatedly and in large quantities over a long period of time without significant adverse effects, such as HAMA reactions or non-specific Cytotoxicity. This is important for the treatment and diagnosis of cancer because it allows these antibodies to be used for a long time. In addition, the scavenging properties of the present human antibodies will allow these antibodies to effectively reach the desired target location, for example, TAG-72 manifests cancerous tumors (due to the effect of serum clearance on compliance efficiency). Therefore, the anthropomorphic antibody of the present invention has a substantial improvement over the previously disclosed antibody specific for TAG-72. Other embodiments of the present invention will be apparent to those skilled in the art < apparent from reference to this specification or implementation of the invention disclosed herein. This description and examples are for illustrative purposes only, and the true scope and spirit of the present invention is indicated by the following patent application scope. (Please read the page on the back: Social Issues page first) — Binding · Order 1 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 4 4 Standard One Standard One Country One Country-China | Use a moderate rule of paper f centimeters 7 9 2

Claims (1)

ABCD VI. Application for Patent Scope No. 861 16293 Patent Reexamination Application Application for Amendment to Patent Scope Amendment Date: January 1992 1. A personification antibody molecule that contains at least one Fab, Fab ', F (ab') 2 or The Fv region can specifically bind TAG-72, and is characterized in that the anthropomorphic antibody molecule contains at least one anthropomorphic variable heavy chain (VH), which has one of the following amino acid sequences One: QVQLQESGPGLVRPSQTLSLTCTVSGYTFTDHAIHWVRQPPGRGLE WIGYFSPGNDDFKYNERFKGRVTMLADTSKNQFSLRLSSVTAADTA VYFCTRSLNMAYWGQGSLVTVSS; or Gln-Val-Gln-Leu-Gln-Glu-Ser-G-Pro-GlyPro-GlyPro Cys-Thr-Ya1-Ser-G1y-Tyr-Thr-Phe ~ Thr-Asp-H i s-A1a-11e-H i s-Trp-Va1-Arg-G1n-Pro-Pro-Gly-Arg-Gly- Leu-Glu-Trp-Ile-Gly-Tyr-Phe-Ser-Pro-Gly-Asn-Asp-Asp-Phe-Lys-Tyr-Asn-Glu-Arg-Phe-Lys-G1y-Arg-Va1-Thr- Met-Leu-A1a-Asp-Thr-Ser-Lys-Asn-G1n-Phe-Ser-Leu-Arg-Leu-Ser-Ser-Va1-Thr-A1a-A1a-Asp-Thr-Ala-Val-Tyr- Phe-Cys-Thr-Arg-Ser-Leu-Asn-Met-Ala-Tyr-Trp-Gly-Gln-Gly-Ser-Leu-Val-Thr-Val-Ser-Ser; And contains at least one anthropomorphic variable light chain (VL), the anthropomorphic variable light chain having one of the following amino acid sequences: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGNQKNYLAWYQQTP GKAPKLLIYWASARESGVPSRFSGSGSGTDYTFTISSLQPEDIATY YCQQYYSYPLTFGQGT-et-M1-M-G-G-G-S -Pro-Ser-Ser-Leu-Ser-A1a-Ser-Va1-G1y-Asp-Arg-Va1-Thr-11e-Thr-Cys-Lys-Ser-Ser-G1n-Ser-Leu-Leu-Tyr-Ser -G1y-Asn-G1n-Lys-Asn-Tyr-Leu-A1a-Trp-Tyr-G1n-G1n-Thr-Pro-G1y-Lys-A1a-Pro-Lys-Leu-Leu-11e-Tyr-Trp-A1a -Ser-A1a-Arg-G1u-Ser-G1y-Va1-Pro-Ser-Arg-Piie-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Asp-Tyr-Thr-Phe-Thr-Ile-Ser -Ser-Leu-Gln-Pro-Glu-Asp-Ile-Ala-Thr-Tyr-Tyr-Cys-G1n-G1n-Tyr-Tyr-Ser-Tyr-Pro-Leu-Thr-Phe- $ Paper Size Universal China National standard (〇?) 8 specifications (210 father 297 mm) -45- 523521 AB c D Six, the scope of patent application Gly-Gln-Gly-Thr-Lys-Leu-Gln-Ile-Thr. 2. A nucleic acid sequence characterized in that a humanoid antibody molecule, as in item 1 of the scope of patent application, can be expressed from the nucleic acid sequence. 3. A vector, characterized in that the anthropomorphic antibody molecule as described in the scope of the patent application can be expressed from the vector, and the vector is a plastid. 4 · A pharmaceutical composition suitable for the treatment of cancer or the in vivo or in vitro measurement of cancer, characterized in that the composition contains an effective treatment or an effective diagnostic amount, such as anthropomorphization of the scope of the patent application Antibody molecule. 5. The pharmaceutical composition according to item 4 of the application, wherein the anthropomorphic antibody molecule is directly or indirectly associated or linked to a therapeutically active subunit moiety, and the composition is suitable for the treatment of cancer. 6. The pharmaceutical composition according to item 5 of the application, wherein the action component is a radiation nucleus, a therapeutic enzyme, an anticancer drug, a cytokine, a cytotoxin or an antiproliferative agent. 7. The pharmaceutical composition of claim 4 in which the anthropomorphic antibody molecule is directly or indirectly associated or linked to a detectable target, and the composition is suitable for the detection of cancer. 8. The pharmaceutical composition according to item 7 of the application, wherein the detectable target is a radiation nucleus or an enzyme. 9. · A detection kit, characterized in that the package contains a pharmaceutical composition as an active ingredient as in item 4 of the patent application scope, and an instruction manual for the composition for treating or detecting cancer, wherein the composition is in use It was restructured before. -46 523521 8 8 8 8 A B c D 6. Scope of Patent Application 10. The anthropomorphic antibody molecule of item 1 of the scope of patent application, which is used to treat or detect cancer. This paper size applies to China National Standard (CNS) A4 (210X297 mm)