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• W2000679973 abstract "HuCC49ΔCH2 is a heavy chain constant domain 2 domain-deleted antibody under development as a radioimmunotherapeutic for treating carcinomas overexpressing the TAG-72 tumor antigen. Mammalian cell culture biosynthesis of HuCC49ΔCH2 produces two isoforms (form A and form B) in an approximate 1:1 ratio, and consequently separation and purification of the desired form A isoform adversely impact process and yield. A protein engineering strategy was used to develop a panel of hinge-engineered HuCC49ΔCH2 antibodies to identify hinge sequences to optimize production of the form A isoform. We found that adding a single proline residue at Kabat position 243, immediately adjacent to the carboxyl end of the core middle hinge CPPC domain, resulted in an increase from 39 to 51% form A isoform relative to the parent HuCC49ΔCH2 antibody. Insertion of the amino acids proline-alanine-proline (PAP) at positions 243-245 enhanced production of the form A isoform to 72%. Insertion of a cysteine-rich 15-amino acid IgG3 hinge motif (CPEPKSCDTPPPCPR) in both of these mutant antibodies resulted in secretion of predominantly form A isoform with little or no detectable form B. Yields exceeding 98% of the form A isoform have been realized. Preliminary peptide mapping and mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present. HuCC49ΔCH2 is a heavy chain constant domain 2 domain-deleted antibody under development as a radioimmunotherapeutic for treating carcinomas overexpressing the TAG-72 tumor antigen. Mammalian cell culture biosynthesis of HuCC49ΔCH2 produces two isoforms (form A and form B) in an approximate 1:1 ratio, and consequently separation and purification of the desired form A isoform adversely impact process and yield. A protein engineering strategy was used to develop a panel of hinge-engineered HuCC49ΔCH2 antibodies to identify hinge sequences to optimize production of the form A isoform. We found that adding a single proline residue at Kabat position 243, immediately adjacent to the carboxyl end of the core middle hinge CPPC domain, resulted in an increase from 39 to 51% form A isoform relative to the parent HuCC49ΔCH2 antibody. Insertion of the amino acids proline-alanine-proline (PAP) at positions 243-245 enhanced production of the form A isoform to 72%. Insertion of a cysteine-rich 15-amino acid IgG3 hinge motif (CPEPKSCDTPPPCPR) in both of these mutant antibodies resulted in secretion of predominantly form A isoform with little or no detectable form B. Yields exceeding 98% of the form A isoform have been realized. Preliminary peptide mapping and mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present. Radioimmunotherapy has been shown to be efficacious in treating hematological diseases, such as relapsed or refractory B cell non-Hodgkin lymphoma, with two radioimmunotherapeutic drugs currently approved for use in humans (1Witzig T.E. Gordon L.I. Cabanillas F. Czuczman M.S. Emmanouilides C. Joyce R. Pohlman B.L. Bartlett N.L. Wiseman G.A. Padre N. Grillo-Lopez A.J. Multani P. White C.A. J. Clin. Oncol. 2002; 20: 2453-2463Crossref PubMed Scopus (1022) Google Scholar, 2Kaminski M.S. Estes J. Zasadny K.R. Francis I.R. Ross C.W. Tuck M. Regan D. Fisher S. Gutierrez J. Kroll S. Stagg R. Tidmarsh G. Wahl R.L. Blood. 2000; 96: 1259-1266Crossref PubMed Google Scholar). However, the treatment of solid tumors with radiolabeled monoclonal antibodies (mAbs) 2The abbreviations used are: mAbmonoclonal antibodyCH2heavy chain constant domain 2ΔCH2CH2 domain-deletedCconstantUHupper hingeMHmiddle hingeLHlower hingeTAG-72tumor associated glycoprotein-72CHOChinese hamster ovaryPAPproline-alanine-prolineHPLChigh pressure liquid chromatographyDTTdithiothreitolCPPCcore middle hinge domain (Cys-Pro-Pro-Cys)SOEsplicing by overlap extensionMSmass spectrometryendoendoproteinaseHuhuman.2The abbreviations used are: mAbmonoclonal antibodyCH2heavy chain constant domain 2ΔCH2CH2 domain-deletedCconstantUHupper hingeMHmiddle hingeLHlower hingeTAG-72tumor associated glycoprotein-72CHOChinese hamster ovaryPAPproline-alanine-prolineHPLChigh pressure liquid chromatographyDTTdithiothreitolCPPCcore middle hinge domain (Cys-Pro-Pro-Cys)SOEsplicing by overlap extensionMSmass spectrometryendoendoproteinaseHuhuman. has achieved only modest clinical responses due, in part, to poor tumor localization and dose-limiting toxicities, primarily bone marrow toxicity, associated with exposure of unbound radiolabeled antibody in the circulatory system (3Liu T. Meredith R.F. Saleh M.N. Wheeler R.H. Khazaeli M.B. Plott W.E. Schlom J. LoBuglio A.F. Cancer Biother. Radiopharm. 1997; 12: 79-87Crossref PubMed Scopus (27) Google Scholar, 4Divgi C.R. Scott A.M. Dantis L. Capitelli P. Siler K. Hilton S. Finn R.D. Kemeny N. Kelsen D. Kostakoglu L. J. Nucl. Med. 1995; 36: 586-592PubMed Google Scholar, 5Meredith R.F. LoBuglio A.F. Plott W.E. Orr R.A. Brezovich I.A. Russell C.D. Harvey E.B. Yester M.V. Wagner A.J. Spencer S.A. J. Nucl. Med. 1991; 32: 1162-1168PubMed Google Scholar). CH2 domain-deleted antibodies are a class of genetically engineered antibody reagents that recently have shown promise for radioimmunotherapy of solid tumors (6Mueller B.M. Reisfeld R.A. Gillies S.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5702-5705Crossref PubMed Scopus (84) Google Scholar, 7Calvo B. Kashmiri S.V. Hutzell P. Hand P.H. Slavin-Chiorini D.C. Schlom J. Zaremba S. Cancer Biother. 1993; 8: 95-109Crossref PubMed Scopus (8) Google Scholar, 8Slavin-Chiorini D.C. Kashmiri S.V. Schlom J. Calvo B. Shu L.M. Schott M.E. Milenic D.E. Snoy P. Carrasquillo J. Anderson K. Cancer Res. 1995; 55 (-S5967): S5957PubMed Google Scholar). HuCC49ΔCH2, a humanized CH2 domain-deleted CC49 mAb (∼122 kDa), has high affinity for the TAG-72 glycoprotein tumor antigen expressed on a majority of human carcinomas, including colorectal, gastric, pancreatic, lung, and ovarian (9Slavin-Chiorini D.C. Kashmiri S.V. Lee H.S. Milenic D.E. Poole D.J. Bernon E. Schlom J. Hand P.H. Cancer Biother. Radiopharm. 1997; 12: 305-316Crossref PubMed Scopus (55) Google Scholar, 10Muraro R. Kuroki M. Wunderlich D. Poole D.J. Colcher D. Thor A. Greiner J.W. Simpson J.F. Molinolo A. Noguchi P. Cancer Res. 1988; 48: 4588-4596PubMed Google Scholar). In human tumor mouse xenograft models, radiolabeled HuCC49ΔCH2 was shown to accumulate and be retained to appreciable levels in tumor, exhibit favorable tumor to normal tissue ratios, but demonstrate rapid serum clearance compared with full-length HuCC49 IgG (9Slavin-Chiorini D.C. Kashmiri S.V. Lee H.S. Milenic D.E. Poole D.J. Bernon E. Schlom J. Hand P.H. Cancer Biother. Radiopharm. 1997; 12: 305-316Crossref PubMed Scopus (55) Google Scholar). Recent clinical studies with low dose 131I-HuCC49ΔCH2 in a small group of patients with meta-static colorectal carcinoma have shown the radioimmunotherapeutic to be well tolerated and to exhibit a demonstrably reduced serum half-life (mean serum half-life of ∼20 h) compared with earlier studies with the 131I-murine CC49 IgG (11Forero A. Meredith R.F. Khazaeli M.B. Carpenter D.M. Shen S. Thornton J. Schlom J. LoBuglio A.F. Cancer Biother. Radiopharm. 2003; 18: 751-759Crossref PubMed Scopus (26) Google Scholar). HuCC49ΔCH2, by virtue of its rapid serum half-life and presumably reduced risk of eliciting a human anti-murine antibody response, holds promise as being clinically useful for both radioimmunotherapeutic and radioimmunodiagnostic applications (12Agnese D.M. Abdessalam S.F. Burak W.E. Arnold M.W. Soble D. Hinkle G.H. Young D. Khazaeli M.B. Martin E.W. Ann. Surg. Oncol. 2004; 11 (Jr.): 197-202Crossref PubMed Scopus (23) Google Scholar, 13Sheikh N. Curr. Opin. Mol. Ther. 2003; 5: 428-432PubMed Google Scholar). monoclonal antibody heavy chain constant domain 2 CH2 domain-deleted constant upper hinge middle hinge lower hinge tumor associated glycoprotein-72 Chinese hamster ovary proline-alanine-proline high pressure liquid chromatography dithiothreitol core middle hinge domain (Cys-Pro-Pro-Cys) splicing by overlap extension mass spectrometry endoproteinase human. monoclonal antibody heavy chain constant domain 2 CH2 domain-deleted constant upper hinge middle hinge lower hinge tumor associated glycoprotein-72 Chinese hamster ovary proline-alanine-proline high pressure liquid chromatography dithiothreitol core middle hinge domain (Cys-Pro-Pro-Cys) splicing by overlap extension mass spectrometry endoproteinase human. The variable light and variable heavy domains of HuCC49ΔCH2 are humanized. The light chain constant domain is a human Cκ, and the human γ1 chain has been genetically modified to produce a heavy chain composed of a CH1 domain followed by a partial IgG1 hinge region tethered to the CH3 heavy chain domain by addition of a flexible 10-amino acid GGGSSGGGSG spacer; thus HuCC49ΔCH2 can be described as containing an atypical hinge region. Native human antibody hinge regions can be structurally defined as consisting of an upper hinge region (UH) extending from the last residue of CH1 up to but not including the first inter-heavy chain cysteine, a middle hinge region (MH) extending from the first inter-heavy chain cysteine to a proline residue adjacent to the carboxyl-end of the last MH cysteine, and a highly conserved 7-8-amino acid lower hinge (LH) (14Roux K.H. Strelets L. Brekke O.H. Sandlie I. Michaelsen T.E. J. Immunol. 1998; 161: 4083-4090PubMed Google Scholar). The atypical hinge region in HuCC49ΔCH2 is similar to that described in the anti-carcinoembryonic antigen minibody (15Hu S. Shively L. Raubitschek A. Sherman M. Williams L.E. Wong J.Y. Shively J.E. Wu A.M. Cancer Res. 1996; 56: 3055-3061PubMed Google Scholar), whereas the MH proline at position 243 (Kabat numbering system (16Kabat E.A. Wu T.T. Perry H.M. Gottesman K.S. Foeller C. Sequences of Proteins of Immunological Interest. 1991; (5 Ed., pp. -lvii, U. S. Department of Health and Human Services, Washington, D. C.): lviGoogle Scholar)) and the entire CH2 domain, including the LH residues, APELLGGP (the first eight amino-terminal residues of the IgG1 CH2 domain), are deleted and replaced by the 10-amino acid peptide Gly/Ser spacer. Biosynthesis of HuCC49ΔCH2 in mammalian Chinese hamster ovary cells has been observed to produce two homodimeric isoforms present in approximately a 50:50 mixture. 3P. Chinn, personal communication.3P. Chinn, personal communication. The presence of two species has also been shown previously with protein G-purified chimeric B72.3 or chimeric CC49 CH2 domain-deleted antibodies (7Calvo B. Kashmiri S.V. Hutzell P. Hand P.H. Slavin-Chiorini D.C. Schlom J. Zaremba S. Cancer Biother. 1993; 8: 95-109Crossref PubMed Scopus (8) Google Scholar, 8Slavin-Chiorini D.C. Kashmiri S.V. Schlom J. Calvo B. Shu L.M. Schott M.E. Milenic D.E. Snoy P. Carrasquillo J. Anderson K. Cancer Res. 1995; 55 (-S5967): S5957PubMed Google Scholar). For HuCC49ΔCH2, one isoform, referred to as form A, contains covalent interchain disulfide bonds at heavy chain MH positions 239 and 242, Kabat numbering system. The second isoform, form B, is held together by noncovalent interactions through the CH3 domains, and it fails to develop an interchain hinge disulfide bond as evidenced by the formation of a 60-kDa product following nonreducing, denaturing gel electrophoresis (Fig. 1). The form B isoform is thought to contain heavy chain intrachain disulfide bonds covalently linking the cysteine residue at position 239 to that at position 242. Compound stability studies support form A HuCC49ΔCH2 as the preferred molecule for therapeutic development, and methods for the separation and purification of form A from form B using hydrophobic interaction chromatography have been described (17Brawslasky G. Glaser S. Yang T.-H. Hopp J. Chinn P. U. S. Patent PCT/US2004/020944. 2005; ((July 28, 2005 U. S. Patent PCT/US2004/020944)Google Scholar). However, we projected that development of a cell line that expressed only form A would avoid synthesis of unwanted antibody by-product and, in turn, eliminate the requirement for physical separation of form A from form B, resulting in a more efficient recombinant protein production process. We hypothesized that generation of the two HuCC49ΔCH2 antibody isoforms is a consequence of hinge heterogeneity because of variation in disulfide bond formation. It follows, therefore, that stabilization of the hinge region should favor production of the desired form A isoform. By using a protein engineering strategy, we describe here a series of variant hinge-connecting peptides that were found to improve significantly the homogeneity and yield of CH2 domain-deleted antibodies. Synthetic oligonucleotides (Sigma Genosys) encoding the various hinge region connecting peptides were introduced into the HuCC49ΔCH2 vector using PCR and splicing by overlap extension (SOE) methods. The HuCC49ΔCH2 vector contains a translation-impaired modified (intron-containing) neomycin phosphotransferase gene to select for transcriptionally active integration events and a murine dihydrofolate reductase gene to permit amplification with methotrexate (18Barnett R.S. Limoli K.L. Huynh T.B. Ople E.A. Reff M.E. Wang H.Y. Imanaka T. Antibody Expression and Engineering. 1995: 27-40Google Scholar). Middle and lower hinge mutations were introduced into the parent HuCC49ΔCH2 heavy chain gene by PCR-based site-directed mutagenesis (19Kadowaki H. Kadowaki T. Wondisford F.E. Taylor S.I. Gene (Amst.). 1989; 76: 161-166Crossref PubMed Scopus (96) Google Scholar). PCR primer sets were designed with 5′ AgeI and 3′ XhoI restriction sites for cloning the hinge region PCR products into HuCC49ΔCH2 as AgeI/XhoI fragments. PCRs consisted of HuCC49ΔCH2 DNA template, the 5′ primer, MB-04F 5′-CTTCCCCGAACCGGTGACGGTG-3′ and the following 3′ primers: Pro-243 (MB-06R, 5′-GATCCGCCTCCACTCGAGCCACCTCCGGGGCACGGTGGGCATGTGTG-3′); PAP (MB-010R, 5′-GATCCGCCTCCACTCGAGCCACCTCCCGGTGCGGGGCACGGTGGGCATGTGTG-3′); C242S + Pro-243 (MB-08R, 5′-GATCCGCCTCCACTCGAGCCACCTCCGGGGGACGGTGGGCATGTGTG-3′); C242S + PAP (MB-09R, 5′-GATCCGCCTCCACTCGAGCCACCTCCCGGTGCGGGGGACGGTGGGCATGTGTG-3′); C239S + Pro-243 (MB-11R, 5′-GATCCGCCTCCACTCGAGCCACCTCCGGGGCACGGTGGGGATGTGTG-3′); and C239S + PAP (MB-012R, 5′-GATCCGCCTCCACTCGAGCCACCTCCAGGTGCTGGGCACGGTGGGGATGTGTG-3′). PCRs were initiated with HotStarTaq™ DNA polymerase (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions. Cycle conditions were of 15 min denaturation at 95 °C followed by 30 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 54 °C, and 1 min of synthesis at 72 °C, followed by a final strand extension for 10 min at 72 °C. Construction of hinge regions containing a cysteine-rich 15-amino acid IgG3 hinge insertion (CPEPKSCDTPPPCPR) in addition to middle and lower hinge mutations were performed using SOE (20Horton R.M. PCR Protocols: Current Methods and Applications. 1993; 15 (White, B. A., ed) Vol., pp., Humana Press Inc., Totowa, NJ: 251-261Crossref Google Scholar). PCR primer sets were also designed with 5′ AgeI and 3′ XhoI restriction sites and consisted of HuCC49ΔCH2 DNA template, the 5′ primer MB-04F, and the following 3′ primers: G1/G3 “SOE overlap” (MB-13R, 5′-GCACCGTGGGCATGGGGGAGGTGTGTCACAAGATTTGGGCTCTGGGCACGGTGGGCATGTG-3′); G1/G3:PAP (MB-14R, 5′-GGATCCGCCTCCACTCGAGCCACCTCCAGGTGCTGGGCACCGTGGGCATGGGGGAG-3′); and G1/G3:P243 (MB-15R, 5′-GGATCCGCCTCCACTCGAGCCACCTCCTGGGCACCGTGGGCATGGGGGAG-3′). Briefly, the first PCR contained DNA template, 0.8 μm of the forward primer MB-04F, and a limiting concentration (0.2 μm) of the reverse SOE primer MB-13R. The reaction mixture was denatured for 15 min at 95 °C followed by 20 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 54 °C, and 1 min of synthesis at 72 °C. The second PCR was performed by adding either 0.8 μm MB-14R or MB-15R reverse primer for an additional 20 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 54 °C, 1 min of synthesis at 72 °C followed by 10-min extension at 72 °C. PCR products were purified, digested with restriction endonucleases, and ligated into AgeI/XhoI-digested HuCC49ΔCH2 vector. Escherichia coli strain XL-1 Blue (Stratagene, La Jolla, CA) was used for plasmid propagation. Correct modifications to the hinge region were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for stable production of antibody protein. The CHO cell line, DG44, was grown in CHO-SSFMII medium supplemented with hypoxanthine and thymidine (Invitrogen). Approximately 1 μg of PacI-linearized plasmid DNA was transfected into 4 × 106 CHO cells by electroporation using a Bio-Rad Gene Pulser II electroporation device (Bio-Rad). Conditions for electroporation were 350 V, 600 microfarads, with high capacitance setting. Each electroporation was plated into a 96-well dish (about 400,000 cells/well). Dishes were fed with media containing 400 μg/ml G418 (Geneticin; Invitrogen) beginning 2 days after electroporation and thereafter every 2nd or 3rd day until colonies arose. Supernatants from colonies were assayed for the presence of antibody by an enzyme-linked immunosorbent assay specific for human antibody. Colonies producing various amounts of antibody were further tested by Western blot analysis to evaluate the secreted isoforms. Briefly, 3 ng of total antibody protein was analyzed by nonreducing 4-20% Tris-glycine SDS-PAGE (Invitrogen) followed by Western blot probed with an anti-human κ antibody (Roche Applied Science), and then an anti-rabbit horseradish peroxidase antibody (Amersham Biosciences) to detect form A and form B isoforms. Membranes were processed with the ECL Western blotting analysis system according to the manufacturer (Amersham Biosciences). Colonies producing the highest amount of antibody were expanded, and supernatant dilutions ranging from 30 to 0.23 ng were tested by Western blot analysis as described above. Band intensities corresponding to the A and B isoforms were semi-quantified by imaging the developed film with a CCD camera (Alpha Innotech Corp., San Leandro, CA). Band intensities from four lanes that fell within the linear range of the exposed film were measured using the spot densitometry function. The relative ratios of the form A and B isoforms were calculated (mean ± S.E.). Select G418-resistant CHO cell lines producing HuCC49ΔCH2 PAP and HuCC49ΔCH2 G1/G3:PAP antibodies were adapted to CD-CHO media (21Nakamura T. Kloetzer W.S. Brams P. Hariharan K. Chamat S. Cao X. LaBarre M.J. Chinn P.C. Morena R.A. Shestowsky W.S. Li Y.P. Chen A. Reff M.E. Int. J. Immunopharmacol. 2000; 22: 131-141Crossref PubMed Scopus (45) Google Scholar). Cell line cultures were scaled up in 25-liter cell bags in a WAVE BIOREACTOR (wave BIOTECH, LLC; Bridgewater, NJ), and supernatants were harvested for affinity purification by protein G-Sepharose (Amersham Biosciences). In some cases protein G-enriched immunoglobulin isoforms were further purified by hydrophobic interaction chromatography. The elution peaks representing form A and form B isoforms were separately pooled, dialyzed, and concentrated, and the amount of protein determined by modified Lowry (Bio-Rad) proteins were analyzed for purity by scanning densitometry (SI 375 personal densitometer; Amersham Biosciences) of reduced and nonreduced SDS-PAGE stained with Coomassie Blue. Size exclusion HPLC was used to analyze the percentage of monomer antibody products. A 96-well assay plate was coated with 8 μg/ml bovine submaxillary mucin, a source of the TAG-72 antigen, and blocked with 1% phosphate-buffered saline:bovine serum albumin. HuCC49ΔCH2, HuCC49ΔCH2 PAP, and HuCC49ΔCH2 G1/G3:PAP antibodies were incubated for 2 h at room temperature starting at 100 μg/ml in 3.5-fold serial dilution with 0.2 μg/ml of Eu3+-labeled HuCC49ΔCH2 tracer, followed by incubation with 200 μl/well DELFIA enhancement solution (PerkinElmer Life Sciences). Time-resolved fluorescence was read using the Europium protocol. IC50 values were calculated using GraphPad Prism 4.0 (San Diego, CA). Trypsin Digestion of HuCC49ΔCH2 and Hinge Variant Antibodies—Samples of the HuCC49ΔCH2, HuCC49ΔCH2 PAP, and HuCC49ΔCH2 G1/G3:PAP antibodies were denatured, reduced, and digested with trypsin as follows. Aliquots of 150 μg were diluted to 100 μl in HPLC water and denatured in 6 m guanidine hydrochloride, 50 mm Tris, pH 8.0. The samples were reduced by the addition of 20 mm DTT and incubated for 30 min at 37 °C. The reduced samples were alkylated with 50 mm iodoacetic acid for 30 min at 37 °C. The alkylation reaction was quenched by the addition of excess DTT. The reduced and alkylated samples were buffer-exchanged into 25 mm Tris, 20 mm CaCl2, pH 7.5, using PD-10 columns. Trypsin was added to each sample in a 1:15 (w/w) ratio and incubated for 4 h at 37°C. The digestion was stopped by the addition of trifluoroacetic acid to a final concentration of 0.1%. Trypsin-digested samples (15 μg) were then analyzed according to the chromatographic procedure described below under “HPLC/Mass Spectrometry Analysis.” Endoproteinase Lys-C Digestion of HuCC49ΔCH2 and Hinge Variant Antibodies (Determination of Disulfide Linkages)—Denatured and reduced samples were prepared by adding a final concentration of 4 m guanidine HCl and 25 mm DTT to 1.5 mg/ml sample. Nonreduced samples were prepared by adding a final concentration of 4 m guanidine HCl to 1.5 mg/ml sample. Samples were incubated for 2 h at 37°C. Digestion buffer (50 mm Tris, pH 7.0, and 0.062 absorbance units/ml endoproteinase Lys-C) was then added to the samples at 1:1 (v/v), and samples were incubated for 15 h at 37 °C. At 15 h, a second aliquot of enzyme (0.29 milli-absorbance units/μg of antibody) was added, and samples were incubated for an additional 6 h at 37°C. To quench the reaction, trifluoroacetic acid was added at 0.1% final concentration. Nonreduced and reduced endoproteinase Lys-C digested samples (12 μg) were then analyzed according to the procedure described below. HPLC/Mass Spectrometry Analysis—Samples were analyzed on an Agilent 1100 HPLC system connected to an Agilent MSD single quadrupole mass spectrometer. A reverse phase C18 column (Vydac catalog number 218TP52) was used with an eluant system of water, 0.1% trifluoroacetic acid (v/v) (Buffer A) and acetonitrile, 0.1% trifluoroacetic acid (v/v) (Buffer B), at a flow rate of 0.2 ml/min. A post-column “trifluoroacetic acid fixative” solution of acetonitrile and acetic acid (1:1 v/v) at 0.1 ml/min was added to enhance ionization. The column temperature was controlled at 45 °C, and the elution profile was monitored by absorbance at 215 and 280 nm. The total ion chromatogram was monitored in positive ion mode. Samples were injected onto the column, and the gradient was held at 0% Buffer B for 5 min. Elution was accomplished with a linear gradient of 0-50% Buffer B over 125 min, followed by a 75% Buffer B wash over 10 min and a 0% Buffer B re-equilibration over 30 min. Hinge-engineered HuCC49ΔCH2 Antibodies, Expression, and Western Blot Analysis—To examine the effect that hinge region modifications have on the secreted fraction of HuCC49ΔCH2 form A and form B isoforms, a series of genetically engineered HuCC49ΔCH2 hinge variants were constructed and the proteins expressed in CHO cells. TABLE ONE shows the UH and MH amino acid sequences for the parent HuCC49ΔCH2 antibody and the engineered hinge variants. Supernatants from ∼10 individually isolated transfected cell lines representing each hinge variant were collected, and the concentration of antibody in the culture supernatants was determined by immunoassay. Expression levels among the entire set of hinge variants ranged from 300 to 1200 ng/ml with no set of variants showing preferentially high or low expression (data not shown). This would imply that the hinge modifications do not have a major effect on protein expression. 3 ng of total antibody protein from each isolate was analyzed by nonreducing SDS-PAGE followed by Western blot with an anti-human κ chain antibody to detect HuCC49ΔCH2 form A and form B isoforms using methods shown in Fig. 1 (data not shown). This preliminary screen showed that differences in the observed ratios of the A:B isoforms among the different engineered antibodies were independent of expression levels and also indicated that the A:B isoform ratios were consistent within the set of approximate 10 clones for each of the hinge-engineered variants. We then performed a more comprehensive Western blot analysis examining a series of supernatant dilutions from representative cell lines for each of the hinge-engineered antibodies (Fig. 2). Under these denaturing conditions, form A antibodies migrate as single 120-kDa disulfide-linked homodimers, and form B antibodies, which fail to form inter-heavy chain disulfide bonds, migrate as two 60-kDa half-molecules. Also visible are disulfide-linked κ dimers and κ chain monomers. A number of lesser intense bands are visible between the A and B antibody isoforms and most likely represent degradation products. The HuCC49ΔCH2 Pro-243 antibody contains an insertion of a single middle hinge proline residue at Kabat position 243, resulting in a modest increase in the form A isoform fraction relative to parent HuCC49ΔCH2 antibody as evidenced by the presence of a 120-kDa band relative to the 60-kDa band at dilute sample concentrations (Fig. 2A). The HuCC49ΔCH2 PAP antibody containing the MH and LH PAP insertion (Kabat positions 243-245) further enhances production of the form A isoform (Fig. 2A). Substituting the cysteine at position 239 with serine in both molecules HuCC49ΔCH2 C239S:Pro-243 and HuCC49ΔCH2 C239S:PAP conversely favored production of the form B isoform (Fig. 2B). Substituting the cysteine at position 242 with serine in HuCC49ΔCH2 C242S:Pro-243 had a marginal effect on the relative quantity of secreted A:B isoforms, yet in contrast it dramatically increased production of the B isoform in HuCC49ΔCH2 C242S:PAP (Fig. 2B). Both antibodies containing the cysteine-rich 15-amino acid IgG3 hinge peptide CPEPKSCDTPPPCPR, HuCC49ΔCH2 G1/G3:Pro-243 and HuCC49ΔCH2 G1/G3:PAP, produced predominantly form A isoform with little or no detectable form B (Fig. 2C). In a similar trend as seen with the HuCC49ΔCH2 PAP antibody, the presence of the PAP addition in the HuCC49ΔCH2 G1/G3:PAP antibody resulted in a greater enhancement of secreted form A isoform compared with HuCC49ΔCH2 G1/G3:Pro-243 containing only the Pro-243 insertion.TABLE ONEParental HuCC49ΔCH2 hinge and variant hinge region amino acid sequences Open table in a new tab Spot densitometry was used to semi-quantify form A and form B band intensities from exposed films shown in Fig. 2 (TABLE TWO). Addition of a single proline residue at position 243 showed modest, albeit statistically insignificant, enhanced production levels of form A relative to the parent HuCC49ΔCH2 molecule (51 ± 7.2% versus 39 ± 7.5%, respectively). Addition of PAP at positions 243-245 increased the fraction of form A to 72 ± 3.1%, whereas the addition of the cysteine-rich 15-amino acid IgG3 hinge peptide sequence plus PAP resulted in up to 98 ± 1.2% secreted form A.TABLE TWOSemi-quantitation of A and B isoforms secreted by CHO cell lines expressing HuCC49ΔCH2 hinge region variant antibodiesConstructFraction form AFraction form B%%HuCC49ΔCH2 (parent)39±7.561±7.5Pro-24351±7.249±7.2PAP72±3.128±3.1C239S:Pro-24323±1.877±1.8C239S:PAP34±0.866±0.8C242S:Pro-24336±2.364±2.3C242S:PAP22±4.578±4.5G1/G3:Pro-24391±1.79±1.7G1/G3:PAP98±1.22±1.2 Open table in a new tab Protein Purification and Characterization—HuCC49ΔCH2 PAP and HuCC49ΔCH2 G1/G3:PAP antibodies were selected for scale-up biosynthesis by mammalian expression in CHO cells. Antibodies from precleared culture supernatants harvested from 10- to 25-liter capacity cell bags were purified by protein G affinity chromatography. HuCC49ΔCH2 G1/G3:PAP form A antibody was efficiently purified using protein G. 41.3 mg of HuCC49ΔCH2 G1/G3:PAP form A antibody was recovered by protein G chromatography from 7.5 liters of supernatant for a yield of 5.5 mg/liter and purity of 98.2% as assessed by scanning densitometry of Coomassie Blue-stained polyacrylamide gels. 213 mg of HuCC49ΔCH2 PAP form A:form B mixture was recovered by protein G chromatography with the form A isoform being further purified by hydrophobic interaction chromatography for a final yield of 138 mg from 25 liters or 5.52 mg/liter. Final purity of HuCC49ΔCH2 PAP A isoform was 98.4%. Nonreducing SDS-PAGE analysis of purified HuCC49ΔCH2, HuCC49ΔCH2 PAP, and HuCC49ΔCH2 G1/G3:PAP antibodies showed that the protein G affinity chromatography step resulted in form A protein yields consistent with the results seen by Western blot analysis (Fig. 3, lanes 1, 4, and 7). HuCC49ΔCH2 yielded ∼45% form A antibody, HuCC49ΔCH2 PAP yielded 83% form A, and HuCC49ΔCH2 G1/G3: PAP yielded 98.2% form A antibody. Form A HuCC49ΔCH2 PAP and HuCC49ΔCH2 G1/G3:PAP antibody elution profiles were analyzed by gel filtration HPLC (Fig. 4). Both antibodies eluted predominantly as single peaks with similar retention times. No aggregates or breakdown products were detected. Competitive Binding Activity—The binding activities of purified HuCC49ΔCH2, HuCC49ΔCH2 PAP, and HuCC49ΔCH2 G1/G3:PAP form A antibodies were compared in a DELFIA time-resolved fluorescence competitive binding assay to bovine submaxillary mucin, a source of the TAG-72 antigen (Fig. 5). In this assay increasing concentrations of unlabeled hinge-engineered antibodies are used to compete with a Eu3+-labeled form A HuCC49ΔCH2 antibody for binding to antigen. The data were analyzed using a one-site competition equilibrium binding model. For both of the hinge-engineered antibodies, HuCC49Δ" @default.
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