Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Anti-EGF-R/anti-CD3 bispecific monoclonal antibody method for i ts production and its use .
• the present invention relates to the construction of a hybrid hybridoma secreting a new anti-EGF-R/anti-CD3 bispecific monoclonal antibody (bimAb) of hybrid isotype ( IgGl/IgG2a) useful in tumor theraphy of cells showing the epidermal growth factor receptor (EGF-R+ ) .
• bimAb bispecific monoclonal antibody
• the present invention relates to such antibody and to i ts purification thereto .
• Such purified antibody was able to trigger the lysis of EGF-R+ tumor cell lines (for example A431 , IGROV-1 , MDA-468 and U-87 ) and of NIH-3T3 transfectant cells expressing the human EGF-R , by cy-tolytic T lymphocytes , but it was uneffective on EGF-R- tumor targets ( where the signs + and - associated to EGF-R indicate respectively the presence or the absence of said receptor on cells ) .
• Anti-tumor/anti-CD3 bispecific monoclonal antibodies are useful reagents able to confer anti-tumor specificity to T lymphocytes .
• the authors of the present invention constructed a hybrid hybridoma secreting an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (bimAb) and they compared such antibody to the above mentioned one (kindly provided by Dr.Ferrini and hereinafter named "Ferrini bimAb”) which, as far as the applicant knows, represents the only anti-EGF-R/anti-CD3 bimAb which has ever been disclosed.
• bimAb anti-EGF-R/anti-CD3 bispecific monoclonal antibody
• a fundamental characteristic of the present invention therefore refers to a hybrid hybridoma deposited at DSM (DSM ACC2151) and to the bispecific monoclonal antibody secreted by such hybrid hybridoma, and that is the anti-EGF-R/anti-CD3 bimAb of hybrid isotype (IgGl/IgG2a) hereinafter named DSM ACC2151 bimAb.
• a further aspect of the present invention refers to the preparation of DSM ACC2151 hybrid hybridoma and to the recovery of bimAb secreted by said hybridoma.
• a further characteristic of the present invention refers to the purification of such DSM ACC2151 bimAb by a Protein-A chromatography followed by a cation exchange chromatography.
• the present invention also refers to anti-EGF-R (DSM ACC2150) and anti-
• the present invention further refers to the production of a pharmaceutical composition comprising a monoclonal antibody or its fragments, for example F(ab' )2 fragment, according to the invention for the treatment of tumors, optionally in combination with a pharmaceutically acceptable carrier and/or excipient.
• FIG. 1 shows the effect of DSM ACC2151 hybrid hybridoma supernatant on the cytolytic activity of CD3+8+ BC1 cytolytic clone against the EGF- R+ A431 target cell line in a 4-hour -> Cr-release test.
• the ordinate reports the % of lysis of the target cells.
• FIG. 2 shows the effect of different concentrations of the purified DSM ACC2151 bimAb on the cytolytic activity of a cytotoxic CD8+ polyclonal cell line against EGF-R+ normal and neoplastic target cells.
• the abscissa reports in logarithmic scale the bimAb concentration (ng/ml).
• the ordinate reports the % of lysis concentration of the target cells.
• FIG. 3 shows the effect of DSM ACC2151 bimAb on the cytolytic activity of different populations of effector cells against the EGF-R+ A431 tumor cell line.
• the abscissa reports the E:T ratio of effector cells/target cells.
• the ordinate reports the % of lysis of target cells.
• FIG. 4 shows the effect of DSM ACC2151 bimAb and DSM ACC2152 mAb on the intracellular free Ca concentration on CD8+ polyclonal cell lines.
• panel A a rabbit anti-mouse Ig antibody was added as negative control.
• panels B and C DSM ACC2152 mAb and DSM ACC2151 bimAb were respectively used, followed by the addition of anti-mouse Ig antiserum as cross-linking agent.
• panel D cells were coated with DSM ACC2151 bimAb and cultured for 24 hours in the absence of soluble DSM ACC2151 bimAb. Before the experiment an anti-mouse IgG was added to the test. The ordinate reports the Ca concentration in nM.
• FIG. 5 shows the capacity of effector cells coated with DSM ACC2151 bimAb to retain the ability to lyse EGF-R+ target cells for a prolonged time.
• a CD8+ polyclonal cell line was coated with DSM ACC2151 bimAb (100 ng/ml for 1 hour at 4°C) and cultured for 24 hours in absence of soluble DSM ACC2151. Data are expressed as % of ⁇ Cr-release from A 31 target cells at a 10:1 effector/target cell ratio.
• Figure 6 shows analytical MonoS chromatography (Pharmacia) of material eluted from Protein A chromatography of DSM ACC2151 hybrid hybridoma conditioned medium.
• FIG. 7 shows analytical MonoS chromatography (Pharmacia) of DSM ACC2151 bimAb purified by cation exchange chromatography on S-Sepharose FF (Pharmacia) .
• FIG. 8 shows an electrophoretic analysis by sodium dodecyl sulphate- polyacrilamide (SDS-PAGE) gel of purified fractions of DSM ACC2151 bimAb.
• FIG. 9 shows the cytolytic activity of DSM ACC2151 bimAb if compared to that of Ferrini bimAb.
• the abscissa reports, in logarithmic scale, the amounts expressed in ng/ml.
• the ordinate reports the percentage of lysis according to an effector cells (lymphocytes)/target cells (tumor cells) ratio of 5/1.
• SEQ ID NO:3 reports the oligonucleotide sequence of the light chain variable (VL) region of DSM ACC2152 mAb .
• - SEQ ID NO : 5 reports the nucleotide sequence of the primer named VH1BACK Hindlll for use in PCR .
• - SEQ ID NO : 6 reports the nucleotide sequence of the primer named VHFOR-LINKER SCA for use in PCR .
• - SEQ ID NO : 7 reports the nucleotide sequence of the primer named VLBACK-LINKER SCA for use in PCR .
• - SEQ ID NO : 8 reports a nucleotide sequence of the primer named VLFOR UNIVERSAL EcoRl for use in PCR .
• the anti-EGF-R/anti-CD3 bispecific monoclonal antibody of IgGl/IgG2a hybrid isotype is secreted by a hybrid hybridoma obtainable by somatic hybridization of a hybridoma secreting an anti-EGF-R monoclonal antibody and a hybridoma secreting an anti-CD3 monoclonal antibody.
• the method for the production of a hybrid hybridoma secreting an anti- EGF-R/anti-CD3 bispecific monoclonal antibody of IgGl / IgG2a hybrid isotype therefore comprises the somatic hybridization of a hybridoma secreting an anti-EGF-R monoclonal antibody and a hybridoma secreting an anti-CD3 monoclonal antibody .
• the method for the production of an anti-EGF-R/ anti -CD3 bispecific monoclonal antibody comprises the somatic hybridization of a hybridoma secreting an anti-CD3 mAb and a hybridoma secreting an anti-EGF-R mAb , and then the purification of such antibody ( preferably , first by Protein-A chromatography and then by cation exchange chromatography) .
• the bimAb according to the invention can be easily and efficiently purified from parental mAbs ( i . e . anti-CD3 mAb and anti-EGF-R mAb ) by Protein-A chromatography followed by cation exchange chromatography .
• the hybridoma secreting the anti-EGF-R mAb is preferably the hybridoma named Mint5 (described in patent application PCT/EP94/02969 of the same applicant) and deposited as DSM ACC2150, while the hybridoma secreting the anti-CD3 mAb is preferably the hybridoma deposited as DSM ACC2152.
• the bispecific antibody according to the invention can be obtained also in form of recombinant single chain by genie fusion of sequences of variable regions of the light and heavy chain of genes encoding an anti- CD3 monoclonal antibody and an anti-EGF-R monoclonal antibody, respectively, according to the known techniques.
• Anti-CD3 mAb is preferably secreted by DSM ACC2152 hybridoma, while anti-EGF-R mAb is preferably secreted by DSM ACC2150 hybridoma.
• the purified bimAb according to the invention was able to trigger the lysis of EGF-R+ tumor cell lines (for example A431, IGROV-1, MDA468, U- 87, etc.) and of NIH-3T3 trasnfectacts expressing human EGF-R by cytolytic T lymphocytes, but it was uneffective to stimulate the lysis of EGF-R- tumor targets.
• EGF-R+ tumor cell lines for example A431, IGROV-1, MDA468, U- 87, etc.
• NIH-3T3 trasnfectacts expressing human EGF-R by cytolytic T lymphocytes
• Normal EGF-R+ cells keratinocytes and endometrial cells
• the ability of the bimAb according to the invention to deliver activation signals to T cells was evaluated by Ca mobilization and lymphokine production experiments.
• the bimAb according to the invention when administered in in vitro studies in soluble form, failed to induce an intracellular Ca increase, which occurred only after cross-linking induced by an anti-mouse IgG antibody (Fig.4).
• Secretion of lymphokines IFN-ga ma, TNF- ⁇ and GM-CSF was induced only by contact of the bimAb coated effector cells with the specific tumor target, according to the invention.
• the data hereinafter reported indicate that the bimAb according to the invention induce efficiently and specifically the secretion of lymphokines from T lymphocytes by contact with EGF-R+ tumor cells.
• T lymphocytes coated with the bimAb according to the invention could be used, for example, for: a) local treatment of tumors expressing EGF-R molecules, provided that tissues surrounding the tumor are EGF-R-; or b) systemic treatment of tumors overexpressing EGF-R.
• Peripheral blood mononuclear cells were isolated from 20 ml of p heparinized peripheral blood by centrifugation on Ficoll Hypaque density gradient. After three washes in RPMI 1640 medium (Seromed, Berlin, Germany) , supplemented with 10% heat-inactivated FCS (Fetal calf serum, PAA , Austria) , penicillin-streptomycin ⁇ 1% vol/vol of a commercial solution ; Seromed , Berlin , Germany ) and 200 mM L-Glutamin ( complete medium) cells were resuspended in the same medium and brought to the concentration of 10 /ml .
• FCS Fetal calf serum, PAA , Austria
• T lymphoblasts for immunization were produced by culture for 3 days in complete medium with 1% vol/vol PHA (Phytohemagglutin, Gibco, NY, USA) and cultured 3 days in 24 well Costar plates . Lymphoblasts were then expanded by culture in complete medium supplemented with 50 Ul/ml of recombinant IL2 (Cetus Corporation, CA, USA) . Immunization fusion and screening
• mice Six-week-old Balb/c female mice were immunized by four weekly intraperitoneal injections of 10 ' cultured lymphoblasts obtained by culture as in the previous step. T lymphoblasts from different cultures were pooled together and washed two times in PBS and resuspended in 0.2 ml PBS for injection. After 10 days mice received a booster injection of 10 ' cells , followed by splenectomy 3 days later . The spleen was
• HAT Hypoxantine Aminopterine Thymidine
• the amplified variable regions were cloned in the Hindlll-EcoRI restriction sites of pUCl ⁇ plasmid for sequencing. To introduce Hindlll and EcoRI restriction sites in the variable regions, they were reamplified and linked together according to Davis et al. , 1991. Bio/Technology 9. I65-I69. using the following primers: 5'VH: VH1BACK Hindlll: (SEQ ID NO: ) 3'VH: VHF0R-LINKER SCA: (SEQ ID NO:6) 5'VL: VLBACK-LINKER SCA: (SEQ ID N0:7) 3'VL: VLFOR UNIVERSAL EcoRl: (SEQ ID NO:8)
• DSM ACC2152 mAb VH nucleotide is reported in SEQ ID N0:1.
• DSM ACC2152 mAB VL nucleotide is reported in SEQ ID NO:3- The DSM ACC2152 VH and VL DNA sequences were analyzed for homology against PCgene data base (Intelligentics 1992) and were confermed as new sequences (being not present in the data bank) .
• Example 2
• Mint-5 ( IgGl ) anti-EGF-R hybridoma (described in the copending patent application PCT/EP94/02969 of the same applicant) was deposited as DSM ACC2150.
• DSM ACC2150 and DSM ACC2152 hybridomas were used for the preparation of a hybrid hybridoma named B/MRS 26.1.17 deposited as DSM ACC2151 secreting the bimAb of interest.
• Hypoxantineguanine phosphoribosyltransferase (HGPRT)-deficient mutants of the DSM ACC2150 hybridoma were selected by culture in the presence of increasing amounts of 8-azaguanine (1-50 ⁇ g/ml) (Sigma, St.Louis, M0).
• Hybrid hybridomas (quadromas) were selected by culture in HAT medium. Hybrid hybridomas were cloned by limiting dilution and tested for antibody production.
• the culture medium for hybrid hybridomas was RPMI 1640 supplemented with 10% FCS and 2mM L- glutamine.
• the hybrid hybridoma supernatants were directly screened for their ability to induce the CD8+ cytolytic T cell clone BC1 to lyse the EGF-R+ A431 tumor target cells (Merlino et al., 1984, Science, 224, 4l7 ⁇ 419). As shown in Figure 1, a 1:100 dilution of the supernatant of the selected hybrid hybridomas, the DSM ACC2151 secreting the bimAb, efficiently induced lysis of the A431 target cells. Parental anti-CD3 anti-EGF-R mAbs used alone or in combination failed to induce A431 cell lysis.
• T cell lines and clones were used as effector cells at effector/target cell ratio ranging from 40:1 to 0.6:1 in a 4h ⁇ C-release assay.
• the following cell lines were used as targets: IGROV-1 (ovarian carcinoma), MeWo (melanoma), Raij (Burkitt's lymphoma) , MDA-468 (breast cancer), A431 (epidermoid carcinoma), U-87 (glioma) , NIH-3T3 and NIH-3T3 EGF-R+ transfectants (kindly provided by Dr. P. Di Fiore, NIH, Bethesda, MD; Di Fiore et al.
• the percentage of lysis was calculated as described in Ferrini et al., 1987. J. Immunol., 138, 1297- 1302. To verify the target specificity, the bimAb DSM ACC2151 was tested for its ability to induce cytolysis of EGF-R+ and EGF-R- tumor target cells,
• EGF-R+ cell lines EGF-R+ cell lines
• EGF-R- cell lines EGF-R- cell lines
• Analysis of both EGF-R+ normal cells (keratinocytes and endothelial cells) required higher amounts of bimAb for half-maximal cytolysis being ED50 12 and 100 ng/ml, respectively, as compared with A431 cells ( Figure 2).
• EGF-R- PHA-induced lymphoblasts were not lysed in the presence of DSM ACC2151 bimAb.
• CD4+, CD8+ or TCRgamma/ ⁇ + T lymphocytes were mitogen-activated and expanded for 2-3 weeks in IL-2 containing medium. It was observed that lysis of A431 target cells could be induced by PHA-activated IL-2 expanded T lymphoblasts but not by resting PBL. Both CD8+ and TCRgamma/ ⁇ + cells efficiently lysed A431 in the presence of DSM ACC2151 bimAb, while CD4+ cells were poorly effective (Figure 3) ⁇ Similar results were obtained using PBL and purified populations isolated from three additional healthy donors.
• the level of cytolytic activity induced by DSM ACC2151 bimAb varied among effector cell populations obtained from the different donors.
• the cytolytic activity of four different CD3+CD8+ populations against A431 cells ranged from 45 to 70% of 3 Cr release at a 10:1 effector to target cell ratio.
• the concentration of cytoplasmic free Ca++ was determined in CD8+ polyclonal cell lines as described previously by Pantaleo et al., 1986, Eur. J. Immunol., 16, l639 ⁇ l644.
• Cells (2.5 10°) were stained with the acetoxymethylester of Fura-2 (1 uM final concentration; Sigma, St. Louis, MO, USA) , and the fluorescence of the cellular suspension was monitored with Spectrofluorimeter (Perkin Elmer Corp., Pomona, CA, USA) using a 2 ml quartz cuvette. Cell suspensions were excited at 345 nm and fluorescence was measured at 4 6 nm.
• Effector cells were stimulated with target cells, with parental antibodies or DSM ACC2151 bimAb in different combination.
• soluble anti-CD3 mAb plus 2 ng/ml of phorbol 1,2-myristate-l,3 ⁇ acetate (PMA) were used as a positive control.
• PMA phorbol 1,2-myristate-l,3 ⁇ acetate
• GM-CSF, IFN-gamma and TNF- ⁇ were measured using the EASIA kits (Enzyme Amplified Sensitivity Immunoassay, Medgenix Diagnostics, Brussels, Belgium) following instructions supplied by the manufacturer.
• anti-CD3 was able to induce secretion of IFN-gamma, TNF- ⁇ and GM-CSF in polyclonal T cell lines in the presence of phorbol esther PMA as a co-stimulus .
• EGF-R+ tumor cells were added to untreated lymphocytes , only low levels of lymphokine secretion could be achieved, while maximal lymphokine production was induced by the simultaneous use of bimAb and relevant target cells .
• EGF-R- tumor cell lines and bimAb did not induce significant lymphokine secretion.
• DSM ACC2151 bimAb represented only a fraction of the antibody molecules secreted by hybrid hybridomas ( Milstein and Cuello , 1984 , Immunol . Today, 5 , 299-304) , the present authors purified the DSM ACC2151 bimAb by protein A chromatography followed by cation exchange chromatography . DSM ACC2151 bimAb was purified from hybrid hybridoma culture supernatant . Cells producing bimAb were grown in serum- free medium in Acusyst R hollow fiber bioreactor (Endotronics) .
• Protein A binding buffer 1.5 M glycine pH 8.9; 150 mM NaCl
• Protein A elution buffer is: 0.1 M sodium citrate pH 3-6.
• Eluted material from Protein A was analyzed by MonoS chromatography as reported in Figure 6 showing the presence of DSM ACC2151 bimAb distributed in two peaks (1 and 2) and the presence of parental ⁇ CD3 DSM ACC2152 and ⁇ EGF-R DSM ACC2150 mAbs.
• eluted material from Protein A was diluted 1:1 in buffer A (25 mM sodium acetate pH 5-0) and located on xk 26 column of S-Sepharose FF (Pharmacia) conditioned with buffer A plus 50 mM NaCl. Elution was performed by a step procedure with buffer B made of buffer A plus 1 M NaCl. The steps were 20%, 25% and 30% of buffer B. Fractions from S-Sepharose chromatography were analyzed by MonoS chromatography and fractions corresponding to peak 1 of Figure 6 were pooled and confirmed to be DSM ACC2151 bimAb according to IEF pattern and functional activity.
• Figure 7 shows the analytical MonoS chromatography of purified DSM ACC2151 bimAb. Purity was further confirmed by SDS-PAGE analysis on Phast gel 4-15% (Pharmacia) ( Figure 8) .
• Figure 8 shows a SDS-PAGE analysis exhibiting the electrophoretic mobility in both reducing and non-reducing conditions.
• Lane 3 contains parental ⁇ CD3 mAb and lanes 4 and 5 contain ⁇ EGF-R and ⁇ CD3 standard in mAbs.
• the presence of the highest concentration of bifunctional molecules of bimAb DSM ACC2151 in purified peak 1 was confirmed by cytolitic assay against A431 target cells. Comparison of the DSM ACC2151 bimAb activity with the Ferrini bimAb activity
• DSM ACC2151 bimAb was prepared and purified as above described.
• Ferrini bimAb was obtained by Dr. Ferrini (as previously specified).
• the activity of the two anti-EGF-R/anti-CD3 bimAbs was analysed testing the cytolitic activity of a CD8+ cytolitic cell line against A431 target cells in the presence of different concentrations of bimAb.
• DSM ACC2151 bimAb was significatively stronger to induce cytolitic activity at low concentration of bimAb if compared to Ferrini bimAb.
• IC50 calculated for the two bimAbs (Fig.9) shows that DSM ACC2151 bimAb is about 10 times more active than Ferrini bimAb. According to the evaluation of the activities of the above cited bimAbs, then it results surprising the different behaviour (that maybe could indicate a different mechanism of action) of the two bimAbs studied.
• a DNA sequence coding for an antibody according to the invention can be prepared. Said sequence, eventually, can be inserted into a plasmid vector, preferably under the control of a promoter which enables its expression in a desired host cell.
• Said host cell is selected from the group consisting of bacterial, yeast, insect, plant or mammalian cells.
• the present invention is therefore directed also to a method for the production of a monoclonal antibody or fragments thereof according to the invention comprising the cultivation of a host cell under conditions appropriate for the expression of said DNA sequence and recovering the product from the culture.
• An antibody according to the present invention is also useful in theraphy.
• An antibody or its fragments [for example F(ab')2_ according to the invention is also used for the preparation of a pharmaceutical composition, useful for the treatment of tumors, in combination with a pharmaceutically acceptable carrier and/or excipient.
• a pharmaceutical composition according to the present invention therefore comprises a monoclonal antibody or its fragments according to the invention for the treatrement of tumors , optionally in combination with a pharmaceutically acceptable carrier and/or excipient .

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Cited By (28)

• 1993
  • 1993-12-01 IT ITFI930246A patent/IT1271461B/it active IP Right Grant
• 1994
  • 1994-12-01 AU AU12417/95A patent/AU1241795A/en not_active Abandoned
  • 1994-12-01 WO PCT/EP1994/003995 patent/WO1995016037A1/en active Application Filing

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