JP2006246896A - Antibody and antibody fragments for inhibiting growth of tumors - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel antitumor agent holding together advantageous characteristics of a monoclonal antibody, antibody fragment and single-stranded antibody. <P>SOLUTION: Disclosed are an anti-EGF receptor monoclonal antibody 225 lacking an invariable region and variable light strand of mouse anti-EGF receptor antibody, and chimerized and humanized versions of fragments thereof. Also disclosed is a molecule having a hypervariable region of monoclonal antibody 225 connecting to an effector molecule which is a cytotoxic agent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  This application is a continuation-in-part of patent application 08 / 573,289 filed on December 15, 1995, which is a continuation-in-part of patent application 08 / 482,982 filed on June 7, 1995. Application, the disclosures of both of which are hereby incorporated by reference.

  The present invention relates to antibodies and antibody fragments useful for inhibiting the growth of certain tumor cells.

Recent studies have elucidated the important role of growth factor receptor tyrosine kinases in the pathogenesis and progression of human malignancies. These biological receptors are anchored by the transmembrane domain in the cell membrane that expresses them. The extracellular domain binds to growth factors. When growth factors bind to the extracellular domain, a signal is transmitted to the intracellular kinase domain. This signaling contributes to events that lead to cell proliferation and differentiation. Members of the epidermal growth factor (EGF) receptor family are important growth factor receptor tyrosine kinases. The first member discovered in the EGF receptor family was a glycoprotein with an apparent molecular weight of about 165 kD. This glycoprotein is described in Patent Document 1 of Mendelsohn et al. And is known as an EGF receptor (EGFR).
Binding of the EGFR ligand to the EGF receptor leads to cell growth. EGF and transforming growth factor alpha (TGF-alpha) are the two known ligands of EGFR.

  Many receptor tyrosine kinases have been found in abnormal numbers in human tumors. For example, many tumors of epithelial origin express high levels of EGF receptors on their cell membranes. Examples of tumors that express the EGF receptor include glioblastoma and lung, breast, head and neck, and bladder cancer. Amplification and / or overexpression of the EGF receptor on the tumor cell membrane is associated with difficulty of prediction.

Antibodies against tumor antigens, particularly monoclonal antibodies, are being pursued as potential anticancer agents. These antibodies suppress tumor growth through a number of mechanisms. For example, antibodies immunologically inhibit tumor growth by antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
Alternatively, the antibody may compete for growth factor binding to the receptor. Such competition suppresses the growth of tumors that express the receptor.
In another approach, the toxin is conjugated to an antibody against the tumor antigen. The antibody moiety directs the conjugate to the tumor and the tumor is killed by the toxin moiety.

For example, U.S. Patent No. 6,057,031 describes a mouse monoclonal antibody called 225 that binds to the EGF receptor. This patent was assigned to the University of California and granted exclusive license to ImClone Systems Incorporated. This 225 antibody can suppress the growth of cultured EGFR-expressing tumor lines, as well as the in vivo growth of these tumors xenografted into nude mice. However, in a phase 1 clinical trial, no clinical response was observed when humans were administered up to 300 mg of mouse 225 antibody. See Non-Patent Document 2. See Non-Patent Document 3. More recently, a therapeutic regimen combining 225 with doxorubicin or cis-platin has shown therapeutic synergistic effects on some of the human xenograft models well established in mice. Non-Patent Document 1.
U.S. Pat. No. 4,943,533 Basalga et al. Natl. CancerInst. 85, 1327-1333 (1993). Divgi et al. Natl. Cancer Inst. 83, 97-104 (1991) Masui et al., Cancer Res. , 44, 5592-5598 (1986)

  A disadvantage of using mouse monoclonal antibodies for human therapy is the potential for human anti-mouse antibody (HAMA) response due to the presence of mouse Ig sequences. This disadvantage can be reduced by replacing the entire constant part of the mouse (or other non-human mammal) antibody with a human constant part. Replacing the constant part of a mouse antibody with a human sequence is usually called chimerization.

  This chimerization process can also be more effectively performed by replacing variable regions other than mouse antibody hypervariables or complementarity determining regions (CDRs) with corresponding human sequences. Variable parts other than CDRs are also known as variable framework regions (FR).

Replacing invariant and non-CDR variable regions with human sequences is commonly referred to as humanization. This humanized antibody becomes less immunogenic (ie, less of a HAMA response) by replacing more mouse sequences with human sequences. Unfortunately, the more mouse antibody regions that are replaced with human sequences, the greater the cost and effort.
Another way to reduce the immunogenicity of an antibody is to use antibody fragments. For example, in an article by Aboud-Pirak et al., Journal of the National Cancer Institute, 80, 1605-1611 (1988), an anti-EGF receptor antibody called 108.4 is compared with the antitumor effect of an antibody fragment. ing. This tumor model is based on xenografts of KB cells in nude mice. KB cells are obtained from human oral epidermoid carcinoma and express high levels of EGF receptors.

Aboud-Pirak et al. Found that both antibodies and divalent F (ab ′) ₂ fragments retarded tumor growth in vivo, whereas F (ab ′) ₂ fragments were less effective. ing. The monovalent Fab fragment of this antibody preserved its ability to bind to cell-related receptors, but did not slow tumor growth.
Therefore, it can be produced effectively and inexpensively, has little or no immunogenicity to humans, has the ability to bind to many receptors expressed on tumor cells, and has the ability to block the binding of these growth factors to the receptor. There is a great need for improved anti-tumor agents. One object of the present invention is the discovery of such new anti-tumor agents that combine the advantageous properties of monoclonal antibodies, antibody fragments and single chain antibodies.

  These and other objectives were achieved by providing a polypeptide lacking the constant region of the antibody and the variable light chain, as will be apparent to those of ordinary skill in the art. It has an amino acid sequence of NYGVH (SEQ ID NO: 1), GVISWSGNTDYNTPFTSR (SEQ ID NO: 2), or VIWSGGNTDYNTPFTS (SEQ ID NO: 3). The polypeptide can be conjugated to an effector molecule such as a molecule that inhibits tumor growth. The invention further relates to DNA encoding these polypeptides.

The present invention also includes polypeptides composed of the amino acid sequence of NYGVH, GVIWSGGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS.
The invention also includes molecules having the constant region of a human antibody and the variable region of a monoclonal antibody 225 conjugated to a cytotoxin agent such as doxorubicin, taxol, or cis-diamine dichloroplatinum (cisplatin). The present invention further relates to a tumor in a human characterized by administering to a human an effective amount of a polypeptide in which the constant part of the variable light chain of the antibody is deleted (this polypeptide has the amino acid sequence NYGVH, GVIWSGGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS) A method of significantly suppressing cell growth is also included. Viewed from another aspect, the present invention relates to a method for significantly suppressing tumor cell growth in humans, comprising administering to the human an effective amount of a polypeptide composed of the amino acid sequence NYGVH, GVIWSGGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS. .

  The present invention further includes a method of significantly inhibiting the growth of tumor cells that express the EGF receptor in humans. This method allows an effective amount of a molecule having a constant part of a human antibody and a variable part of a monoclonal antibody 225 to be administered to a human both in the presence and absence (particularly in the absence) of a cytotoxic molecule such as a chemotherapeutic agent. It is characterized by administering.

In one aspect, the present invention relates to a polypeptide in which the constant region of the antibody and the variable light chain are deleted, the polypeptide comprising the complementarity determining regions of the first and second heavy chains of monoclonal antibody 225. These complementarity determining regions have the following amino acid sequences:
CDR-1: NYGVH (SEQ ID NO: 1)
CDR-2: GVIWSGGNTDYNTPFTSR (SEQ ID NO: 2)
Peptides containing the first and second complementarity determining regions can be obtained by methods known in the art. For example, the polypeptides can be isolated by expressing the DNA encoding the polypeptide in a suitable host. This DNA can be chemically synthesized from all or part of the four nucleotides by methods well known in the art. As such a method, there is a method described in Science, 230, 281-285 (1985) by Caruthers.

  This DNA can also be obtained from mouse monoclonal antibody 225, which is described in US Pat. No. 4,943,533 to Mendelsorn et al. This antibody was deposited with the American Standards Stock Conservation Organization (ATCC), Bethesda, Maryland, on June 7, 1995 (deposit number 11935). Methods for obtaining the variable heavy chain region of an antibody are known in the art. Such methods include, for example, those described in US patents by Boss (Celltech) and Cabilly (Genentech). See U.S. Pat. Nos. 4,816,397 and 4,816,567, respectively.

DNA encoding the protein of the present invention can be used to express replication and recombinant proteins by inserting into a wide variety of host cells with various cloning and expression vectors. The host may be prokaryotic or eukaryotic.
The polypeptide includes either NYGVH, GVIWSGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS. Alternatively, the polypeptide may comprise a NYGVH sequence and either the GVISWSGGNTDYNTPFTSR or VIWSGGNTDYNTPFTS sequence.
The polypeptide can also be conjugated to an effector molecule. An effector molecule is a molecule that performs various useful functions, such as inhibiting tumor growth, allowing a polypeptide to enter a cell, such as a tumor cell, or directing a polypeptide to an appropriate location in the cell.

Examples of the effector molecule include a cytotoxic molecule. The cytotoxic molecule can be a protein or a non-protein organic chemotherapeutic agent. Examples of suitable chemotherapeutic agents include, for example, doxorubicin, taxol, and cisplatin.
Examples of some other effector molecules suitable for conjugation to the polypeptides of the invention include signal transduction inhibitors, ras inhibitors, and cell cycle inhibitors. Examples of signal transduction inhibitors include quercetin (Grazieri et al., Biochem. Biophs. Acta, 714, 415 (1981)); lavendastin A [lavendustin A] (Onoda et al., J. MoI. Nat. Prod., 52, 1252 (1989)); and herbimycin A (Ushara et al., Biochem. Int., 41, 831 (1988)). Las inhibitors include Las farnesylation inhibitors such as the benzodiazepine peptide mimetics described by James et al., Science, 260, 1937 (1993), which has the following formula:

(In the formula, R is H or CH ₃ , and X is methionine, serine, leucine, or an ester or amide derivative thereof.)
Protein and non-protein chemotherapeutic agents can be conjugated to polypeptides by methods known in the art. Such methods include, for example, those described in Greenfield et al., Cancer Research, 50, 6600-6607 (1990) for doxorubicin conjugation, and Arnon et al., Adv. Exp. Med. Biol. , 303, 79-90 (1991) and Kiseleva et al., Mol. Biol. (USSR), 25, 508-514 (1991).

The invention further includes modified antibodies having the constant region of a human antibody as well as the hypervariable region of monoclonal antibody 225. These modified antibodies may optionally be conjugated to an effector molecule such as a cytotoxin agent. Variable regions other than hypervariable regions can also be derived from the variable regions of human antibodies. Such antibodies are called humanized. Methods for making humanized antibodies are known in the art. The method is described, for example, in US Pat. No. 5,225,539 by Winter.
The most complete method for humanizing the 225 antibody is CDR grafting. As described in Example IV, the complement determining region or CDR of a mouse antibody that is directly involved in binding to the antigen is grafted into the human variable region, creating a “reshaped human” variable region. These fully humanized variable regions are then joined to the human invariant region, creating a complete antibody that is “all humanized”. In order to create fully humanized antibodies that bind well to antigens, it is necessary to carefully design reshaped human variable regions. The human variable region into which the 225 antibody CDRs are grafted must be carefully selected and usually requires the creation of several amino acid changes at key positions within the framework region (FR) of the human variable region. is there.
The designed reshaped human H225 variable region contains no more than 1 amino acid change in the FR of the selected human kappa light chain variable region and 12 amino acid changes in the FR of the selected human heavy chain variable region. The DNA sequences encoding these reshaped human H225 heavy chain and kappa light chain variable region genes are linked to the DNA sequences encoding the human γ1 and human κ constant region genes, respectively. This reshaped human H225 antibody is then expressed in mammalian cells and tested for binding to the human EGF receptor expressed on the surface of A431 cells compared to the murine M225 antibody as well as the chimeric C225 antibody.

Variable regions outside the hypervariable region of the antibody are also derived from monoclonal antibody 225. In that case, the entire variable region is derived from mouse monoclonal antibody 225, which is referred to as the chimerized antibody, or C225. Methods for making chimerized antibodies are known in the art. Such methods include, for example, those described in the US patents of Boss (Celltech) and Cabilly (Genentech). See U.S. Pat. Nos. 4,816,397 and 4,816,567, respectively.
The invariant part of these modified antibodies can be of any human class, ie IgG, IgA, IgM, IgD, and IgE. Subclasses of the above classes such as IgG1, IgG2, IgG3, and IgG4 are all suitable, of which IgG1 is preferred.

  Any of the effector molecules listed above for conjugation to a polypeptide can also be conjugated to the chimerized or humanized antibody of the present invention. Doxorubicin, taxol and cisplatin are preferred.

The polypeptides and antibodies of the present invention significantly inhibit tumor cell growth when effective amounts are administered to humans. The optimal dose can be determined by a physician taking into account many parameters, such as age, sex, weight, degree of symptom to be treated, active ingredient to be administered, and method of administration. In general, serum concentrations of polypeptides and antibodies capable of saturating the EGF receptor are desirable. A concentration of about 0.1 nM or more is usually sufficient. For example, a C225 dose of 100 mg / m ² results in a serum concentration of about 20 nM for about 8 days.
As a rough guideline, an antibody dose of 10-300 mg / m ² is given weekly. Corresponding doses of antibody fragments must be used at more frequent intervals to maintain serum levels above the concentration that saturates the EGF receptor.
Suitable routes for administration are intravenous, subcutaneous and intramuscular administration. Intravenous administration is preferred. The peptides and antibodies of the invention can be administered with additional pharmaceutically acceptable ingredients. Such components include, for example, immune system stimulants and chemotherapeutic agents as described above.

  The chimerized and humanized antibodies of the present invention are surprisingly different from the mouse 225 antibody in the absence of other antitumor agents including chemotherapeutic agents such as cisplatin, doxorubicin, taxol and their derivatives. Even so, it has been found that tumor growth in humans is significantly suppressed. Significant suppression means at least 20%, preferably 30%, and more preferably 50% tumor regression. In the best case, 90% and as much as 100% regression of the tumor is achieved. Alternatively, significant suppression may mean RI of 0.3 or higher, preferably 0.4 or higher, and more preferably 0.5 or higher.

A marked suppression of tumor growth and / or an increase in RI appears in many ways. For example, extending lifespan and / or stabilizing tumor growth that has been severely advanced.
In cases where the side effects are too severe for a patient to continue treatment with a chemotherapeutic agent, C225 can be replaced with a chemotherapeutic agent and comparable results can be obtained.
For example, the results shown in Example III-1 show that although the in vitro inhibitory properties of 225 and C225 are comparable, the in vivo effects of these antibodies are significantly different. The antibody isotype does not play an important role in the difference seen between 225 and C225 (eg, mouse IgG1 vs. human IgG1). Recent reports report that neither 225 nor C225 elicit complement-mediated elution and that the ADCC reactivity of these antibodies appears to be species specific. Naramura et al., Immunol. Immunother. 37, 343-349 (1993). Thus, if suppression of A431 xenografts was mediated by an immune response, 225 should have acted more as an antibody because of its ability to activate mouse effector cells involved in ADCC. But that is not the case.

  In addition, there is a difference in response in individual animals from the same group to treatment with 225 or C225. C225 alone is very effective at inducing complete tumor regression at the 1 mg dose, whereas this dose level of 225 shows very little effect. In experiments 2 and 3 of Example III-1, about 40% of the animals had no tumor at the end of each study. The animals that responded in these groups were usually small tumors at the beginning of the treatment protocol, but again the initial tumor burden has been shown to play a role in the biological effects of C225. Of note, animals treated with either 225 or C225 showed greater survival characteristics in all studies compared to the PBS control group.

  As shown in Example III-2, prostate cancer is also a suitable target for mediating anti-EGFR immunotherapy with C225. Since metastatic prostate cancer cells express both TGF-α and EGFR, terminal prostate cancer is a particularly suitable target.

Example III-2 illustrates the biological effects of C225 on the activation of EGFR in biohuman prostate cancer cells and the growth of prostate xenografts in nude mice. The in vitro experiment was designed to be able to determine the expression level of EGFR against 3 human prostate cancer cell lines, as well as the effect of C225 blocking the functional activation of the receptor. FIG. 7 shows the results of FACS analysis comparing EGFR expression levels in A431 cells found in LNCaP (androgen dependent) and PC-3 and DU145 (androgen independent cells). Both PC-3 (MFI = 135) and DU-145 (MFI = 124) cells had about 7-fold less receptor expression than A431 cells (MFI = 715). Since MFI is an indirect indicator of antigen density, it is considered that both PC-3 and DU145 cells expressed about 10 ⁵ receptors, respectively. LNCaP cells, on the other hand, had very low surface receptor expression (MFI = 12).

As described above, EGFR expressed by A431 cells is stimulated by extracellularly added ligand (EGF), whereas C225 can eliminate receptor activation. FIG. 8 shows a similar study result in the prostate system. Addition of EGF to LNCaP, PC-3 and DU145 induced EGFR phosphorylation, which was blocked very effectively by C225. These data indicate that C225 effectively represses the EGFR signaling pathway activated by the ligand and has antitumor activity against EGFR activation required for growth in vivo.
The ability of C225 to suppress tumor growth in vivo was tested on DU145 xenografts established in athymic nude mice. DU145 cells were inoculated with 10 ⁶ cells per animal in combination with Matrigel. 100% of the animals developed tumors within 20 days. Preliminary experiments have been shown to induce significant tumor suppression at a dose level of 1 mg (10 ×). In these studies, C225 was injected at a 0.5 mg (10 ×) dose level.

  As shown in FIG. 9, only C225 was effective in significantly suppressing the growth of established DU145 xenografts (p <0.5). The overall therapeutic effect was evident by day 34 and became significant by day 36 compared to the control group (FIG. 9A). All tumors in the mimic injection group grew throughout the study period (FIG. 9B), but the anti-tumor effect of the antibody was observed throughout the study period (FIG. 9C). Spontaneous remission in the PBS-treated animals was not seen in this model at all, but 60% of the C225-treated animals had no tumor until day 60 (FIG. 10A), and there was no tumor even 90 days after the end of antibody infusion. Continued. In addition, tumors that did not disappear in the C225 group grew very slowly after treatment cessation (day 55; FIG. 9C), suggesting a long-lasting effect of this antibody. There was no significant difference in survival curves during the treatment period (Figure 10B).

Example III-2 clearly shows that C225 is capable of inhibiting the growth of established EGFR-positive DU145 xenografts and can induce long-term tumor regression in treated animals at a high rate . These results were unpredictable with in vitro data.
Not all cell lines that express EGFR at levels similar to DU145 cells responded to C225 in vivo. For example, KB cells (human epidermoid carcinoma) expressed about 2 × 10 ⁵ EGFR per cell, and receptor activation by EGF was blocked in vitro by C225. However, KB xenografts did not respond to treatment with a 1 mg dose (x10) of C225, a level that could induce complete remission in 100% of the animals with established A431 tumors. Surprisingly, as shown in Example III-2, 0.5 mg dose (x10) C225 monotherapy on mice inoculated with DU145 tumor cells resulted in significant tumor regression in all treated animals. It was. 60% of the mice showed complete remission after the end of treatment. Blocking receptor activation by C225 may also have clinical effects in the treatment of metastatic prostate cancer in humans, particularly in the late stages of cancer.

Example I Materials
Example I-1 Cell Line and Medium A431 cells were routinely used in a 1: 1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and antibiotics. Allowed to grow.
Androgen-independent and dependent human prostate cancer cell lines (DU145, PC-3 and LNaP) were obtained from ATCC (Rockville, MD) and 10% fetal calf serum (Intergen, Purchase, NY) and Maintained as usual in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine (Sigma). Cells were checked regularly to confirm the presence of mycoplasma.

Example I-2 Preparation and purification of M225 and C225 The 225 antibody was grown as ascites in pristane-primed Balb / c mice. Ascites fluid was purified by HPLC (ABX and protein G) and judged to have a purity of> 95% by SDS PAGE.
Human clinical grade C225 was grown in 300 liter lots in a proprietary medium without serum. After separation, the concentrated broth was purified through a series of chromatography columns and packed into small bottles under aseptic conditions. The purity was determined to be> 99% by SDS PAGE.

Example 1-3 Preparation of Doxorubicin-C225 Conjugate C225 doxorubicin conjugate (C225-DOX) was prepared using a modification of the method described by Greenfield et al., Cancer Research, 50, 6600-6607 (1990). did. That is, doxorubicin is reacted with a cross-linking agent PDPH (3- [2-pyridylthio] propionylhydrazide) (Pierrece Chemical Co.) to produce doxorubicin 13- [3- (2-pyridyldithiol) propionyl] hydrazone hydro An acyl hydrazone derivative of chloride was formed. C225 was thiolated with N-succinimidyl 3- (pyridyldithio) propionate reagent and reacted with doxorubicin hydrazone to form conjugates containing hydrazide as well as disulfide bonds. The complex was purified by gel filtration at neutral pH. The C225-doxorubicin conjugate was stable at neutral to alkaline pH (pH 7-8) and stored at 4 ° C. This conjugate was easily hydrolyzed at pH 6 to release active doxorubicin.

Example I-4 Chimerization of Antibody 225
Example I-4A Cloning of H and L chain cDNAs The medium containing the 225 mouse hybridoma cell line was increased to 1 liter in a tissue culture flask. Elute a solution of total cellular RNA washed with guanidine isothiocyanate containing 2-mercaptoethanol, shear the solution with a dounce homogenizer to degrade cellular DNA, and place the preparation on 10 mL of cesium chloride buffer. Prepared by spreading. After centrifugation at 24,000 rpm for 16 hours, the pellet was resuspended in Tris-EDTA (TE) buffer and precipitated with ethanol. The poly A (+) mRNA fraction was isolated by binding to and eluting oligo dT cellulose. A cDNA library was prepared using poly A (+) mRNA as a template and oligo dT as a primer. The second strand was synthesized by nick translation using RNase H and DNA polymerase I. Double stranded DNA was passed through a 2 mL Sepharose G75 column to remove oligo dT and trace contaminants. The purified DNA was ligated to a polylinker of the following sequence:

5′-AATTTCCGAGTCTAGA-3 ′ (SEQ ID NO: 31)
However, this sequence encodes an Eco RI 4-base sticky end for ligation to a cloning vector and Xho I and Xba I restriction sites for subsequent manipulation of cDNAs. The ligated cDNA was then size selected by electrophoresis on a 5% polyacrylamide gel. Appropriately sized fractions (˜1500 bp for heavy chain and ˜900 bp for light chain cDNA) were electrically eluted from the gel slice and ligated to Eco RI digested lambda gt10 phage DNA. The library was created by packaging the ligation product in vitro and spreading the recombinant phage onto the E. coli strain C600 HFL strain. Phages containing H and L cDNAs were hybridized with mouse kappa and gamma invariant radiolabeled oligonucleotides and identified by phage filter lift. The identified phage was restriction mapped.

Isolates with the longest cDNA inserts were cloned into plasmid vectors (Eco RI-Bam HI fragment for the heavy (H) chain variable (V) region and Eco RI-Hpa for the light (L) chain variable (V) region. I fragment) and DNA was sequenced. This subcloned fragment contains a complete V region and a small portion of the associated mouse invariant (C) region. A total of 8 light chain cDNAs were sequenced, representing 4 different mRNAs. Three full-length heavy chain cDNAs encoding the same V region and the correct gamma 1C region were sequenced. Three other isolates containing the gamma 2a sequence were also identified but were not examined further. In order to identify the correct L chain cDNA, a sample of mouse 225 antibody was first sequenced by automated Edman degradation after first separating the H and L chains by SDS reducing gel electrophoresis and blotting to a membrane.
The sequence obtained for the light chain was compatible with one of the cDNAs. This isolate was rearranged to J5 and was found to be 91% homologous to VkT2. The H chain V region was found to be 96% homologous to VH 101 subgroup VII-1.

Example I-4B Adaptation of cDNA and Construction of Expression Vector V regions are synthetic DNA duplexes that encode the sequence between the unique restriction site closest to the V / C junction and the boundary of the V region itself. To make it suitable for expression. This was ligated with a second short intron sequence which restored the functional splice donor site for the V region after ligation. The end of the L chain intron is the Bam HI site and the end of the H chain intron is the Hind III site. The compatible V region was then isolated as an Xba I-Bam HI fragment (the Xba I site was in the original linker used for cDNA cloning), while the compatible H chain V region was isolated as a Xho I-Hind III fragment. Isolated.
The expression vector pdHL2 containing human kappa and human gamma 1 constant region was used for insertion of the compatible light chain V region. The resulting plasmid pdHL2-Vk (225) was then digested with Xba I and Bam HI and used to insert a compatible L chain V region. The resulting plasmid pdHL2-Vk (225) was then digested with Xho I and Hind III and used to insert a compatible heavy chain V region. The final vector was identified as pdHL2-ch225 by restriction mapping.

Example I-4C Expression of Chimera 225 into Transfected Hybridoma Cells The pdHL2-ch225 plasmid was introduced into hybridoma Sp2 / 0 Ag14 cells by protoplast fusion. Bacteria parasitizing the plasmid were grown to an optimal density of 0.5 at 600 nm, at which point chloramphenicol was added to stop growth and amplify the number of plasmid replications. The next day, the bacteria were treated with lysozyme, the cell walls were removed, and the resulting protoplasts were fused to hybridoma cells with polyethylene glycol 1500. After fusion, the cells were grown in antibody to kill viable bacteria and spread on 96-well plates. Selection medium (containing 0.1 μM methotrexate (MTX)) was added 24-48 hours later, and only transfected cells were exploited using the expression of the marker gene (dehydrofolate reductase) present on the expression plasmid. Was grown.
Two weeks later, several MTX resistant clones were obtained and tested for expression of these antibodies. Culture supernatant was added to wells coated with anti-human Ig (Fc specific) antibody as capture reagent. The detection system was an HRP-conjugated goat anti-human kappa antibody. Although most of the clones were found to secrete human antibody determinants, the three with the highest production capacity were made suitable for growth with an additional 1 μM and then 5 μM methotrexate. Two of these lines, designated SdER6 and SdER14, continued to grow well at high levels of MTX and were subcloned by limiting dilution. Subclone productivity was tested by inoculating 2 × 10 ⁵ cells per mL in growth medium and measuring accumulated antibody on day 7. The two lines with the highest production capacity from the first subcloning were SdER6.25 and SdER14.10. These were subcloned again and the final three candidate lines were named SdER6.25.8, SdER6.2.49, and SdER14.10.1. Clone SdER6.25.8 was selected on the basis of antibody expression.

Example I-5 Analysis of C225 Expressed from SdER6.25.8 The antibody produced from clone SdER6.25.8 was examined to characterize the nature of the antibody. Culture supernatants from transfected cell clones expressing C225 antibody were tested for their ability to bind to human tumor cells expressing different levels of EGF receptor. A431 epithelial cancer cells (highly expressed) were strongly stained, while M24 melanoma cells (expressing 1/10 less receptor) were moderately stained. Neuroblastoma IMR-32, which does not express the EGF receptor, was not stained.

Example I-6 Effect of C225 Antibody Chimerization Apparent Kd was determined by ELISA and SPR methods to be 0.1 and 0.201 nM for C225 and 1.17 and 0.808 nM for 225, respectively (Table 1). These results were comparable to the previously published C225 data (Kd = 0.39 nM) and 225 data (Kd = 0.79 nM, Kd = 1 nM) shown in Table 1. These antibodies were found to inhibit the growth of cultured A431 cells to the same extent (Table 2). In addition, 225 and C225 had the ability to block EGF-induced phosphorylation of EGFR in A431 cells. These results indicate that 225 chimerization did not affect the biological properties of the antibody and increased the relative binding affinity of C225 to EGFR.

Example II Methods and Assays
Example II-1 Measurement of Relative Affinity by ELISA The relative binding affinity of an antibody has already been determined by Lokker et al. Immunol. 146, 893-898 (1991). That is, A431 cells (10 ⁴ or 10 ⁵ per well) are grown overnight at 37 ° C. in 96-well microtiter plates. Cells are fixed with 3.7% neutral buffered formalin for 10 minutes at room temperature. After washing 3 times with PBS, the wells are blocked with 1% fetal bovine serum in Hank's balanced salt solution for 2 hours at room temperature. C225 or 225 is added to the wells at various concentrations (continuous dilution starting from 50 nM). After 2 hours incubation at 37 ° C, the plates are washed thoroughly with PBS and incubated for 1 hour at 37 ° C with goat anti-human antibody (Sigma, St. Louis, MO; 1: 1000). Plates are washed and Chromogen TMB (Kirkegaard and Perry, Gaitherberg, MD) is added for 30 minutes in the dark. The color reaction is stopped with 1N sulfuric acid and the plate is read at 450 nm with an ELISA reader. Relative binding affinity was taken as the concentration that gave 1/2 maximum OD.

Example II-2 Apparent binding affinities of 225 and C225 affinity constants M225 and C225 using surface plasmon resonance (SPR) were also measured using InAcore ^™ (PharmaciaBiosensor, Pharmacia Biosensor, Piscataway, NJ; The user's precautions 301 and O'Shannessy et al., Anal. Biochem., 212, 457-468 (1993)). That is, the soluble recombinant EGFR is immobilized on the sensor chip through the amino group as described by the manufacturer. Real-time binding parameters for 225 and C225 EGFR were established at various antibody concentrations and the apparent Kd was calculated from the binding rate constants obtained by non-linear fitting using Biavaluation ^™ 2.0 software.

Example II-3 In Vitro Inhibition of Cell Growth by 225 and C225 In Vitro 225 and C225 were applied to 96-well microtiter plates using A431 cells (300-500 / well) in complete growth medium. Measured. After adding various concentrations of C225 or 225 (4 replicates / 1 concentration), the plates were incubated for 48 hours at 37 ° C. and pulsed with 3H-thymidine for 24 hours. Cells were harvested, collected on a filter mat and counted with a Wallace Microbeta scintillation counter to determine percent inhibition. The percent inhibition was defined as the degree of reduction when comparing 3H thymidine uptake of antibody treated cells with 3H thymidine uptake of cells grown in the absence of antibody.

Example II-4 Experimental Animal Study Athymic nude mice (nu / nu; 6-8 weeks old, female) were obtained from Charles River Laboratories. The right flank of animals (10 per treatment group) was inoculated with 10 ⁷ A431 cells added to 0.5 mL Hanks balanced salt solution. Observations were made until the tumor was visible (approximately 7-12 days) and the mean volume reached 150-300 mm ³ . At that time, antibody therapy was started. Treatment was given intraperitoneal injections twice a week (at various concentrations in 0.5 mL PBS) for 5 weeks. U1 animals were injected with PBS. Tumors were measured twice a week and their volume was calculated using the following formula: π / 6 × large diameter × (small diameter) ² . At the time of the last antibody treatment (8 weeks after the start of treatment), U1 and extremely large tumor test animals were euthanized, and the animals were subsequently observed for at least 3 weeks. For tumor free and small tumor animals continued for an additional 2-3 months. Statistical analysis of tumor growth in each study was performed using Student's two-tailed T-test. In addition to showing tumor growth-inhibitory effects, the antibody was in complete remission (ie, no tumor) in many animals. This biological effect was expressed quantitatively as the remission index RI, ie the number of mice without tumor / total number of animals in the treatment group. The study was terminated at the time of euthanasia for animals with large tumors and 2-3 months later for other animals. Animals that died during treatment were excluded from the analysis. For example, when there is one complete remission among 8 surviving animals, RI = 0.125.

Example III Biological Activity of C225
Example III-1 Antibody Ability to Suppress Growth of A431 Xenografts in Nude Mice Animals were inoculated with A431 cells on the flank. 150-300 mm ³ tumors appeared by 7-10 days. (See Experiments 1-4 in Table 3) The experimental animals were then randomized and injected with PBS or 225 (Experiment 1), PBS, 225, or C225 (Experiment 2); and PBS or C225 (Experiment). 3 and 4) were injected. In Experiment 1-3, experimental animals were administered 1 mg of antibody (in 0.5 mL PBS) twice a week for 5 weeks and the total antibody dose per animal was 10 mg. In Experiment 4, experimental animals were administered one of three doses of 1, 0.5, and 0.25 mg / injection for a total dose of 10, 5 and 2.5 mg, respectively. Tumors were measured twice a week over the entire treatment period. For tumor free and small tumor animals, large tumor animals were continuously monitored for 2-3 months after sacrifice.
FIG. 1 shows the effect of 225 on the growth of A431 tumors in nude mice (Experiment 1). Although the average tumor volume in the experimental group and the U1 group was comparable (FIG. 1A), only one complete tumor remission was observed (remission index (RI) 0.17; FIG. 1B and Table 3). A comparison between 225 and C225 is shown in FIG. 2 (Experiment 2 in Table 3). There was no significant difference in mean tumor size between groups, but animals treated with C225 had a RI of 0.44 (ie, 4/9 complete remission), whereas in the case of 225 the RI was 0. 11 (FIG. 2B and Table 3). The apparent tumor regression on day 37 in the PBS U1 group (FIG. 2A) is due to the death of 3/10 animals at this time and the concomitant reduction in total tumor volume. A similar RI for C225 was found in Experiment 3 (FIG. 3B; RI = 0.4). In addition, inhibition of tumor growth by C225 was also significant when compared to the growth of xenografts in PBS treated mice (FIG. 3A; p <0.02 after day 32).

Since many animals receiving C225 showed tumor regression at the 1 mg / single injection level, the lowest biologically effective dose was determined. FIG. 4 shows the results of a dose response experiment (Experiment 4). All animals receiving 1 mg / injection were in complete remission and became tumor-free for over 100 days after the end of antibody injection (FIGS. 4A and B; Table 3). These results were very remarkable, with the p value changing from p <0.006 on the 33rd day to p <0.0139 on the 59th day. C225 showed significant tumor regression in experiment 3 (FIG. 3), but in experiments 2 and 3, about 40% of the animals that received the 1 mg dose of C225 were in complete remission. The effect of the 1 mg dose in Experiments 3 and 4 significantly reduced the mean tumor volume compared to U1 due to the use of small tumor mice at the beginning of the treatment protocol for these experiments ( 152 mm ³ [Experiment 4] and 185 mm ³ [Experiment 3] vs. 267 mm ³ [Experiment 2]). These data suggest that the clinical effect of C225 may be related to tumor burden.

At the 0.5 mg dose in Experiment 4, the overall suppression of tumor growth was not statistically significant as the tumor volume in both animals in the PBS and 0.5 mg groups varied greatly. However, the RI was high for the 0.5 mg group (RI = 0.63; FIG. 4B and Table 2), indicating that the antibody induced a high tumor response in individual experimental animals. Interestingly, the 0.5 mg dose group of experiment 4 had a higher RI than the 1 mg dose group of experiment 3. This result could probably be attributed to the effect of tumor burden. The average volume at the start of the tumor in the 0.5 mg dose group was 160 mm ³ , but there was a large variation in tumor size in individual animals. Many experimental animals with small tumors (<100 mm ³ ) were most sensitive to the biological effects of C225. At the 0.25 mg dose, average tumor growth appeared to be greater than PBS U1. This was due to the inclusion of two animals with large tumors (760 and 1040 mm ³ ) at the start of treatment, which increased the mean tumor volume over the course of experiment 4. Overall, there were no significant differences between these groups, but it was noted that one of the 0.25 mg dose groups (1/8) had no tumor at the end of the study ( RI = 0.13). On day 47, a decrease in RI was observed. At this point, the tumor recurred in one mouse that was clearly in complete remission. In this only case, C225 only showed a transient biological effect. This animal was not included in Table 3. Similar to the 1 mg dose group, tumor-free animals in the 0.5 and 0.25 mg groups remained tumor-free for a minimum of 2-3 months after sacrifice of the PBS control mice.

The results shown in Table 2 show typical experiments that tested the ability of 225 and C225 to inhibit A431 growth in vitro. Details are as described above. Percent inhibition is defined as the decrease in uptake of antibody treated samples (4 replications / concentration) relative to the uptake of 3-H thymidine in cell growth in the absence of antibody.
Table 3 compares complete tumor regression after treatment with PBS, 225, or C225 twice weekly for 5 weeks in athymic nude mice with established A431 tumors. Experimental animals other than Experiment 4 were treated by intraperitoneal route with 1 mg of antibody in 0.5 mL PBS, whereas Experiment 4 was a dose response experiment in which mice were injected with 1, 0.5, or 0.25 mg / time. It is. This table shows the RI at the time of euthanizing experimental animals with a large tumor (PBS control test group). All animals that showed complete remission or small tumors continued treatment for 2-3 months. The difference in the total number of experimental animals is due to the death of mice in these treatment groups in this experiment.

Example III-2 Growth inhibition of human prostate cancer xenografts established in nude mice
Example III-2A FACS analysis of C225 binding to DU145, PC-3 and LNCaP The relative expression level of the EGF receptor on DU145, PC-3 and LNCaP cells was determined by FACS analysis. Cells are grown to near confluency in complete medium, removed from the flask using non-enzymatic separation buffer (Sigma), and 100 ul cold H-BSA (Hanks balanced salt solution containing 1% BSA) ) 5-10 × 10 ⁵ per test tube. 10 μg C225 or irrelevant myeloma-derived human IgG1 (Tago, Burlingame, Calif.) Was added to the test tube and incubated for 60 minutes on ice. After washing with cold H-BSA, goat anti-human IgG conjugated to FITC (Tago, Burlingame, Calif.) Was added and kept on ice for an additional 30 minutes. Cells were washed twice with cold H-BSA, resuspended in 1 mL H-BSA, and analyzed using a Coulter Epix Elite cell sorter (Coulter, Hialeah, Fla.). Basal fluorescence intensity was determined using only FITC labeled secondary antibody and non-specific fluorescence was defined by an irrelevant isotype control. Data were expressed as mean fluorescence intensity MFI, which is an indirect measure of antigen density. MFI is the average channel fluorescence multiplied by the percentage of positive cells in each sample.

Example III-2B Phosphorylation assay of PC-3, DU145 and LNCaP cells Phosphorylation assay was performed on PC-3, DU145 and LNCaP cells, and the EGF receptor expressed by these cells is functional It was determined whether it was suppressed by C225. This assay and Western blot analysis were performed as described by Gill et al., Nature, 293, 305-307 (1981). That is, DU145, PC-3 and LNCaP cells were grown to 90% confluency in complete media and then nutrient deficient in DMEM-0.5% bovine serum 24 hours prior to the experiment. Cells were stimulated with EGF for 15 minutes at room temperature in the presence or absence of C225. The monolayer was then washed with ice-cold PBS containing 1 mM sodium vanadate. Cells were eluted and subjected to SDS PAGE followed by Western blot analysis. The pattern of phosphorylation was determined by probing the blot with a monoclonal antibody against phosphotyrosine (UBI, Lake Placid, NY) and then detecting using the ECL method (Amersham).

Example III-2C Animal Experiments Athymic nude mice (nu / nu; 6-8 weeks old, male; 0.2 mL of Matrigel on the right flank of Charles River Laboratories, Wilmington, Mass.) Inoculated with 10 ⁶ DU 145 added to mixed 0.2 mL Hanks balanced salt solution Mice were visible until tumors were visible (approximately 14-20 days after administration) and average volume reached 100 mm ^3. The animals were weighed and randomly divided into treatment groups (10 per group), and antibody therapy was started with intraperitoneal injections of 0.5 mg C225 twice weekly for 5 weeks. The animals were injected with PBS.In a preliminary study, there was no significant increase in DU145 xenograft growth between animals treated with polyclonal DU145-absorbed human IgG and those treated with PBS. Measured such difference is it is confirmed not tumor twice a week, its volume was calculated using the equation of the following Symbol:.. Π / 6 × diameter × (diameter) ² final antibody injection (after the start of treatment At 8 weeks), the control animals were euthanized, and the animals were then observed for at least 3 weeks, with an additional 2-3 months for tumor-free animals and animals with small tumors. Statistical analysis of growth was determined by Student's two-tailed T-test using a computer program from Sigma Must (Jandel, San Rafael, Calif.) If the p-value is <0.05, Considered significant.

Example III-3 Biological Activity of Peptides Containing 225 CDR Regions This example demonstrates that peptides constructed using the 225-CDR sequence have biological activity against cell lines expressing the EGF receptor. Is. Six series of peptides were made with the following sequence:
Heavy chain CDR-1 NYGVH
CDR-2 GVIWSGGNTDYNTPFTSR
CDR-3 RALTYYDYEFAYW (SEQ ID NO: 32)
Light chain CDR-1 RASQSIGNIH (SEQ ID NO: 33)
CDR-2 YASESIS (SEQ ID NO: 34)
CDR-3 QQNNWP (SEQ ID NO: 35)

These peptides were dissolved in PBS at a concentration of 1 mg / mL. A431 cells were placed in a 96-well plate at 1,000 cells per well. Peptides were added at various concentrations. Chimeric C225 antibody and an irrelevant isotype matched immunoglobulin were used as positive and negative U1, respectively. Plates were incubated for 72 hours at 37 ° C. and pulsed with ³ H-thymidine overnight. Cells were collected and counted with a liquid scintillation counter. Percent inhibition was defined as the reduction in 3-H thymidine incorporation of antibody or peptide treated cells compared to 3-H thymidine incorporation of cells grown without the presence of antibody or peptide.

  As is apparent from FIG. 5, A431 cells were suppressed by C225, and heavy chain CDR-1 and heavy chain CDR-2 of monoclonal antibody 225. In contrast, isotype-matched irrelevant antibodies and U1 peptides did not suppress A431 cells. These results indicate that heavy chain CDR-1 and -2 can suppress the growth of A431 cells by interfering with ligand binding to EGFR.

Example III-4 Biological activity of C225- doxorubicin conjugate (C225-DOX) In vitro evaluation was performed using MEL-28. EGF receptor expression was verified by FACS analysis using C225 and C225-DOX conjugates. The assay was performed using a detected value of [ ³ H] -thymidine and WST-1 with a 72 hour incubation period. In all assays of EGFRc expressing cell lines, namely A431, KB and MDA-468 cells, C225-DOX showed a high suppression of cell proliferation compared to no treatment or hIgG1 U1 class treatment. Comparison of doxorubicin alone or a mixture of C225 and doxorubicin with equimolar concentrations of C225-DOX showed 4-5 times higher inhibition when using the C225-DOX conjugate. Inhibition of proliferation by C225-DOX was also seen in high dose EGFRc non-expressing cell lines. C225-DOX suppression in the EGFRc negative cell line was 5-15 times lower than that of the EGFRc-positive cell line, comparable to the suppression seen at equimolar concentrations of doxorubicin alone. Representative results for the activity of C225-DOX on 431 cells are shown in Table 6.

Example IV Humanization of M225
Example IV-1 Modified Eagle Medium (DMEM) Abbreviated Dulbecco; Fetal Bovine Serum (FCS); Ribonucleic Acid (RNA); Messenger RNA (mRNA); Deoxyribonucleic Acid (DNA); Double-Stranded DNA (ds-DNA); Polymerase chain reaction (PCR); enzyme-linked immunosorbent assay (ELISA); hours (hr); minutes (min); seconds (sec); human cytomegalovirus (HCMV); polyadenylation (poly (A) ⁺ ); Globulin (IgG); monoclonal antibody (mAb); complementarity determining region (CDR); framework region (FR); tris-borate buffer (TBE); bovine serum albumin (BSA); phosphate buffered saline ( PBS): room temperature (RT); nanometer (nm); epidermal growth factor receptor (EGFR).

Example IV-2 Material Medium Composition and all other tissue culture materials were purchased from JRH Biosciences [JRH
Except for FCS purchased from Biosciences (USA), it was obtained from Life Technologies (UK). RNA isolation kits were obtained from Stratgene (USA) and first strand cDNA synthesis kits were purchased from Pharmacia (UK). All components and equipment for the PCR reaction were purchased from Perkin Elmer (USA), including AmpliTaq ^™ DNA polymerase. The TA Cloning ^™ kit was obtained from Invitrogen (USA) and the Sequenase ^™ DNA sequence kit was purchased from Amarsham International (UK). Agarose (UltraPure ^™ ) was obtained from Life Technologies (UK). Wizard ^™ PCR Preps DNA purification kit, Magic ^™ DNA cleanup system and XL1 blue competent cells were purchased from Promega (USA). All other molecular biological products were purchased from New England Biolabs (USA). Nunc-Immuno Plate MaxiSorp ^™ immunoplates were obtained from Life Technologies (UK). Both Fcγ fragment specific goat anti-human IgG antibody and goat anti-human IgG (H + L) / horseradish peroxidase conjugate were purchased from Jackson ImmunoResearch Laboratories Inc. (USA). TMB substrate A and substrate B were obtained from Kirkegaard-Pery (USA). All other products for ELISA were obtained from Sigma (UK). Microplate Manager ^™ data analysis software package was purchased from Bio-Rad (UK). The molecular modeling package QUANTA was obtained from Polygen Corporation (USA) and the IRIS 4D workstation was purchased from Silicon Graphics (USA).

Example IV-3 PCR cloning and sequencing of mouse variable region genes Mice using DMEM supplemented with 10% (v / v) FCS, 50 units / mL penicillin, 50 μg / mL streptomycin and 580 μg / mL L-glutamine The M225 hybridoma cell line was grown in suspension. Approximately 10 ⁸ viable cells were harvested while supernatant from hybridoma cells was assayed by ELISA to confirm that they produced mouse antibodies. Total RNA was isolated from 10 ⁸ cells using an RNA isolation kit according to the manufacturer's instructions. This kit utilizes a guanidine thiocyanate phenol-chloroform one-step extraction method as described in Chomczynski and Sacchi (6). A single strand copy of M225 hybridoma mRNA was made using the NotI- (dT) ₁₈ primer supplied with the kit, also using the first strand cDNA synthesis kit as directed by the manufacturer. Approximately 5 μg of total RNA was used in a final reaction volume of 33 μl. Then, before the reaction completion mixture was ice-cooled, the RNA-cDNA duplex was denatured by heating to 90 ° C. for 5 minutes to inactivate the reverse transcriptase.
In order to PCR amplify the mouse variable region gene, the method described in Jones and Bendig (7) was followed. Basically, to PCR clone the mouse variable region gene of M225 antibody, two series of degenerate primers, ie one series annealed to the leader sequence of mouse kappa light chain gene (ie MKV1-11; Table 4) And those designed to anneal to the leader sequence of the mouse heavy chain gene (ie, MHV1-12; Table 5), and one series, the mouse kappa light chain constant region gene (MKC; Used together with primers designed to anneal to the 5 ′ end of Table 4) and the 5 ′ end of the mouse γ1 heavy chain constant region gene (MHCG1; Table 5), respectively. Separate reactions were prepared using MKV and MHV degenerate primers with the respective constant region primers. The PCR reaction tube was placed in a Perkin Elmer 480 DNA thermal cycler and subjected to a total of 25 cycles at 94 ° C for 1 minute, 50 ° C for 1 minute and 70 ° C for 1 minute (initial melting at 94 ° C for 1.5 minutes). rear). At the end of the last cycle, a final extension step was performed at 72 ° C. for 10 minutes before cooling the reaction to 4 ° C. Except during annealing (50 ° C.) and extension step (72 ° C.), a longer ramp time was 2.5 minutes, with a 30 second ramp time between each step of the cycle.

A 20 μl aliquot from each PCR reaction was run on an agarose gel to determine which produced the correct size PCR product. The PCR reaction that appeared to have amplified the full-length variable domain gene was repeated to produce independent PCR clones, thereby reducing the effects of PCR errors. A 6 μl aliquot of these PCR products of the correct size was cloned directly into the pCR ^™ II vector provided in the TA Cloning ^™ kit and transformed into INVαF ′ competent cells as described in the manufacturer's instructions. Colonies containing plasmids inserted at the correct size were treated with the pCR ^™ II forward and pCR ^™ II reverse oligonucleotide primers described in Table 6, based on the method of Gussow and Clackson (8). Used to identify colonies by PCR screening. The identified putative positive clones were finally double stranded plasmid DNA sequenced using the Sequenase ^™ DNA sequence kit based on the method of Redston and Kern (9).

Example IV-4 Construction of Chimeric Genes The cloned mouse leader variable region gene was modified at both the 5 ′ and 3 ′ ends with PCR primers, and immunoglobulin chains for correct RNA splicing of the variable and constant region genes. An expression vector for efficient eukaryotic translation of mRNA encoding each of (10) and the splice donor site, ie, a restriction enzyme site suitable for insertion into the Kozak sequence, was constructed. A HindIII site is added to the 5 ′ end of both mouse variable region genes, but another restriction site is added to the 3 ′ end of the mouse variable region gene. That is, a BamHI site is added to the 3 ′ end of the V _H gene and an XbaI site is added to the 3 ′ end of the V _K gene.
PCR reactions were based on the chimeric gene construction method of Kettleborough et al. (11) with primers C225V _H 5 ′ and C225V _H 3 ′ for heavy chains and C225V _K 5 ′ and C225V _K 3 for copper light chains. (Table 7). Following the initial 90 second melting step at 94 ° C., the mixture was PCR amplified in 25 cycles of 94 ° C. for 2 hours and 72 ° C. for 4 hours. This two-step PCR cycle is possible for a typical three-step cycle, but each primer is designed to anneal the template DNA to 24 bases, which allows a relatively high temperature of 72 ° C. This is because annealing can be performed at a high temperature. There was a 30 second ramp time between each step and the end of the last cycle, and the PCR reaction was completed with a final extension step at 72 ° C. for 10 minutes before cooling to 4 ° C. The PCR product is column purified using the Wizard ^™ PCR Preps DNA Purification Kit according to the manufacturer's instructions, digested with appropriate restriction enzymes such as plasmid pUC19, and run on a 1% agarose / TBE buffer (pH 8.8) gel. Separated. The heavy and kappa light chain variable region genes were excised from the agarose gel and purified using the wizard's PCR Preps DNA purification kit. pUC19 was also excised from the agarose gel and purified using a Magic ^™ DNA cleanup system according to the manufacturer's instructions. The heavy chain and kappa light chain variable region genes were then ligated separately to purified pUC19 to generate pUC-C225V _H and pUC-C225V _K plasmids, respectively, and transformed into XL1 blue competent cells. Positive colonies that appear to contain the appropriate plasmid were identified by PCR screening using oligonucleotide primers RSP and UP (Table 6), and finally ds-DNA was sequenced to confirm the introduction of sequence modifications, It was also confirmed that no undesirable changes occurred in the DNA sequence as a result of the PCR reaction.

PCR mutagenesis was utilized to modify the signal peptide sequence at the 5 ′ end of the kappa light chain variable region according to the protocol of Kettleborough et al. (11). PCR primers C225V _K 5′sp and C225V _K 3′sp (Table 7) were used for pUC-C225V _K template DNA, and a modified gene (C225V _K sp) was made using a modified two-step PCR amplification protocol. This PCR product was then column purified before digesting the purified PCR product and pUC-C225C * K with HindIII and PstI. This PCR fragment and plasmid DNA were then agarose gel purified, ligated and cloned together to create plasmid pUC-C225V _K sp. As described above, putative positive transformants were identified by PCR screening (using RSP and CP primers) and then ds-DNA sequencing was performed to confirm the presence of the modified signal peptide and the absence of PCR errors.
The matched mice kappa light chain and heavy chain leader variable region gene then, as HindIII-BamHI fragment in the case of a mouse V _H, and as HindIII-XbaI fragment in the case of a mouse V _K, chimeric light into mammalian cells It was inserted into a vector designed to express the heavy and heavy chains. These vectors contain an HCMV enhancer and promoter, and polyadenylate the immunoglobulin chain, MCS for insertion of immunoglobulin variable region genes, cDNA clones of appropriate human kappa light chain and heavy chain constant regions, and immunoglobulin chain mRNA. Synthetic poly (A ⁺ ) sequences to produce, artificial sequences designed to terminate transcription of immunoglobulin chains, genes for selecting transformed stable cell lines such as dhfr or neo, and transients into COS cells HCMV enhancer and promoter to drive the SV40 replication origin for efficient DNA replication. The human kappa light chain mammalian expression vector is called pKN100 (FIG. 11) and the human gamma 1 heavy chain mammalian expression vector is called pG1D105 (FIG. 12). For putative positive colonies, the primers HCMVi and New. PCR screening using Huk and the chimeric heavy chain vector using primers HCMVi and HuC gamma 1 (Table 6) and performing restriction analysis to confirm the presence of the correct insertion in the expression vector construct. It was. The new constructs containing the mouse variable region genes of the M225 antibody are called pKN100-C225C _K (or pKN100-C225V _K sp) and pG1D105-C225V _H respectively.

Example IV-5 Molecular modeling of the murine M225 antibody variable region As an aid to the design of the CDR grafting variable region of the H225 antibody, a murine M225 antibody variable region molecular model was created. Structural modeling of well-characterized protein families such as immunoglobulins is done using established methods of homologous modeling. This was done on an IRIS 4D workstation operating on the UNIX® operating system using the molecular modeling package QUANTA and Brookhaven's elucidated protein structure crystal database (12).
The FR of the M225 variable region is modeled on the basis of a similar structurally resolved immunoglobulin variable region FR. Identical amino acid side chains maintain their original orientation, but incompatible side chains are replaced using the maximum overlap method to preserve the chi angle of the original mouse M225 antibody. Most CDRs of the M225 variable region are modeled on the standard structure of a hypervariable loop corresponding to the structure level CDR. However, in the case of CDR3 or the like of the heavy chain variable region, the standard structure is unknown, so the CDR loop is modeled on the basis of a similar loop structure present in a structurally elucidated protein. Finally, the CHARMm potential (17) as implemented in QUANTA is used to minimize energy in order to eliminate unwanted atomic contacts and optimize van der Waals and electrostatic effects.

  The FR of the light chain variable region of the M225 antibody is modeled based on the FR from the Fab fragment (18) of the mouse monoclonal antibody HyHel-10. The FR of the heavy chain variable region is modeled based on the FR of the Fab fragment (19) of mouse monoclonal antibody D1.3. The side chains of different amino acids between the mouse M225 antibody and the modeled variable region are first substituted. Next, the light chain of the Fab HyHel-10 antibody is obtained by adapting residues 35-39, 43-47, 84-88 and 98-102 as defined by Kabat et al. (20). Overlay on 3 light chains. The aim is to have the two heterogeneous variable regions in the correct orientation with respect to each other, namely the HyHel-10 based copper light chain variable region and the D1.3 based heavy chain variable region.

The CDR1 (L1) of the light chain variable region of mAb M225 is the L1 standard except that isoleucine is present in place of the normal leucine at standard residue position 33, as suggested by Chothia et al. (14). Fits type group 2. However, this substitution is thought to be too conserved to gain significant benefits in assigning a standard loop structure to this hypervariable loop. The L1 loop of mouse Fab HyHel-10 is identical in amino acid length and fits the same standard group with leucine at position 33 as the L1 loop of M225 mAb.
This hypervariable loop is therefore used to model the L1 loop of the M225 kappa light chain variable region. Similarly, CDR2 (L2) and CDR3 (L3) of the M225 mAb are both compatible with their respective standard group 1 loop structures. In addition, the corresponding hypervariable loop structures of the HyHel-10 Fab fragment are both group 1. Therefore, the L2 and L3 loops of the M225 kappa light chain variable region are modeled on L2 and L3 of Fab HyHel-10.
Similarly, both the CDR1 (H1) and CDR2 (H2) hypervariable loops of the heavy chain variable region of mAb M225 fit into their respective standard group 1 loop structures as defined by Chosia et al. (14). Moreover, the corresponding H1 and H2 hypervariable loops of the mouse D1.3 Fab fragment are also compatible with the respective standard group 1 loop structure. Thus, as with the light chain, these hypervariable loops are modeled on the H1 and H2 loops of the heavy chain variable region of the base model. To identify the loop structure fit to the CDR3 (H3) hypervariable loop of the heavy chain variable region of M225, the Brookhaven database is searched for amino acid sequences similar to loops of the same length. By this analysis, the H3 loop of murine Fab26 / 9 (21) was very close to that of M225 mAb and was therefore used as the basis for the hypervariable loop of this murine M225 variable region model. In order to adjust the apparent steric collisions of the entire model, as is done with QUANTA, it eliminates unwanted atomic contact and ultimately energy to optimize van der Waals and electrostatics. Minimize.

Example IV-6 Design of Reshaped Human H225 Antibody Variants The first step in designing CDR grafting variable regions of H225 antibodies is the selection of the human light and heavy chain variable regions that serve as the basis for the humanized variable region. . To assist in this process, the consensus sequences of the four subgroups of the human kappa light chain variable region as well as the human heavy chain, as Kabat et al. (20) defined the light and heavy chain variable regions of the M225 antibody. The consensus sequences of the three subgroups of the variable part are compared. The murine M225 light chain variable region is generally 61.68% identical with the human kappa light chain subgroup I consensus sequence and 65.00% identity with FR alone, and also the subgroup III consensus sequence. Generally has 61.68% identity and FR alone has 68.75% identity, both of which are most similar. The mouse M225 heavy chain variable region is most similar to the human heavy chain subgroup II consensus sequence with 52.10% overall identity and 57.47% FR alone. This analysis is used to show which subgroups of human variable regions can be a good basis for human variable regions to serve as templates for CDR grafting, but this is a few of these artificially constructed Due to the diversity found in the individual sequences of the subgroups, it is not always applicable.
For these reasons, the murine M225 variable region was also compared with all published examples of individual sequences of human variable regions. With respect to the human antibody sequence, the mouse M225 light chain variable region is very similar to the sequence of the human kappa light chain variable region of human antibody LS7′CL (22) —but this is not related to the mouse L7′CL sequence. . The kappa light chain variable region of human LS7′CL is a member of subgroup III of the human kappa light chain variable region. The overall sequence identity between mouse M225 and human LS7′CL light chain variable region is calculated as 64.42% overall and 71.25% for FR alone. The mouse M225 heavy chain variable region is very similar to the human heavy chain variable region sequence of the human antibody 38P1′CL (23). Surprisingly, the heavy chain variable region of human 38P1′CL is a member of subgroup III but not subgroup II of the human heavy chain variable region. The overall sequence identity between mouse M225 and human 38P1′CL heavy chain variable region is 48.74%, and FR alone is calculated to be 58.62% identity. Based on these comparisons, the human LS7′CL light chain variable region was selected as the human FR donor template for the design of the reshaped human M225 light chain variable region, and the human 38P1′CL heavy chain variable region was reshaped. Selected as a human FR donor template for the design of the human M225 heavy chain variable region.
As is common practice, the human light and heavy chain variable regions selected for humanization of the M225 antibody are derived from two different human antibodies. Such a selection process can use the human variable region that exhibits the highest identity with the M225 variable region. Furthermore, there are many successful examples of CDR-grafted antibodies based on variable regions obtained from two different human antibodies. The most well-studied example is the reshaped human CAMPATH-1 antibody (24). Nevertheless, such strategies also require careful analysis of the packing residues between domains between the kappa light chain and the heavy chain variable region. Incorrect packing in this region can dramatically affect antigen binding, even if the CDR loop structure of the reshaped human antibody is identical. Thus, as specified by Chosia et al. (25), it is checked whether the amino acids present at the V _K / V _H interface are unusual or rare residues. If such a residue is identified, then mutagenesis must be considered that changes the particular residue at that consideration position to the normally found amino acid.

The second step in the design process is to insert the M225 CDRs into selected human light and heavy chain variable regions FR, as defined by Kabat et al. (20), to create a simple CDR graft. Mouse antibodies humanized by such simple CDR grafting usually show little or no binding power to the antigen. Therefore, to determine whether these amino acid residues are negatively affecting the binding power to the antigen either directly by acting with the antigen or indirectly by changing the position of the CDR loop In addition, it is important to examine the amino acid sequence of human FR.
The third step in this design process is to determine whether to change amino acids in human donor FRs to their corresponding murine M225 residues in order to obtain good binding capacity for antigen. This is a difficult and critical step in the humanization method and it is at this stage that the M225 variable part model is most useful in the design process. The following points are discussed regarding this model. It is very important that the standard structure (13-16) for the hypervariable loop is preserved. It is therefore crucial to conserve all mouse FR residues that are part of these canonical structures in the humanized H225 variable region. It is also useful to compare the sequence of the M225 antibody with similar sequences of other mouse antibodies to determine whether the amino acid sequence is unusual or rare. This is because in such a sequence, mouse residues play an important role in antigen-binding ability. By studying the model of the M225 variable region, it is possible to predict which of these amino acids, or any other residue at a particular position, may or may not affect antigen binding capacity. Become. It is also important to compare the human donor sequence of an individual kappa light chain and heavy chain variable region with the consensus sequence of the human variable region subgroup to which the donor sequence belongs and to identify particularly unusual amino acids. By following this design process, a number of amino acids in the human FR that are to be changed from an amino acid at a position in the human variable region to an amino acid at that position in the mouse M225 variable region are identified.
Table 8 describes how to design the first version (225RK _A ) of the reshaped human H225 kappa light chain variable region. There is only one residue in the reshaped human FR that may need to change the amino acid present in the human FR to the amino acid present in the original mouse FR. This change is at position 49 in FR2, as defined by Kabat et al. (20). The tyrosine in the human LS7′CL kappa light chain variable region is changed to lysine as in the mouse M225 kappa light chain variable region. From the model, it appears that the lysine in M225 is in a position close to the CDR3 (H3) of the heavy chain variable region and interacts with it. This residue will also be in a position adjacent to CDR2 (L2) of the kappa light chain variable region, which, as defined by Kabat et al. (20), is the mouse copper to which the M225 kappa light chain variable region belongs. This position is rarely found in light chain subgroup V members. For these reasons, it is advisable to conserve mouse lysine residues at 225RK _A.

As a second version, a reshaped human kappa light chain (225RK _B ) was also created that reversed the FR2 modification made in 225RK _A by replacing the lysine at position 49 with the original human tyrosine amino acid. Thus, this version of the reshaped human kappa light chain will not contain any mouse residues in the FR.
For the design of the reshaped human H225 heavy chain variable region, Table 9 shows the first version (225RH _A ). There are a total of 8 residues in the reshaped human FR, assuming that the amino acid in human 38P1′CL FR must be changed to the amino acid in the original mouse M225FR (ie A24V, T28S, F29L, S30T, V48L, S49G, F67L, and R71K). Since the amino acid residues at positions 24, 28, 29 and 30 of FR1 of the mouse sequence are several standard residues important for the H1 hypervariable loop structure (14), the reshaped human H225 heavy chain variable Maintained in the department. Since canonical residues are critical to the correct orientation and structure of the hypervariable loop, they are almost always conserved in the reshaped variable. In addition, residue positions 24-30 are considered to be part of the H1 hypervariable loop itself, and preserving them is considered more important for the correct conformation and orientation of this loop. Similarly, residue position 71 in FR3 is another position in the heavy chain variable that is important for the correct orientation and structure of the H2 hypervariable loop, as identified by Chosia et al. (14), This is similar to one of the standard amino acids of CDR2. Thus, mouse FR lysine replaces arginine in human FRs at this residue position. At positions 48 and 49 of FR2, and 67 in FR3, the valine, serine and phenylalanine residues (respectively) present in the human 38P1′CL V _H sequence are leucine, glycine, present in the mouse M225 V _H sequence, respectively. And leucine (respectively). This cleavage is based on a model in which all these three residues are buried under the H2 loop, affecting the conformation of the hypervariable loop and thus interfering with antibody binding capacity. These then become mouse residues conserved in the first version of the reshaped human H225 heavy chain variable region.

Reshaped human H225 heavy chain variable region version B (225RH _B ) incorporates all substitutions made at 225RH _A and also contains additional mouse residues. The human threonine residue at position 41 of FR2 is replaced with a proline that is necessarily found at this position in mouse subgroup IB and is very commonly found in human subgroup III. In contrast, threonine is not normally found at this position in human subgroup III (only 11/87 times), and from the model this residue is present in the turn on the surface of the M225V _H region. appear. It is unclear what effect this has on the hypervariable loop structure, but this version of the reshaped human H225 heavy chain variable region will reveal this point.
Reshaped human H225 heavy chain variable region version C (225RH _C ) incorporates all substitutions made at 225RH _A and also contains two mouse residues at positions 68 and 70 of FR3. From the mouse M225 variable region model, it is believed that both serine at position 68 and asparagine at position 70 are on the surface and end of the antigen binding site. Because either or both of these amino acids may react directly with EGFR, the threonine at position 68 and the serine at position 70 in human FRs are replaced with the corresponding 225RH _C mouse residue.
Reshaped human H225 heavy chain variable region version D (225RH _D ) simply incorporates all mouse FR substitutions made at 225RH _A , 225RH _B and 225RH _C and looks at the combined effect of these changes.
Reshaped human H225 heavy chain variable region version E (225RHE) takes all substitutions made in 225RH _A, also incorporates further position 78 Another residues changes FR3. The model gives some evidence that the mouse amino acid at position 78 (valine) affects the conformation of the H1 hypervariable loop from the position buried under CDR1. As a result, in 225RH _E , the human residue (leucine) replaces the mouse amino acid.

Example IV-7 Construction of Humanized Antibody Variable Region Gene The first version of the reshaped human H225V _K region (225RK _A ) construct was performed essentially as described by Sato et al. (26). That is, using a two-step PCR amplification protocol to synthesize a reshaped human variable region gene, a PCR primer encoding the FR modification is annealed to the DNA template of the chimeric C225V _K gene. As a result, the FR DNA sequence of the chimeric C225V _K was modified with the primer to the sequence of the reshaped human kappa light chain variable region gene 225RK _A. The newly synthesized reshaped variable region gene was digested with HindIII and XbaI after column purification, purified on agarose gel, and subcloned into pUC19 (digested and agarose gel purified in the same manner). The newly constructed plasmid pUC-225RK _A was then transformed into XL1 blue competent cells. Putative positive clones were identified by PCR screening (using primers RSP and UP), and finally ds-DNA sequencing was performed both to confirm their integrity and the absence of PCR errors. Select individual clones from confirmed positive clones were inserted directly human kappa light chain mammalian expression vector (pKN100) in a HindIII-XbaI fragment to produce plasmid pKN100-225RK _A. The integrity of this vector construct was confirmed by PCR screening (using primers HCMVi and NEW.HUK) and restriction digest analysis.

Reshaped human H225KV _K version B (225RK _B ) contains oligonucleotide primer 225RK _B. Constructed using K49Y and APCR40 (Table 11). 65.5 μl sterile distilled / deionized water, 5 μl 2 ng / μl plasmid pUC-225RK _A template DNA, 10 μl 10 × PCR buffer II, 6 μl 25 mM MgCl ₂ , 2 μl each of 10 mM stock solution of dNTP, 225RK _B. A 100 μl PCR reaction mixture containing 2.5 μl aliquots of K49Y and primer APCR40 (10 μM each) and 0.5 μl AmpliTaq ^™ DNA polymerase was overlaid with 50 μl mineral oil and loaded into a DNA thermal cycler. The PCR reaction was PCR amplified by repeating the 2-step protocol for 25 cycles, and the PCR product was column purified before cleaving with MscI. Plasmid pUC-225RK _A was also cut with MscI and both the digested PCR product and this plasmid fragment were agarose gel purified. This PCR product was then cloned into pUC-225RK _A to create pUC-225RK _B prior to transformation into XL1 blue competent cells. The putative positive transformant was first primer 225RK _B. Identified using UP in K49Y and PCR screening assay, then confirmed by ds-DNA sequence. Individual clones selected were finally subcloned into pKN100 to produce plasmid pKN100-225RK _B , primers HCMVi and NEW. The correct construct was confirmed by both PCR screening assay and restriction analysis using HUK (Table 6).

Construction of the first version of the reshaped human H225V _H region (225RH _A ) was also performed essentially as described by Sato et al. (26). In the case of the reshaped human 225 RH _A gene, PCR primers (Table 2) were recombined with the DNA template of the mAb previously humanized to create the 5 ′ half of the reshaped human kappa light chain variable region gene. Annealed to both of the chimeric C225V _H genes to synthesize the 3 ′ half of the formed human kappa light chain variable region gene. Using Again two step PCR amplification protocol, the reshaped variable region genes were created as HindIII-BamHI fragments agarose gel purified and cloned into pUC19 vector, it produced the plasmid pUC-225RH _A. Putative positive clones are identified by PCR screening (using primers RSP and UP), then their DNA sequences are confirmed and finally ds-DNA sequencing is performed for both purposes to confirm that there are no PCR errors It was. Select individual clones from confirmed positive clones, inserted directly into the human γ heavy chain mammalian expression vector pG1D105 as HindIII-XbaI fragment, produced a plasmid pG1D105-225RH _A. The construction of this plasmid was then confirmed by both PCR screening assay and restriction analysis using primers HCMVi and γAS (Table 6).
Reshaped human H225V _H version B (225RH _B ) was synthesized by two-step PCR mutagenesis as follows. First two separate 100 [mu] l PCR reaction mixture, sterile distilled / deionized water 65.5Myueru, 5 [mu] l of 2 ng / [mu] l plasmid, pUC-225RH _A template DNA, 10 [mu] l of 10 × PCR buffer II, 25 mM MgCl ₂ in 6μl , 2 μl each of a 10 mM stock solution of dNTP, and primers APCR10 and 225RH _B. T41P-AS, in the second PCR reaction, primers APCR40 and 225RH _B. Prepared by mixing 2.5 μl aliquots (10 μM each) of T41P-S (Table 13) and finally 0.5 μl of AmpliTaq ^™ DNA polymerase. Each of the two PCR reaction mixtures was overlaid with 50 μl mineral oil, loaded into a DNA thermal cycler and PCR amplified 25 cycles using a two-step protocol. Prior to resuspension in 50 μl of distilled / deionized water, the two PCR products were agarose gel purified to separate any remaining template DNA from the PCR reaction and their concentrations were determined.

In the second PCR reaction, a 20 pmol aliquot of each of the two PCR products from the first PCR reaction (8 μl APCR10 / 225RH _B .T41P-AS PCR product and 10 μl APCR40 / 225RH _B .T41P-S PCR product To 57.5 μl of sterile distilled / deionized water, 10 μl of 10 × PCR buffer II, 6 μl of 25 mM MgCl ₂ , 2 μl each of 10 mM stock solution of dNTP, and 0.5 μl of AmpliTaq ^™ DNA polymerase It was. The PCR reaction was overlaid with mineral oil and PCR amplified by performing the two-step protocol for only 7 cycles. The third PCR reaction was then performed with 1 μl of the second PCR reaction product, 69.5 μl of sterile distilled / deionized water, 10 μl of 10 × PCR buffer II, 6 μl of 25 mM MgCl ₂ , 10 mM stock solution of dNTP. 2 μl each, 2.5 μl aliquots of nested primer RSP and UP (10 μM each) and 0.5 μl AmpliTap ^™ DNA polymerase. The PCR reaction was overlaid with mineral oil and the last 25 cycles were amplified using a two-step protocol. The PCR product was then column purified, isolated as an agarose gel purified HindIII-BamHI fragment, subcloned into HindIII-BamHI digestion as well as the agarose gel purified plasmid pUC19 and finally transformed into XL1 blue competent cells. Putative positive transformants were first identified and then confirmed as described above. Subcloned selected the individual clones PG1D105, it was produced a plasmid pG1D105-225RH _B. This was confirmed by PCR screening assay and restriction analysis using primers HCMVi and γAS (Table 6).
Reshaped human H225V _H version C (225RH _B ) was synthesized in the same manner as 225RK _C. 65.5 μl sterile distilled / deionized water, 5 μl 2 mg / μl plasmid pUC-225RH _A template DNA, 10 μl 10 × PCR buffer II, 6 μl 25 mM MgCl ₂ , 2 μl each of 10 mM stock solution of dNTP, primer APCR40 And 225RH _C. A 100 μl PCR reaction mixture containing 2.5 μl aliquots (10 μM each) of T68S / S70N (Table 13) and 0.5 μl AmpliTaq ^™ DNA polymerase was prepared. The PCR reaction was overlaid with mineral oil and PCR amplified using a two-step protocol using 25 cycles and column purified prior to digestion with SalI and BamHI. Plasmid pUC-225RH _A was also cut with SalI and BamHI and both the digested PCR product and the plasmid were agarose gel purified. This PCR product was then cloned into pUC-225RH _A to create pUC-225RH _C before transformation into XL1 blue competent cells. Putative positive transformants were first identified by PCR screening assay using primers RSP and UP and then confirmed by ds-DNA sequencing. It was produced a plasmid PG1D105-225RH _C was subcloned individual clones selected then in PG1D105. The correct construction of this vector was finally verified by PCR screening assay and restriction analysis using primers HCMVi and γAS (Table 6).
Reshaped human H225V _H version D (225RH _D ) is a product incorporating changes to versions B and C of the reshaped human heavy chain of the H225 antibody. Unexpectedly, it is possible to fuse changes made to these heavy chain variable region genes by digesting both pUC-225RH _B and pUC-225RH _C with SalI and BamHI. agarose gel purified prior to coupling the insertion fragment of about 180bp from 2.95kb vector fragment and pUC-225RH _C from pUC-225RH _B, were transformed into XL1 Blue competent cells. Identified positive transformants individual, before the production of plasmid PG1D105-225RH _D was subcloned individual clones selected in PG1D105, and ds-DNA sequences. The correct construction of this vector was finally confirmed as described above.

Reshaped human H225V _H version E (225RH _E ) is a derivative of 225RH _A and primers APCR40 and 225RH _E. Synthesized in the same way as 225RH _C using L78V (Table 13). The 225RH _E clones selected from the plasmid pUC-225RH _E subcloned into PG1D105, vector PG1D105-225RH _E were produced, it was verified in the usual way that this is the correct construct.

Example IV-8 Transfection of DNA into COS cells COS cells were transfected with a mammalian expression vector using the method of Kettleborough et al. (11).

Example IV-9 Protein A Purification of Recombinant 225 Antibody Both the chimeric C225 antibody and various reshaped human H225 antibody constructs were subjected to protein A purification based on the protocol (27) described by Kolbinger et al.

Example IV-10 Mouse Antibody ELISA
Each well of a 96-well Nunc-Immuno Plate MaxiSorp ^™ immunoplate was first diluted with a coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6), 0.5 ng / μl goat anti-mouse IgG. (Gamma chain specificity) Coated with 100 μl aliquots of antibody and incubated overnight at 4 ° C. Wells were blocked with 200 μl / well mouse blocking (blocking) buffer (2.5% (w / v) BSA in PBS) for 1 hour at 37 ° C., then 200 μl / well washing buffer (PBS / 0. It was washed three times with 05% (v / v) Toine-20 [Tween-20]) aliquots. 100 [mu] l / well experimental sample (i.e., media removed from the M225 hybridoma cell line was centrifuged to remove cell debris) aliquot and sample-enzyme conjugate buffer (0.1 M Tris-HCl (pH 7.0)) A 1: 2 sample dilution diluted to 0.1 M NaCl, 0.02% (v / v) Toine-20 and 0.2% (w / v) BSA) was dispensed onto the immunoplate. Furthermore, a purified mouse IgG standard solution serially diluted 1: 2 from a concentration of 1000 ng / mL was added to the immunoplate. The immun plate was incubated for 1 hour at 37 ° C. and washed 3 times with 200 μl / well wash buffer. 100 μl of goat anti-mouse IgG / horseradish peroxidase conjugate diluted 1000-fold with sample-enzyme conjugate buffer was added to each well, and the immune plate was incubated at 37 ° C. for 1 hour, as before Washed. A 100 μl aliquot of TMB peroxidase substrate A: peroxidase substrate B (1: 1) was added to each well and incubated for 10 minutes in the dark at RT. The reaction was stopped by adding 50 μl of 1N H ₂ SO ₄ to each well. The optical density at 450 nm was finally determined using a combination of Bio-Rad 3550 microplate reader and Microplate Manager ^™ .

Example IV-11 Quantification of full-length human γ1 / κ antibody by ELISA Each well of a 96-well Nunc-Immuno Plate MaxiSorp ^™ immunoplate was first coated with a coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9). Coat with 100 μl aliquots of 0.5 ng / μl goat anti-mouse IgG (γ chain specific) antibody diluted in .6) and incubate overnight at 4 ° C. Wells were blocked with 200 μl / well human blocking buffer (2.0% (w / v) BSA in PBS) for 2 hours at RT, followed by 200 μl / well washing buffer (PBS / 0.05% (v / V) Toyon-20 [Tween-20]) Washed 3 times with aliquots. An aliquot of 100 μl / well experimental sample (ie, supernatant from which collected COS cells were centrifuged to remove cell debris), and sample-enzyme conjugation buffer (0.1 M Tris-HCl (pH 7.0), 0 A 1: 2 sample dilution diluted to 1 M NaCl, 0.02% (v / v) Toine-20 and 0.2% (w / v) BSA) was dispensed onto the immunoplate. In addition, 1: 2 serial diluted purified human γ1 / κ antibody used as a standard was added to the immunoplate. The immun plate was incubated at 37 ° C. for 1 hour and then washed 3 times with 200 μl / well wash buffer. After adding 100 μl of goat anti-human kappa light chain / horseradish peroxidase conjugate diluted 5000-fold with sample-enzyme conjugate buffer to each well, the immun plate was incubated at 37 ° C. for 1 hour, and Washed in the same way. The subsequent protocol was the same as in the case of the mouse antibody ELISA.

Example IV-12 ELISA of A431 cells for detection of EGFR antigen binding
The method for determining the relative binding ability of the recombinant 225 antibody construct to EGFR expressed on the surface of A431 cells was based on the method provided by ImClone Systems Inc. A431 cells were placed on 96-well flat bottom tissue culture plates and incubated overnight at 37 ° C. and 5% CO ₂ in DMEM medium supplemented with 10% (v / v) FBS. The next day the medium was removed and the cells were washed once in PBS and then fixed with 100 μl / well of 0.25% (v / v) glutaraldehyde in PBS. This was removed and the plates were washed again with PBS and blocked with 1% (w / v) BSA in 200 μl / well PBS for 2 hours at 37 ° C. Remove blocking solution, 100 μl / well aliquot of experimental sample (ie, centrifuged supernatant of harvested COS cells to remove cell debris) and their 1: 2 sample dilutions (1% in PBS (W / v) diluted with BSA) was spread on a tissue culture plate. In addition, an 80 μl / well aliquot of purified human γ1 / κ antibody serially diluted 1: 5 from a starting concentration of 20 μg / l used as a standard was also added to the plate. The immun plate was incubated at 37 ° C. for 1 hour and then washed 3 times with 200 μl / well of 0.5% (v / v) Tween 20 in PBS. 100 μl of goat anti-human IgG (H + L) / horseradish peroxidase conjugate diluted 5000-fold with 1% (w / v) BSA in PBS was added to each well, and then the immunoplate was incubated at 37 ° C. for 1 hour. Incubate and wash first with 200 μl / well 0.5% (v / v) Tween 20 in PBS (3 times) and then with distilled deionized water (2 times). The subsequent protocol was the same as in the mouse antibody ELISA.

Example IV-13 M225 Antibody Variable Region Cloning and Sequencing The presence of mouse antibody in the medium from M225 hybridoma cells at the time of harvesting cells for RNA purification was verified using mouse antibody ELISA. . Following the first strand synthesis, the single-stranded cDNA template is divided into two series of degenerate primers, ie, one series is kappa light chain signal peptide / variable gene specific (Table 4) and the second series is heavy chain. PCR amplification was performed using signal peptide / variable region gene specific ones (Table 5). Both V _K gene and the V _H gene of these primers from M225 hybridoma cell line using M225 antibody was PCR cloned.
Using primers MKV4 (annealed to the 5 'end of the DNA sequence of the kappa light chain signal peptide) and MKC (designed to anneal to the 5' end of the mouse kappa constant region gene), the M225 kappa light chain variable region The gene was PCR cloned as an approximately 416 bp fragment. Similarly, using primers MHV6 (annealed to the 5 ′ end of the DNA sequence of the heavy chain signal peptide) and MHCG1 (designed to anneal to the 5 ′ end of the CH ₁ domain of the mouse γ1 heavy chain gene) The M225 heavy chain variable region gene was PCR cloned as an approximately 446 bp fragment.
To reduce the possibility of error in introducing the mouse M225 variable region gene into the wild type sequence, ie, the change introduced by AmpliTaq ^™ DNA polymerase itself or by reverse transcription (the error frequency is about 1/10 of AmpliTaq ^™ ). A strict protocol was used. At least two PCR products from each of the separate total RNA preparations and subsequent first strand cDNA synthesis reactions were PCR cloned and then fully DNA sequenced for both the M225 mAb kappa light chain and heavy chain variable region DNA strands Was done.

From DNA sequence analysis of several individual clones from each of these PCR reactions, the mouse M225 antibody V _K and V _H genes were determined as shown in FIGS. 13 and 14, respectively. The amino acid sequences of the M225V _K and V _H regions were also compared to other mouse variable regions and the consensus sequences of variable region subgroups subdivided in the Kabat database (20). This analysis revealed that the M225V _K region most closely resembled the mouse kappa subgroup V consensus sequence and had 62.62% identity and 76.64% similarity to this subgroup. However, this kappa light chain variable region also showed very high suitability with mouse kappa subgroup III with 61.68% identity and 76.64% similarity to the consensus sequence. When only the M225κ light chain variable region FRs (ie, excluding amino acids in the CDRs) were compared to mouse subgroups III and V, identity increased to 66.25% for both subgroups, while similar Sex increased to 78.75% for subgroup III and just 80.00% for subgroup V. Similar analysis of the M225V _H region showed that this region was very close to the consensus sequence of mouse heavy chain subgroup IB in the Kabat database (20). The identity of the M225 mouse heavy chain variable region amino acid sequence and the subgroup IB consensus sequence was determined to be 78.15%, while the similarity was calculated to be 84.87%, the other consensus sequences far exceeding these values. It did not reach. From these results, it was confirmed that this mouse M225 variable region is a typical mouse variable region.

Example IV-14 Construction and Expression of Chimeric C225 Antibody PCR products from the two PCR reactions prepared to construct the C225V _K and V _H genes were subcloned into pUC19 separately as HindIII-BamHI fragments and putatively positive PCR screening was performed to identify transformants. The identified transformants were then ds-DNA sequenced to confirm that they were synthesized and then subcloned into the respective mammalian expression vectors. The DNA and amino acid sequences of the chimeric C225κ light chain and heavy chain variable regions are shown in FIGS. 15 and 16, respectively. After the integrity of the expression vectors was confirmed by PCR screening and restriction analysis to confirm that the correct insertion was present, these vectors were co-transfected into COS cells. After 72 hours of incubation, media was collected, cell debris was removed by centrifugation, and antibody production and binding to EGFR were analyzed by ELISA. Unfortunately, no chimeric antibody was detected in the supernatant of this COS cell cotransfection.
Analysis of the leader sequence of C225V _K revealed that it was abnormal compared to the leader sequence of other kappa light chain variable regions of mouse kappa light chain subgroups III and V (20). In order to find a more suitable leader sequence, the Kabat database was analyzed to identify individual kappa light chains that matched the C225V _K amino acid sequence and whose signal peptide sequence was known. This study showed that the murine antibody L7′CL (28) showed 94.79% identity and 94.79% similarity to the C225V _K region and was a perfect match for FR1 and signal during secretion. It was found that it plays an important role in peptide excision. The amino acid sequence of the L7′CLκ light chain signal peptide (ie, MVSTQFLVFFLLFWIPASRG (SEQ ID NO: 36)) shows all the properties that are considered important in the signal sequence such as the hydrophobic nucleus, and thus the signal peptide of PCR cloning 225V _K We decided to replace it with this new sequence. Another interesting point is that almost all the differences between M225V _K and the L7'CL signal peptide were all annealed to the first 11 amino acids of the signal peptide when the M225V _K gene was first PCR cloned. It was happening at the 5 'end (ie the first 33 bases, which corresponds to the first 11 amino acids of the signal peptide). Therefore, these differences are considered to be errors in the DNA sequence of the signal peptide induced by the primer. PCR mutagenesis of the C225V _K template produced a product of approximately 390 bp. The fragment digested and purified with HindIII-PstI was then subcloned into the same digested and agarose gel purified plasmid pUC-C225V _K and transformed into XL1 blue competent cells. Putative positive transformants were identified and then ds-DNA sequenced. The C225V _K sp gene (FIG. 17) was subcloned into pKN100, and the resulting expression vector (pKN100-C225V _K sp) was subjected to PCR selection and restriction digest to confirm the presence of the correct insert. This vector was finally cotransfected into pOS cells with pG1D105-C225V _H , the medium was collected after 72 hours of incubation, cell debris was removed by centrifugation, and antibody production and binding to EGFR were analyzed by ELISA. . In this case, the chimeric C225 antibody was detected in the supernatant of co-transfection of COS cells at a concentration of about 150 ng / mL and this antibody bound to EGFR in the cell ELISA. FIG. 18 shows one typical example of such an experiment.

Example IV-15 Construction and Expression of Reshaped H225 Antibody (225RK _K / 225RH _A ) Construction of the first version of the reshaped human H225κ light chain variable region (225RK _A ) produced a product of approximately 416 bp, which was Was subcloned into pUC19 as a HindII-BamHI fragment. Putative positive transformants were identified by PCR screening assay followed by ds-DNA sequence. The 225RK _A gene (FIG. 19) was subcloned into pKN100, and the resulting expression vector (pKN100-225RK _A ) was PCR screened and restriction digested to confirm the presence of the correct insert. Similarly, construction of the first version of the reshaped human H225 heavy chain variable region (225RH _A ) produced an approximately 446 bp product, which was then subcloned into pUC19 as a HindII-BamHI fragment. Again, putative positive transformants were identified by PCR screening assays followed by ds-DNA sequences. The 225RH _A gene (FIG. 20) was subcloned into pG1D105, and the resulting expression vector (pG1D105-225RH _A ) was PCR screened and restriction digested to confirm the presence of the correct insert.
These vectors were then co-transfected into COS cells, and after 72 hours of incubation, the medium was collected, cell debris was removed by centrifugation, and antibody production and binding to EGFR were analyzed by ELISA. The concentration of reshaped human antibody in the COS cell supernatant was slightly higher than that following transient expression of the C225 chimeric antibody (approximately 200 ng / mL). In addition, a high level of binding to EGFR was shown in cell ELISA. FIG. 8 shows a typical example of such an experiment, in which reshaped human H225 antibody (225RK _A / 225RH _A ) is expressed on the surface of A431 cells with approximately 65% relative affinity of the chimeric C225 antibody. It shows that it is combined.

  The amino acid sequences of the two constructed kappa light chain reshaped human H225 variable regions are shown in FIG. 21, while the amino acid sequences of the five versions of the constructed heavy chain reshaped human H225 variable region are shown in FIG. .

225 shows the effect of 225 on the growth of xenografts of A431 tumors established in nude mice. 10 ⁷ cells were injected into the flank of the experimental animals. When the tumors reached an average volume of 2-300 mm ³ , treatment was started with PBS or 1 mg / specimen 225 administered twice a week for 5 weeks. Tumor volume and remission index (RI) were determined as described in the "Examples" section. FIG. 6 shows the effect of 225 and chimerization 225 (C225) on the growth of xenografts of A431 tumors established in nude mice. Experimental animals received 1 mg / mouse PBS twice a week for 5 weeks. A: mean tumor volume; B: remission index. The apparent tumor regression in the PBS control group on day 37 is due to the death of 3 of the 10 control groups and the concomitant reduction in total tumor volume. Figure 2 shows the effect of C225 on the growth of xenografts of A431 tumors established in nude mice. The experimental animals were given 1 mg C225 or PBS twice a week for 5 weeks. The mean tumor volume of group C225 showed a statistically significant biological effect compared to the control (see text). A: mean tumor volume (asterisk indicates statistically significant difference from control), B: remission index. FIG. 4 is a dose response of C225 to the growth of A431 xenografts established in nude mice. Experimental animals received PBS, 1, 0.5, or 0.25 mg / animal twice weekly for 5 weeks as described in Materials and Methods. Animals treated with 1 mg / dose of C225 showed a statistically significant biological effect compared to controls (see text). A: mean tumor volume (asterisk indicates statistically significant difference from control), B: remission index. The decrease in RI on day 47 in the 250 μg dose group is due to tumor recurrence in animals that were clearly tumor free (in this case the effect of C225 was temporary). FIG. 6 shows suppression of A431 cells by C225 and monoclonal antibody 225 heavy chain CDR-1 and heavy chain CDR-2. FIG. 5 shows in vivo suppression as a function of the concentration of C225-doxorubicin conjugate in A431 cells. FACS analysis of EGFR expression on human prostate cancer cells. LNCaP (androgen-dependent human prostate cancer), DU145 and PC-3 (androgen-independent human prostate cancer), and A431 (human epidermoid carcinoma) cells were removed from the growth flask with EDTA and stained with C225. Data were expressed as MFI (mean fluorescence intensity), an indirect measure of antigen expression. The results shown in this figure represent the results of at least 5 experiments. FIG. 6 shows suppression of EGF-induced phosphorylation of EGFR by C225. LNCaP, DU145, and PC-3 monolayers were stimulated with EGF in the presence or absence of C225. Cells were eluted, subjected to SDS PAGE, blotted, and screened with a mouse monoclonal antibody against PTyr (Lake Placid UBI). Lane A: No addition (basal level of EGFR phosphorylation); Lane B: Stimulated EGFR with 10 ng / mL EGF for 15 minutes at room temperature in the absence of C225; Lane C: EGF in the presence of 10 μg / mL C225 Stimulated EGFR by. FIG. 6 shows growth inhibition by C225 of established DU145 xenografts. Nude mice (male, nu / nu) were inoculated with 1 million DU145 cells added to Matrigel. After the tumors reached an average volume of about 100 mm ³ (day 20), animals were randomized (10 per group) and either PBS (control) or C225 (0.5 mg / dose, 10 ×) Processed. The animals were treated for 35 days and followed for an additional 3 weeks. Mice without tumors or with small tumors were maintained for an additional 3 months. Significance (indicated by an asterisk in FIG. 3A) was determined by Student's T test and was found to be significant at a p value <0.5. A: Average tumor volume; B: Tumor growth characteristics in PBS group; C: Tumor growth characteristics in C225 treatment group. Effect of C225 on tumor disappearance and survival. The complete disappearance of the tumor during this study was defined by the remission index (RI). Animal deaths during this study were considered treatment failures and were included in this analysis. A: remission index; B: survival curve. The white circles and black circles in FIG. 10 have the same meaning as in FIG. 1 is a schematic representation of the pKN100 mammalian expression vector used for expression of the kappa (κ) light chain of chimeric C225 and reshaped human H225 antibody. FIG. 2 is a schematic of the pG1D105 mammalian expression vector used for expression of the heavy chain of chimeric C225 and reshaped human H225 antibodies. It is the DNA sequence (SEQ ID NO: 4) and peptide sequence (SEQ ID NO: 5) of the kappa light chain variable region of the M225 antibody. The PCR clones from which this information was obtained were obtained by amplification using the degenerate primer MKV4 (SEQ ID NO: 6) (7). These are the DNA sequence (SEQ ID NO: 7) and peptide sequence (SEQ ID NO: 8) of the heavy chain variable region of the M225 antibody. The PCR clones from which this information was obtained were obtained by amplification using the degenerate primer MHV6 (SEQ ID NO: 9) (7). It is the DNA sequence (SEQ ID NO: 10) and peptide sequence (SEQ ID NO: 11) of the k225 light chain variable region of the C225 antibody. The DNA sequence (SEQ ID NO: 12) and peptide sequence (SEQ ID NO: 13) of the heavy chain variable region of the C225 antibody. These are the DNA sequence (SEQ ID NO: 14) and peptide sequence (SEQ ID NO: 15) of the k225 light chain variable region of the C225 antibody having a modified leader sequence from the K7 light chain of the L7'CL antibody (28). FIG. 5 is a typical example of the results of a cell ELISA to measure the binding affinity of chimeric C225 and reshaped human H225 (225RK _A / 225RH _A ) to the epidermal growth factor receptor expressed on the A431 cell surface. Figure 2 is the DNA sequence (SEQ ID NO: 16) and peptide sequence (SEQ ID NO: 17) of the first version (225RK _A ) of the kappa light chain variable region of the reshaped human H225 antibody. The DNA sequence (SEQ ID NO: 18) and peptide sequence (SEQ ID NO: 19) of the first version (225RH _A ) of the heavy chain variable region of the reshaped human H225 antibody. The amino acid sequences (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 22) of the two versions (225RK _A and 225RK _B ) of the kappa light chain variable region of the reshaped human H225 antibody. 23). Residues were numbered according to Kabat et al. (20). Mouse framework residues conserved in the reshaped human framework are shown in bold. Amino acid sequence (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 25) of five versions (225RH _A , 225RH _B , 225RH _C , 225RH _D , 225RH _E ) of the heavy chain variable region of the reshaped human H225 antibody : 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), and (SEQ ID NO: 30). Residues were numbered according to Kabat et al. (20). Mouse framework residues conserved in the reshaped human framework are shown in bold. Amino acid sequence (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 25) of five versions (225RH _A , 225RH _B , 225RH _C , 225RH _D , 225RH _E ) of the heavy chain variable region of the reshaped human H225 antibody : 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), and (SEQ ID NO: 30). Residues were numbered according to Kabat et al. (20). Mouse framework residues conserved in the reshaped human framework are shown in bold.

Claims (22)

  A polypeptide comprising an amino acid sequence represented by NYGVH, GVIWSGGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS, which is a polypeptide in which the constant part and variable light chain of an antibody are deleted.   The polypeptide according to claim 1, comprising an amino acid sequence represented by NYGVH and an amino acid sequence represented by GVIWSGGNTDYNTPFTSR or VIWSGNTDYNTPFTS.   A polypeptide comprising an amino acid sequence represented by NYGVH or GVIWSGGNTDYNTPFTSR.   A polypeptide comprising an amino acid sequence represented by NYGVH or VIWSGNTDYNTPFTS.   The polypeptide of claim 1, which is conjugated to an effector molecule.   The polypeptide according to claim 5, wherein the effector molecule suppresses tumor growth.   6. The polypeptide of claim 5, wherein the effector molecule is cytotoxic.   6. A polypeptide according to claim 5, wherein the effector molecule is doxorubicin.   6. A polypeptide according to claim 5, wherein the effector molecule is cisplatin.   6. A polypeptide according to claim 5, wherein the effector molecule is taxol.   6. The polypeptide of claim 5, wherein the effector molecule is a signal transduction inhibitor.   6. A polypeptide according to claim 5, wherein the effector molecule is a ras inhibitor.   6. The polypeptide of claim 5, wherein the effector molecule is a cell cycle inhibitor.   DNA encoding a polypeptide from which the constant part of an antibody and the variable light chain have been deleted, wherein the polypeptide comprises an amino acid sequence represented by NYGVH, GVIWSGGNTDYNTPFTSR, or VIWSGGNTDYNTPFTS.   The DNA encoding the polypeptide according to claim 14, comprising an amino acid sequence represented by NYGVH and an amino acid sequence represented by GVISWSGNTDYNTPFTSR or VIWSGGNTDYNTPFTS.   The DNA encoding the polypeptide according to claim 14, which is conjugated to an effector molecule.   The DNA encoding the polypeptide according to claim 16, wherein the effector molecule suppresses tumor growth.   A molecule having the constant part of a human antibody and the hypervariable part of a monoclonal antibody 225 conjugated to an effector molecule.   19. A molecule according to claim 18, wherein the effector molecule is a cytotoxic agent.   20. A molecule according to claim 19, wherein the cytotoxic agent is doxorubicin.   20. A molecule according to claim 19, wherein the cytotoxic agent is taxol.   20. A molecule according to claim 19, wherein the cytotoxic agent is cisplatin.