JPH07507440A - Antibody constructs with increased binding affinity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Antibody constructs with increased binding affinity Conventional (QiiJ The present invention relates to cancer diagnostic imaging reagents and cancer therapeutic agents, and more specifically, to imaging diagnostic reagents and cancer therapeutic agents. Title: L relates to the preparation of antibody constructs useful as therapeutic agents for cancer. Special In addition, the present invention provides biochemicals with increased antigen binding affinity and short half-life in vivo. Concerning synthetic antibody constructs.

Systemic drug therapy is a universally used treatment modality. Ilr & or organ The same applies to the administration of 45 identification reagents such as radioisotopes for image diagnosis. death However, the administration of such drugs and radioactive labels does not, by itself, It is dangerous because it usually has only one or no target selection. cancer limit the effectiveness of chemical therapeutics and radioisotopes used in the treatment of This is particularly difficult. Because these chemicals are harmful to most cells, especially during the proliferative phase. This is because they have the ability to interfere with the metabolic processes of certain cells. Therefore, this field Often targeting such drugs to specific tissues or cancer cell types It is suggested that it be conjugated to a reagent that can be used.

Antibody molecules can be linked to various drugs, enzymes, toxins, and radioisotopes. Ghose and Blair (197B) J, Natl .

Cancer In5L, see the review on G: 657-676) 0 For example, Antibodies were analyzed using magnetic resonance imaging, X-ray analysis, and computerized tomography. i! detects hidden tumors that cannot be detected using conventional detection methods such as ! I need to diagnose the image. It is widely used (Goldenberg (198B) ) Δarch, PaLho1. Lab, Med, 112+580-587).

However, there are many problems with the use of antibody molecules as targeting reagents. A radiolabeled antibody with specificity for the common tumor jHb'LI Showing an adhesion rate of only 0.01% to 0.001% in the targeted tumor There are also (Goldenberg, supra), in addition, antigens associated with certain &lIw& Despite being specific for Different combinations may also occur. Some of this non-specific binding is due to the binding This is due to the adhesion of the antibody by its Fc region rather than the main one. Sara Intact antibodies have a faster removal rate than smaller molecules such as fragments of antibody molecules. It's slow.

In an attempt to alleviate these problems, previous studies have shown that specificity and antigen binding ability The use of retained antibody fragments has been suggested. Antibody fragments are better than intact antibodies. exhibiting rapid specific targeting ability (Wahl et al. (1983)). J.

Nuclear Med, 24:3L7-325), currently produced by enzymes. F(ab“)2 fragments are the most widely used for such applications. . These fragments remove potentially problematic Fc portions of intact antibody molecules. Due to the lack of nonspecific antibodies in the liver and 1lIpW& For example, radiolabeled antibodies have been shown to have low accumulation and fast removal rates1. A few days after administering 1gG of a tactile antibody, circulating radioactivity and non-specific The level of bound radioactivity is high and removal is gradual over 4 days or more. (Goldenberg, supra), whereas antibodies lacking an Fc domain Utilization of body fragments allows tumor imaging within 24-48 hours, thus 1ffi 3 It becomes possible to use radioisotopes with short half-lives such as 1 and Tc. (Goldenberg, supra).

However, the use of F(ab')t fragments is not without problems.

In addition to being difficult to produce enzymatically, these fragments have reduced binding properties. (e.g., see Wahl et al., supra) and therefore have specificity and binding activity. increased, non-specific binding capacity was minimized, and once Targeting molecules with short half-6Ji periods are needed.

For this purpose, monoclonal clones with mutations in the constant region and increased antigen-binding activity are used. null antibodies have been identified (e.g., PolLock et al., (19BB) Proc, NaLl, Acad, Sci, USA 85:2298-2 302), further modified the antibody by genetic engineering and transfected it. By being able to express it again in cells that have It is now possible to combine these parts as a fusion protein. Neuberger et al. (1984) Nature 3:61 4; Morrison (M.

rrison) et al., (1987) 8nn, N. Y. Hecad, Sci, 50 Uni 187; and Morrison et al. (1988) CI in, Chern, 34 :166B).

Also, Fab (Horwit CI (orwiLz) et al., (198B) Proc, Na t1. 8cad, Sct, U, S, Δ, 85-1687) or F( Genetically encoded immunoglobulin deletion mutations such as ab’)*-like 7-7 In one study, a truncated form of the immunoglobulin heavyweight An Fd' fragment was expressed in E. coli (Cabil y) et al. (1984) Pr.

c, NaLl, Hecad, Sci, US 81:3273-3277), Separate In their study, the specific constant region of the human) Cr3 heavy chain was used for antibody synthesis and assembly. (Moririn et al. (1999)). 87) Ann, N.Y., Acad, Sci.-507:1873).

Therefore, it is useful for second recognition of tumor antigens and therefore for tumor imaging and/or destruction. One objective of the present invention is to co-introduce antibodies that are considered to be Also Fc receptor Reduced binding to the body and therefore non-specific accumulation in m and other parts of the human body Provided are antibody constructs useful for tumor imaging and/or cancer therapy that have reduced This is also an object of the present invention. Yet another purpose is to quickly bind to tumor antigens. Providing antibody constructs that can be used and antibodies that have a shorter half-life than the whole intact antibody molecule Includes provision of constructs.

HatsuhoΦ Overview Antibodies that retain CHl,i; and CH3 domains but lack the CH2 domain The construct surprisingly has a short half-life compared to the intact antibody molecule. The binding affinity is increased, and non-specific binding is eliminated while maintaining the same specificity. It was discovered that the levels were unexpectedly low. This discovery is based on a variety of tumor antigens. used in the development of the present invention regarding antibody constructs with specificity for epitopes. ing.

+ [Native antibody molecules of the G and IgD classes have two heavy (H) chains and two These consist of light (Cuff) bonds and non-covalent interactions. connected to each other by purpose. Variable (v) domain at the amino terminus of the two chains together form the antigen-binding 8U region. The first constant (C) domain of the H chain ( CHI) also interacts with the C region of the chain through hydrophobic interactions and disulfide bonds. are interacting. The next domain of the heavy chain is called CH2, next to the hinge region.

According to some reports, this domain contributes to complement fixation and Fc receptor-mediated antibody dependence. AnLibody-dependent cellular disorder Antibody effector containing a sequence responsible for ytoLoxicity; ADCC) It contains many of the functions and is the only N-linked glycosyl chain in the rl chain. This is the part where it changes. The carboxy-terminal CH3 domain of the H chain is involved in Hffl assembly. It is believed that it plays an important role.

Antibody constructs of the invention have immunoglobulin binding regions of predetermined specificity. Contains the first part. This binding region has specificity for the same epitope. A zero-piled body construct with multiple hypervisible regions homologous to the hypervisible regions of the native antibody is In addition, it is essentially the CHI domain and the hinge region (the sheep C terminus is the N terminus of the CH3 domain). (linked), i.e., the C Contains a hybrid constant domain with the H2 domain removed. CH2 domain The antibody construct lacking the ΔCH protein is referred to herein as the IjinΔCH2tl construct. The 2l1 construct is a combination of antibody constructs with the same specificity but intact constant domains. It is characterized by a binding activity that is at least twice as high as the synthetic activity.

One suitable one! In some cases, the binding region of the ΔCH2 antibody construct is a murine immunoglobulin. Some embodiments of the present invention include binding regions for non-human immunoglobulins such as lobulins. In the phase, the present immunoglobulin binding region is the binding region of monoclonal antibody 872.3. with predetermined specificity for mucins, such as A predetermined binding region for Get ganglioside, such as the binding region of antibody 14.18. In another embodiment of the invention, the immunoglobulin has specificity. The binding region has predetermined specificity for melanoma-specific proteoglycans. Preferred modified constant regions with human immunoglobulin constant domains include human immunoglobulin constant domains. I'm here.

The antibody construct typically comprises an immunoglobulin disulfide bonded to an immunoglobulin light chain. Contains Brin heavyweights.

The antibody construct further includes a carboxylic acid in the second portion that includes a tumor-destroying protein. It may also include a tight third portion at the distal end. This third protein part is a lymphoprotein. Toxin, interleukin 2, epidermal growth factor (EGF), and its similar active substances , or an active fragment thereof. In a preferred embodiment of the present invention, The third part is formed through amino acid residues that can be cleaved by endopeptidases, such as lysine. This is a peptide linked to the carboxy terminus of the CH3 domain of the second part.

Alternatively, the third portion may be linked to the second portion by, for example, a chemical crosslink. good.

In yet another embodiment, the ΔCH2 antibody for use as a diagnostic imaging reagent The construct includes a radioactive label attached to it.

concave history The foregoing and other objects of the invention, its various features, as well as its invention, are described below. The following explanation can be fully understood when read in conjunction with the accompanying drawings.

Figure 1 is a schematic diagram showing the construction of the ΔC112 deletion mutant of the human) CT1 gene. :(A) Hind l I containing Cyl genomic gene segment and restriction site Map of the I-PvuII fragment; (B) is the Af l after restriction enzyme and ligation treatment ΔCH2 deletion gene lacking I-Ava 11 fragment (CH2 exon) child; (C) does not show the Sma1 site introduced into the CH3 gene; (D) shows the I used to ligate the Pvull-Xho1 fragment (E) containing the L2ijl gene. is a map of Sma[-F”tu I I linker; and (F) is The remainder of the vector contains the hol site and the polyASII region.

Figure 2 is a graph of antigen binding activity of various hemiCH2 chimera constructs = (A) Δ Ct (2ch14.1 body ms product; (B) ΔCH2 chimera B72.3 ( chB, 72.3) Antibody constructs.

Figure 3 shows (A) 37”C after 12 hours incubation and (B) 4°C1 Intact ch14.12 antibody and ΔCH2 after 18 hours incubation Figure 2 is a graph of competitive antigen binding analysis of ch14.18 antibody constructs.

Figure 4 shows intact antibody and ΔCH2CH2 antibody construct-dependent cytotoxicity (AD CC) is a graph.

Figure 5 shows the complement-dependent cytotoxicity (CDC) of intact antibodies and ΔCH2 antibody constructs. ).

Figure 6 shows the time in an athymic nude mouse bearing an M21 xenograft tumor. Fresh (A) intact ch14.1 body and (B) ch14.18 ΔC Figure 2 is a graph depicting tissue distribution of H2 antibody constructs.

Figure 7 shows F(ab')! in nude mice bearing M21 xenograft tumors. Fragme FIG.

Figure 8 shows Δ from the direct fluid circulation of athymic nude mice bearing M21 xenograft tumors. CH2 antibody constructs, intact antibodies, and removal of F(ab')t fragments This is a graph representing the future.

Archetype 9 theory The present invention is based on [11m1. with specificity for antigens related to or other antigens; Enhanced avidity, relatively short half-life, and relatively low levels of nonspecific binding Mainly uses truncated antibody constructs that exhibit the characteristics of will be handled.

These ΔCH21l constructs are useful for tumor imaging and treatment with systemic drug therapy. It is suitable as a targeting reagent.

The present invention provides antibody constructs with binding specificity for epitopes on human tumor antigens. The specificity of the 0-pillar body; allows for selective targeting of specific tumors. IIrgI antigens are preferred, i.e. the constructs are directed against tumor-unique markers. Join to car. Unfortunately, there are few antigen-specific markers for human cancer. only human tumor cells have been identified, however, human tumor cells are similar to normal cells such as embryonic cells. It also has tumor-associated antigens, which are present in small amounts. For such antigens, alphafe These include toprotein (AFP) and carcinoembryonic antigen (CEA). These antigens and whips (872,3 antibody), dialloganglioside G, (14,18 antibody), etc. Antibody constructs that recognize antigens associated with tumors are included in the invention.

The ΔCH2 construct of the present invention is a gene encoding DNA encoding antibody-like domains. Nat. ure312:604; Moririn et al. (1987) Ann, N. Y., Acad. Sci, 50 Uni 187; Moririn et al. (198B) CI in Chem, 3 4: 1668; Horwitz et al. (1988) Proc, Nat 1. Aca d, Sci, ■: 8687).

To reduce the immunogenicity of the ΔCH2 construct in humans, the present ΔCH2 construct is Designed to minimize human protein content. This means that the stationary part is small. Chimeric antibody molecules that are both human and have mammalian (e.g., mouse) variable regions This can also be achieved by creating.

Production of recombinant chimeric antibodies with predetermined specificity is based on cloned genomes. For example, coding for H and Lii was typically made using DNA fragments. in a rearranged form (i.e., recombinant expression during B cell maturation). (in the form of DNA sequences originating from elephants). These games The genome sequence itself contains information necessary for expression (i.e., 5° untranslated sequences, promoter , enhancers, protein coding regions, and donor splice sites). I'm here. ■The donor splice signal at the 3° end of the gene segment It is compatible with the splice acceptor signal at 5° in the 1gl region of the species.

That is, the splice product from the two maintains the correct reading frame. Connect the S and human C segments and transfect into the appropriate host cell type. If so, the primary transcript is spliced correctly and spans both the ■ and C regions. Mature Metsenger RNA (mRNA) with an open reading frame ) occurs.

Chimeram CH21! A preferred method of producing constructs includes co-coating immunoglobulin constant regions. jJim of the cassette to be loaded is included. This cassette is used for this immunoglobulin determination. The immunoglobulin variable region of the constant-encoding segment (compatible splice sequences) DNA that enables splicing into DNA segments encoding sequence and thus the subsequent transcription and translation of the immunoglobulin heavy or light chain is possible. This method is described in co-pending U.S. Patent Application No. 409,889 ( Title of the invention: "Method for producing fusion proteins", filed on September 20, 1989, herein (Incorporated as a bibliography in the book).

Briefly, the DNA cassette/cell reconstructs the 3° end of one splice donor site. and the DNA sequence encoding the variable CV) 14 region or at the 3° end of the exon. It is prepared by adding . Install the cassette with a compatible splice on the 5' end. Expression possible NA for constant (C) Si region genes with an acceptor structure (structural gene).

of the sequence of events leading to expression of the fusion protein in transfected cells. Inside, the mRNA derived from the two DNA sequences undergoes splicing to form the complete ■, or ■, and the 5' end encoding the human C, or cL domain. The resulting single-chain polypeptide, which yields an adult mRNA with a 3° end, is It is a fusion product of the ■ region encoded by the exon of a structural gene and a constant domain.

Furthermore, the cassette encoding the tumor killing factor was spliced into IgC8l. The IgcsJt region may be connected to the VSi region of immunoglobulin. ``Magic bullet'' type treatment is completed.

This cDNA contains H or L chain VM region (often non-human) specific to II tumor antigen. (e.g., mouse), whereas this cl region exon encodes another (e.g., mouse); 18 species (7) Ht or L-region C6I region (CHI, CH2 and CH3 domains) code). Areas of use include many neuroblastomas, melanomas, and disialogangliosides on the surface of cell lines and tissues of tumors and small lung cancers. Murine monoclonal antibody that recognizes G1 14.18 Mujoo (19B?) Cancer Res, Sat Uni 1098-11071) V, and vL domains. Other useful VSI ranges include many breast cancers and Murine monoclonal antibody 872.3 (Zinin) recognizes mucin-like structures on intestinal cancer. Johnson et al. (1986) Cancer Res, 46:850) It contains the V@6 and vL domains of . These ■regions are 1g human gamma ( Combine with various CHI regions such as the Cfil region of the H) chain and the force 7B (L) chain. Is possible. In addition, this VSI region may be of human origin, and may be of human origin. Expression of the H and L constructs in competent host cells is an vector with claimed specificity. Production of intact chimeric immunoglobulins and intact human constant regions, e.g. bring about.

To produce the ΔCH2 construct, one VSI region cassette was constructed and the C region It is introduced into a suitable vector along with the encoding DNA sequence. This CSI area is specifically lacking the CH2 domain and is designed as shown in Figure 1. A is a Hindlll-Pvull fragment containing the human genome Cγl gene segment Figure IB shows the map of the synthetic linker (to flll11) treated with restriction enzymes. The ΔCH21l construct after joining the two fragments at The DNA encoding the C89 region is shown.

The vector encoding this mutant Ce1l region has the ability to express this vector. transfect into a suitable host. Upon co-expression of two DNAs, the nucleus of the masonry Endoenzymes produce mRNA by carrying out normal splicing events and mRNA encoding the combined protein is produced. In the case of chimeric binding proteins In this case, this mRNA encodes the vo or vL domain (in the 5° to 3° direction). It can also be coded, which is identical to the native arrangement up to the VC junk at least one Cs, other polypeptides such as at least part of the if region domain The C6i region may be directly connected to the C6i sequence, for example, even if it is a human sequence. The 3' half of the donor splice site (and downstream nucleotides) and The 5° half of the scepter splice site (and upstream nucleotides) The protein is removed as a protein, yielding mRNA encoding the appropriate fusion protein.

Typically, the two exons are placed on the same vector, both under the control of a single regulatory sequence. It will be destroyed. Additionally, Lti It is also suitable to co-express both the H and H constructs. The use of host cells that have enzymes that recognize the signal and carry out appropriate splicing. I need something to do.

Specific Hector fM construction, host cell selection, transformation, and expression methods were originally described in the book. Although it does not constitute an aspect of the invention, it may be used in this field according to the preference or convenience of each person. Techniques that can be used in the present invention may be selected and implemented by the person being asked to do so. For example, Current ProLocols in Mo1ecular Bi.

L9JLL (Green Publishing As5ociates, 43 0Fourth 5LreeL, Brooklyn, N.Y., 1989) It is shown. Useful vectors include transcription and translation vectors of the gene of interest. A number of plasmids known to contain the correct sogenals are included. enhancer Elements may be present, including additions for polyadenylation and splicing. Additional signals must be present if not provided by the gene itself. For example, a signal for the expression of a functionally rearranged Igifl gene. All of the genes are located on a series of DNA trenches, including transcription promoters, splicing signals and terminators for the exclusion of intronic sequences and polyadenylation. Contains the knot site. Furthermore, the information that must be provided by the vector is It is a selective marker gene.

This gene is used to express selective phenotypes (usually the inability of toxic drugs, such as methotrexate) It must also contain a signal for the expression of (resistance to death effects); Therefore, if the vector encodes both the first and second polypeptide, provides sequence information for three independent transcription units in a limited amount of space Must.

The vector containing the recombinant cassette is transfected into appropriate seed cells. inn The choice of principal cell selection is based on the criteria noted above, as well as their ability to proliferate in the growth medium. commercially available serum-free products as well as ease of selection after transformation. is preferable. For the production of chimeric antibodies, useful host cells include, for example, Zumi■g non-producing 5p210Ag14 hybrid monomer cell line, etc. myellow This includes yeast, hyperidoma, yeast, bacteria, and plant cells. Useful cloth types Cells are widely available in various warehouses and from commercial sources, and are available to those skilled in the field. It can also be easily separated from natural sources.

One method for introducing recombinant DN A into cells is electroporation. (e.g., Botter et al. (1984) Proc, NaL, A cad.

Sci, USA (see Dan 1 Near 161-7165), this is a special device and highly purified DNA must be obtained, however, certain specific This method can be used to treat many different cell lineages if conditions are optimally set for the cell type. It is possible to transform.

Another transfection method is protoplast (spheroplast) fusion (e.g. , Sandry Golgin et al. (1981) Molec, Ce1l, Biol, 1nia 43-752). Bacteria carrying recombinant plasmids are treated with chemical reagents (typically 45-50% fuse with lymphoid cells in an isotonic buffered solution of lyethylene glycol). this method is simple and does not require extensive purification of plasmid DNA, and in addition, has a very high conversion efficiency can be obtained, and this transfection method can be used to This is a highly productive strain as it has a high possibility of giving transfectants containing lasmids. The time it takes to obtain transfected cell clones is reduced.

Cells that are successfully transformed with this vector are then separated from those that are not. There are many methods for selecting transfected cells that have to be For example, guanine phosphoribosyltransferase (gpc) and neo Mycin (neo) resistance markers can be used for lymphoid cell selection purposes. Ru. The gene encoding the marker is included in the vector encoding the v region. Will.

Resistant forms of dihydrofolate reductase (DHFR) are also present in hybridoma cell transformants. for selection and further amplification of the marker and its adjacent product genes. can do.

The transfected cells are then cultured to obtain the polypeptide encoded in the cassette. Express. Culture may be performed once in vitro, or recombinant In the case of antibodies, other strategies such as upper viv+) culture in ascites fluid can be used. It can also be achieved by

The CH2 domain of the human Cr1 heavyweight deleted from the intact antibody molecule is Contains sequences: sequences that provide long half-life; Fc receptors on effector cells A sequence responsible for binding to the body; a sequence with a single N-linked sugar chain; and a fourth capture component, Sequence bound by C1q.

To determine the extent to which these sequences are required for various antibody functions, Δ The CH2CH2 construct was tested and the amount of cells expressing intact antibody and First, the supernatant of cells expressing the ΔCH2 antibody was measured for bound human chains. The number of antigen-binding domains (antibody molecules are determined per antigen-binding site) is quantified by (containing a light chain) and then a direct antigen binding assay. As shown in Figure 2, the heCH2 construct showed much higher activity. showed his sexuality. Ch14.18 ΔCH241! The dyed material is pre-coated with CIIK. On the other hand, the chB72.3ΔCH2fX construct mucin-coated plate assayed on a plate.

To eliminate the possibility of non-specific interactions on the ELISA plate, ΔCH The 2CH2 construct binding activity was further assayed in a competitive binding format as seen in Figure 3. As shown, ch14.18 ΔCH2 constructs allow normal antibodies to compete with themselves. cells also competed much more effectively with the labeled intact ch14.1B construct. . Increasing the length of the binding assay from 2 hours (Figure 3A) to 18 hours (Figure 3B) Differences in binding are not as dramatic; dirt may be due to removal of the CH2 domain. This suggests an increase in antigen binding rate, but not overall affinity.

Increased antigen binding of the ΔCH2 antibody construct is due to conformational changes in the antibody molecule. It is assumed that this reflects the By removing the C112 domain, FabjJ region (including the V region, CL and CHI sequences) no longer interacts with the CH2 domain. 11iIllll depending on the purpose! ! These inter-domain interactions that are never appears to increase the rate of association with antibodies. Furthermore, CL ( Removal of the carbohydrate moiety of the 2-domain (without reducing the spatial interaction between nine bodies and 12 It may happen.

Effector function by the C82M region is no longer possessed by the ΔCH2 antibody To confirm this, an intact ch14.18 antibody was used as a control. This can be done by ADCC assay. In ADCC Afusei, the unfractionated end Antibodies with the melanoma-targeting lytic ability of peripheral vascular leukocytes (PBLS) Measure against. Typical results are shown in Figure 4. In the 0 diagram, ch14.1 M21 human membranes under conditions where B antibody and ΔCH2 construct were given at certain concentrations. Regarding Norma's special data points, the average value of three experiments was taken. in Tactful ch14.1B antibody has high AD even at low concentration of lng/ml It has CC activity. Remarkably, the ΔCH2 construct has a very low ΔDCC The inactivation of ADCC activity, which only showed activity, reflects the loss of binding ability to Fc receptors. are doing. CH whose binding site for high affinity receptor (FCR) is adjacent to the hinge region Since the binding activity is limited to the 261 region, it is unlikely that the binding activity will be lost due to the ΔCH2 mutation. Not surprisingly, what is important in radioactive imaging diagnosis of tumors is the activation of Fc receptors. By reducing the binding, cells with Fc receptors are localized [non-specific to The point of the ΔCH2CH2 construct described here, which is to suppress heterogeneous masonry. It is also useful.

Furthermore, as expected, in the ΔCH2Iji construct, complement It was observed that the ability to induce lysis of melanoma target cysts was decreased. , the lytic ability of hi217 in the presence of human complement was reduced using the ΔCH2 mutation. amorous enough to detect ch14.1B antibody has extremely high lytic ability, whereas it is not shown in agriculture. It is shown.

Anti-tumor CH2 antibodies are therapeutic because they can localize specifically and rapidly to tumor cells. It can be an ideal vehicle for delivering therapeutic drugs in vivo. For example, Figure 1 (C) - As shown in (E), encodes human interleukin-2 (IL2) The DNA sequence was tethered to the carboxyl terminus of the IaftiΔC142 construct and the ΔCH 2/I Deliver tL2 to the 1m site by expressing the L2 double protein The product can activate T cells in the tumor area; 0 local II, 2 T; If! Promotes destruction of tumors. ΔC1 (2 antibodies have a shorter half-life than whole antibodies, but ΔC1 The half-life of CH2/IL,2 fusion protein should be longer than IL2.

Another example is anti-117B cytokine lymphotoxin or tumor Necrosis factor β (TNFβ) fused to the carboxyl terminus of the ΔC112 antibody is possible. This fusion protein also targets these cytotoxic proteins to tumors. It is effective in differentially localizing and reducing potential systemic side effects. as well as other poisons This is the case by fusing the sex protein to the ΔCH2 antibody. As these proteins The ones that come to mind are Rin (r.i.c.in), diphtheria element, and pseudomo. Eggplant (Pseudorn.

It is an exotoxin of nas). In addition, l\DP ribosylation enzyme activity alone is sufficient for breastfeeding. The toxin is present in the entire toxin molecule so that elongation factor 2 can be inactivated in cell-like cells. Even just the parts that are important to sex are effective.

The in vitro tissue specificity and half-life of various antibody constructs are It can be determined by examining the distribution of Dynamics with exogenous M211111 An intact antibody, ΔCH2 or F(ab'), is injected into the seed's body. Each construct in organs and tissues is measured during or at a certain time. Figure 6 and Table 1 show that (A) intact ch14.1B antibody and (B) ΔCH2 The biodistribution of the 411i construct is shown over time, with the latter rapidly ( (within 4 hours) has been demonstrated to specifically target tumors.

Table 1 Kimi □ Antibody sample set w8 4 hours 24 hours 96 hours (^) ch14.1B Mll,/Blood 0.18 0.56 1.08 Liver N/Blood 0.79 2.0 2 4.55<8) ΔClI2 Tumor/Blood fi O, 862, 201, 19 Liver/Blood 2.98 7.37 2 ．． 28 Figure 7 shows human TgF(ab’)tM kataishi and ch14.18ΔCH2 horizontal This figure shows the biodistribution of the construct 24 hours after injection, and ΔCH2tll The specificity of the construct's targeting action has been demonstrated.

Determination of the half-life of the ΔCH2 antibody construct in the bloodstream of animals with II tumors. to measure the abundance of these constructs in blood over time using aJI. Figure 8 shows that in the bloodstream of nude mice bearing exogenous M21 tumors. , ΔCH2CH2 construct i phase compared to F(ab')□ fragment and intact antibody. This shows that the length is short.

The ΔCH2 antibody rapidly binds to the Lt epitope and prevents nonspecific binding to other tissues. Since the amount of cancer is relatively small, it is difficult to use various therapeutic drugs to target tumors. I can do it. Particularly effective therapeutic agents include interleukin-2 and lymphotoxin. Drugs with direct or indirect tumor-killing activity, such as epithelial growth factor, are listed. It will be done. For example, EGF is fused with an antibody that binds to and activates cytotoxic T cells. This allows T cells and cells with EGF receptors to interact with each other. Matako Partial fragments and biosynthetic analogs of proteins with m-killing activity, such as, are also useful. this These proteins are chemically linked to the CHJ SI region using various cross-linking agents known in the field. It is possible to bind to Encoding 0 e.g. ΔCH2 constructs that can also be peptide linked to the carboxy terminus In addition, a tt tumor-killing protein can be inserted into the vector constructed to do so. is possible.

The increased avidity of the ΔCH2fJll construct is due to its short half-life in vivo. This, combined with reduced levels of non-specific binding and For example, low-energy Iodine-123, Indium-Ill, Technetium-99m, which are Rugie nuclides , and radioisotopes such as iodine-125 that do not change the binding activity when incorporated into the construct. If labeled with a topical element, radiation can be detected externally and ΔCH2 anti- It is also possible to use tumors using body constructs. In addition, the construct of ΔCH2 antibody Concentration of antibodies labeled with ferromagnetic substances such as gadolinium and localized around the tumor It is also possible to image the difference using nuclear magnetic resonance imaging.

Lateral structures fused with tumor-killing substances and/or loaded with radionuclides For targeted effects, constructs have a longer half-life than required for imaging at high concentrations. It is necessary to have Also, the determining factors of the level of radioactivity required for diagnostic imaging. The ability of the antibody construct to selectively label the tumor relative to the surrounding &lIw& , the size of the tumor, and the distance from the injection site to the tumor.

, e.g. 2-20 residues at either the 3" or 5' end of the ΔCH2 antibody construct. By chemically bonding polypeptide sequences rich in lysine (Iysin), , as mentioned above, as a remotely detectable component uptake site. . Gamma-ray or positron-emitting ions interact with the ti-separated amino groups on lysine residues. Incorporation into these linkers by electrical interaction or chelation is preferred.

For example, noethylenetriaminepentaacetic acid (DTPA) can be converted into Tc-99 or In- It can be used for labels according to 111. The isotope of In and ■ is oxine It can be incorporated using a salt (8-hydroxyquinoline). iodogen (1,3,4,6tetrachloro3α,6αdiphenylglucoryl ceChcm, Co., Roskville, III) is labeled by +zsl. (e.g. Thakur et at, Throm b.Res.

9:345 (1976), McFarlane, J., CI in, Inves. t, 42:346 (1963), Knight eL al, Radioph. armaceuLicals, N, Y, Soc, of Nuclear Med icene.

1975, pp. 149 and Harwig et al., InL, J. Ap. pl, Radiat, l5oL, 27:5.1976), and several others. methods are known to those skilled in the art.

Based on the above-mentioned concept, the other 11 known reagents can also be converted into ΔCH2 structure by conventional means. It should be obvious that it can be combined with a building, here an example of healing facilitation. Calcitonin, epidermal growth factor, tumor growth factor α and β, platelet-derived growth factor, insulin-like growth factor (somatomedin-C), Representative examples include connective tissue activating peptides and human collagenase inhibitors.

The concentration and method of administration of ΔCH2 constructs produced according to the present invention may be The properties of the conjugate, the degree of decrease in physiological activity, if any, and the individual This needs to be decided based on the patient's immune tolerance and the symptoms of the disease being treated. If the polypeptide is conjugated to a drug for diagnostic imaging, scintillation – tumor-localized radioactivity, such as site scanning or autoradiography. Sufficient amount to be detected using a radiation detection method that allows the radiation to be detected externally. generally need to be administered between 5 and 20 microcuries. There is. Most preferably about 10 microcuries. Actual dose It is well known in the art that tumor-associated antigen localization determines It is. If necessary, radioactive peptide in an amount within the safety standard range. Imaging and treatment of tumors that can be further injected with ΔCH2CH by intravenous injection This can be done with two construct administrations. The preferred dose in this case is about 0.01-10 mg/kg body weight.

The following non-limiting examples may provide a better understanding of the invention. Example (1989) J, Immuno, MCth, 125:191-202 to pBR3 22 was subcloned and used to create an 11-chain mutant strain. CH2Si area The deletion mutant lacking (ΔCH2) is Hindllll-AtllllFr fragment (C1 (containing exons 2 and hinge) and AvalI-Pvul+ fragment (CH (including 3 exons) using a synthetic double-stranded oligonucleotide fragment. Created. The CH2 construct was confirmed by DNA 1% analysis. Then Mau monoclonal antibody 14.18 VeIMCMujoo eL al, (19 87) pdHL2-VC71C containing Cancer Res, 47:190B) Mutation modification of z (Gillies et al, above) near the chimelpoxy terminus The Sma1 site was introduced by (Figure 1 (c)), and this mutation resulted in 2373 th base (Huck eL al, (1986) Nucl, ΔcidS, R cs, 14:1779-1789) is replaced by T to C. However, this corresponds to the wobble position of the serine codon, so there is no change in the amino acid sequence. The sequence of the L2 protein is attached to this Sma1 site. (Figure ID-Sen), this sequence has Sma [single sequence of sites] from 5° to 3°. Includes up to the first Pvul [site. The remaining [L2 sequence is further pvu[1 part link to the single Xho1 site located 3' to the translation termination signal. This includes the code array. Ligate the Xhor end of the connected DNA to Hector. The 3” untranslated region of the mouse χ constant region and the addition of polyA The constructed construct has been added to the ΔCH2 human gamma 1 chain (Figure IF). IL2 is bound to the end of Lubokin.

Transformation and selection were essentially carried out by G11lies et at , CBio/Techno1. (19B9) 7:799-804) I went there. Briefly, the non-1g producing murine hybridoma line 5p210 Ag14 was added to Dulbenko's modified Eagle's medium (DME) containing 10% fetal bovine serum. M) was cultured. G11lies l! L al-((1983) Cell, 3 Plasmids were transformed into protoplasts substantially according to the method of 3 Near 17-728). It was introduced into cells using the fusion method. Transformants were treated with methotrex at an initial concentration of 0.1 μH. Selection was made in growth medium containing Sate (MTX). About MTX-resistant clones Secretion of human antibodies was examined by ELISA assay. microquiter plate Goat anti-human [gG (H and L specific - Jackson Laborato ries), then add conditioned medium from the transformants and incubate. did. Goat anti-human IgG (Fc specific - Jackson Laboratory Human antibodies were detected using es) conjugated to horseradish peroxidase. Ta. The clones producing the highest amount of antibodies were further treated with increasing concentrations of MTX. (061μH to 1μH, further after acclimatization 5#M to final concentration 10μM MTX) Medium cultivated inside. 10μ) under IMTX for several generations (after getting used to it, the limit dilution method was used) A noclonal cell line was obtained.

3. Protein purification The specific ch14.1B antibody was prepared using Protein A Sepharose (Replige). Purified by binding and elution to n). ΔCH2 mutant antibody is anti-human monochrome null antibody - purified by immunoaffinity chromatography on a Sepharose column did. As a result of analysis by 5DS-PAGE or HPLC, both proteins were 90% % pure.

4. Antigen binding assay Direct binding assays and competitive binding assays to antigens are Do G! antigen (14,18 antibody) or submandibular gland mucin (Sigma) (872, G11lies et al. using a microtiter plate coated with 3 antibodies) Al, (1990 ditto) method was used in both cases, secondary (detection) As antibodies, goat anti-human) IgG, Fc-specific polyclonal antiserum (Jackson horseradish peroxidase (HRP) Simply put, a plate coated with Got was first coated with 5% Contains bovine albumin (BSA) and 5% goat serum [BS for 2 hours at 37°C. Blocked. After covering the label plate and incubating overnight at 4 °C , washed 6 times with PBS, and developed color using 〇-phenylenediamine (OPD) as a substrate. I set it.

5.11 cell disorder! 1a7say ADCC Afsay uses Cr-labeled M21 human melanoma as the target cell. Chimeric antibody diluted with 50 µl of peripheral vasculature (from the mother) Mixed in a 10 μm microtiter plate. 37°C for 4 hours After incubation, centrifuge the plate to collect 1ootI+ and use it for LKB model. Counts were measured using a Le 1272 gamma counter.

Complement lysis of labeled M21 melanoma was performed using 50 ul of cells (2XlOS This was carried out by mixing an equal amount of each diluted antibody with cells/ml). 37° After a 15 minute incubation, add 100 μl of human complement (4x with fresh human serum). diluted) was added to each well. Ink the plate for another hour at 37°C. After cubating, centrifuge and supernatant 1OOOK! ■The amount of Cr released using measured. What is the percentage of KJ cytotoxicity?

It was decided by.

6. Examination of biodistribution and disappearance from blood 8 ~ lOs female? ! ! thymus mouse (nu/nu) (Lhe　NaLionalCancer　In5LiLuLe , Hesesda, N-D) 2X10' h4zt! 1lffl cells were injected subcutaneously. . Tumors grew to a size of 50-150 mg within 10 days. At this point the mouse Inject 25 μg and 3 to 4 μC1 of +21-labeled antibody into the tail vein of the patient intravenously! I did 1 . Groups of three mice were anesthetized with halothane at designated time points after injection. , blood samples were collected by posterior U1 fossa bleeding method. In vivo preparation of radiolabeled antibodies To examine the cloth, groups of three animals were killed at the end of the main side.

Tumors and major organs (heart, skin, muscles, bones, lungs, liver, spleen, thyroid, kidneys) , 1Lii) was taken out and its weight was measured. 1 for all tissue samples! $1 live The sex was assayed by gamma counter. Results are per gram of tissue injected. Calculated as % of concentration and localization ratio (cpm/g tumor: cpm/g tissue) did.

7. Radioactive labeling Ch14.1B, ch14.1B-ΔCH2, F(ab'), fragment of Hinocular gG Labeled with USr. Briefly, lOOtlg's 1odo-Gen reagent (Pierce Chemical Co, Oy Quaford, IL) In a polystyrene tube, add 500 uz of antibody to 0.5 mCl of "ST" (100mci or 3.75GBq/m1.Amersham Corp,, (Arlington Heights, IL) for 25 minutes on ice. take The 11S■ that was not loaded was a PDIO column (Pharmacia Fine C hemical, Piscataway.

NJ) was removed by gel filtration. The specific activity is 1.0% per body for nIJ. 5-0 ．． 5 nCi was a typical value.

The invention may be carried out in other specific forms without departing from its spirit or essence. It is also possible to Therefore, the embodiments described herein are limited in all respects. It should be understood as illustrative rather than specific, and the scope of the invention is therefore clearly defined. It is determined by the scope of claims rather than the description of the specification, and the scope is equivalent to the scope of claims. All changes that may occur within are included within the scope of the invention.

h’l/11 n is 1 1ρ 91 Ichiζl "Nonon, 5% ”　!Jlllt/　G　　　1% 3/1/’G’+% il　FjlΦIf/1':'XY':! !　%Supplement i [translation submission form (Special Act 5' [Law Article 184-8) 9112, 1992 Special r ``Agency Commissioner Aso Watari-dono p 匍 1. Display of patent application PCT/US91100633 2. Name of the invention Antibody constructs with increased binding affinity 3. Patent applicant Address: Massachusetts, USA 02139. Cambridge.

One Kentor Square, Building 700 Name Lebriken Co. poration 4, agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyo IJJ-ku, Tokyo Phone: 3270-6641~6646 Name (2770) Patent attorney Kyozo Yuasa “Wo “2′・2 5. Submission portal for supplementary IL documents Replacement of full text of claims The scope of the claims 1. An antibody construct specific for an epitope on an antigen, comprising <a) said epitope; It consists of a hypervariable 6-region of a tope-specific natural antibody and a homologous hypervariable region, an immunoglobulin binding region with a predetermined specificity; and <b) substantially a CI (Consists of 1 domain, hinge and CH3 domain, C comprising an immunoglobulin constant region lacking the H2 domain, The above-mentioned constant 1g region has the binding ability of the above-mentioned antibody construct) and the antigen-binding ability of the above natural antibody. The antibody construct as described above, characterized in that it is increased by at least 2 times in comparison.

2. The structure according to claim 1, wherein the binding region comprises a binding region of a non-human immunoglobulin molecule. construction.

3. The binding region of claim 2, wherein the binding region comprises a murine immunoglobulin molecule binding region. construct.

4. The construct according to claim 1, wherein the binding region has specificity for mucin.

5. The binding region according to claim 1, wherein the binding region has specificity for GD2 ganglioside. construct.

6. The binding region has specificity for melanoma-specific proteoglycans; A construct according to claim 1.

7. The construct according to claim 1, wherein the constant region comprises a constant region of a human immunoglobulin. .

8. A claim containing an immunoglobulin heavy chain disulfide-bonded to an immunoglobulin light chain. The construct according to claim 1.

9. A protein with tumor-killing activity covalently linked to the carboxy terminus of the constant region. The construct of claim 1, further comprising:

10. i protein is cleaved by endopeptidase through an amino acid sequence, 10. The peptide of claim 9, wherein the iCI+3 domain is peptide-bonded to the carboxy terminus of the iCI+3 domain. construct.

i+, i protein attaches peptide to the carboxy terminus of 1cH3 domain via lysine residue. 11. The antibody construct of claim 10, wherein the antibody construct is tide-conjugated.

12. The protein is selected from the group consisting of lymphotoxin and its active mu form. 10. The construct of claim 9, comprising the I bee killing protein.

13. The protein is selected from the group consisting of interleukin-2 and its active analogues. 10. The construct of claim 9, comprising a selected II tumor killing protein.

+4. The protein is epidermal growth factor, lf! Tumor necrosis factor sin, diphtheria toxin , Shutomonas exotoxin and their active analogues. 10. The construct of claim 9, comprising a tumor killing protein.

15. The construct of claim 1, which also includes a radioactive label.

16. The W white matter is an active flag of lymphotoxin or lymphotoxin analogs. 10. The construct of claim 9, comprising II tumor killing protein which is a protein.

17. The protein is interleukin-2 or an interleukin-2 analogue. 10. The construct of claim 9, comprising an active fragment of the tumor killing protein.

18. The protein contains epidermal growth factor, tumor necrosis factor, ricin, diphtheria toxin, a protein selected from the group consisting of Eudomonas exotoxins and their active analogues; 10. The construct of claim 9, comprising an active fragment of l [I tumor killing protein.

international search report Continuation of front page (51) Int, C1,' Identification symbol Internal office reference number A61K 39/39 5 V 9284-4CY 9284-4C CO7K 14152 16/46 8318-4H // C12N 15109 (C12P 21108 C12R1:91) 8314-4C 9051-4C 9281-4B I A61K 37/24 //　C12N　15100　A

Claims (15)

[Claims] 1. An antibody construct specific for an epitope on an antigen: (a) Contains a hypervariable region homologous to that of a native antibody specific for an immunoglobulin binding region with specificity; and (b) a xylemally CH1 domain It consists of a hinge region, a hinge region, and a CH3 domain, and is usually Immunoglobulin constant region with existing CH2 domain removed (The constant region controls the binding activity of the antibody construct described above, and the binding activity of the native antibody described above.) at least 2-fold compared to sex). 2. Claim 1, wherein the binding region comprises a binding region for a non-human immunoglobulin molecule. construct. 3. Claim 2, wherein the binding region comprises a binding region for a mouse immunoglobulin molecule. construct. 4. 2. The construct of claim 1, wherein said binding region has specificity for mucin. 5. Construction of claim 1, wherein the binding region has specificity for GD2 ganglioside. thing. 6. The above binding region has specificity for melanoma-specific proteoglycans. Construct of requirement 1. 7. The construction of claim 1, wherein the constant region comprises a human immunoglobulin constant domain. thing. 8. Immunoglobulin heavy chains crosslinked by disulfide bonds to immunoglobulin light chains 2. The construct of claim 1, comprising a strand. 9. Added to the carboxy terminus of the above constant region and has tumor-destroying activity. The construct of claim 1 further comprising a protein. 10. The above protein passes through an amino acid sequence that can be cleaved by endopeptidase. Accordingly, the claimed peptide is a peptide truncated at the carboxy end of the CH3 domain. Construct of item 9. 11. The above protein connects to the carboxy terminal of the CH3 domain through a lysine residue. 11. The antibody construct of claim 10, which is a truncated peptide. 12. The above proteins include lymphotoxin, its similar active substances, and its active activators. a tumor-destroying protein selected from the group consisting of Construct of requirement 9. 13. The above proteins include interleukin 2, its similar active substances, and its active substances. containing tumor-destroying proteins selected from the group consisting of 10. The construct of claim 9. 14. The above proteins include epidermal growth factor, tumor necrosis factor, ricin, and diphtheria toxin. consisting of the elementary Pseudomona exotoxin, its similar active substances and its active fragments The anti-inflammatory agent according to claim 9, comprising a tumor-destroying protein selected from the group body construct. 15. The construct of claim 1 further comprising a radioactive label.