Universal glycoform compositions and methods for enhancing antibody efficacy

Abstract

This application relates to general glycoform compositions and methods for enhancing antibody efficacy. The present invention relates to compositions comprising an antibody or a binding fragment thereof, further comprising a general Fc glycoform, and methods of use.

Description

[0001] This application is a divisional application of the invention patent application with the application date of January 30, 2016, the application number of "201680008450.2", and the title of "Universal Glycoform Compositions and Methods for Enhancing Antibody Efficacy". Technical Field

[0002] The present invention relates to selected universal Fc glycoforms that are modulated according to the desired binding / effector activity and are used to enhance the therapeutic efficacy of antibodies against many diseases, including cancer, inflammatory disorders, and infectious diseases. Specifically, for enhancing therapeutic efficacy, selected and / or directionally optimized universal Fc glycoforms can be generated and / or incorporated into the design and / or production of monoclonal antibodies. Background Art

[0003] The efficacy of antibody-based therapies against many diseases, including inflammatory disorders, cancer, infectious diseases, and solid organ transplant rejection, has a proven record. Currently, more than 40 therapeutic monoclonal antibodies (mAbs) have been approved for clinical use in the United States (USA), the European Union (EU), and several other countries. Most of these are used to treat cancer and immune diseases. Examples of therapeutic antibodies with anti-tumor activity include anti-CD20, anti-Her2, anti-EGFR, anti-CD40, anti-CTLA-4, and anti-PD-1 antibodies.

[0004] Most of the approved biopharmaceuticals are produced in mammalian cell culture systems to deliver proteins with the desired glycosylation patterns and thereby ensure reduced immunogenicity as well as higher in vivo efficacy and stability. Non-human mammalian expression systems, such as CHO or NS0 cells, have the machinery necessary to add complex human-type polysaccharides. However, the polysaccharides produced in these systems may be different from those produced in the human body. Their glycosylation machinery often adds unwanted carbohydrate determinants, which can alter protein folding, induce immunogenicity, and shorten the circulating life of the drug.

[0005] In addition, mammalian cell cultures deliver a heterogeneous mixture of glycosylation patterns that do not all have the same properties. These glycosylation patterns can affect the properties of therapeutic proteins, such as safety, efficacy, and serum half-life. Mammalian cell culture systems deliver a heterogeneous mixture of glycosylation patterns that do not all have the same properties. Summary of the Invention

[0006] Fc glycosylation has become an important topic in the field of therapeutic monoclonal antibodies. Fc glycosylation can significantly alter Fc effector functions, such as Fc receptor binding and complement activation, and thus affect the in vivo safety and efficacy profiles of therapeutic antibodies. The diversity of Fc glycosylation within an antibody will correspond to the diversity of Fc effector functions. Therefore, this heterogeneity in Fc glycans has functional consequences as it affects the binding of IgG molecules to Fc receptors and thereby affects antibody effector functions, and may trigger undesirable effects in patients, thus raising safety concerns.

[0007] There is a need to improve monoclonal antibody therapies for many diseases, including inflammatory disorders, cancer, and infectious diseases. Some specific glycoforms in Fc can confer improved effector functions for desired biological functions, such as antibody-dependent cellular cytotoxicity (ADCC). Therefore, it is applicable to generate therapeutic antibodies with optimized Fc glycoforms.

[0008] Accordingly, the present invention provides selected general Fc glycoforms modulated according to desired binding / effector activities, which are used to enhance the efficacy of therapeutic antibodies against many diseases, including cancer, inflammatory disorders, and infectious diseases. To enhance therapeutic efficacy, the selected and / or directed-optimized general Fc glycoforms can be applied and / or incorporated into the design and / or generation of monoclonal antibodies, preferably therapeutic monoclonal antibodies.

[0009] In one aspect, the present invention provides an Fc glycoform for enhancing the binding / effector activity of a monoclonal antibody, wherein the antibody comprises a glycoform having the following formula:

[0010] Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2

[0011] In some embodiments, the present invention provides a pharmaceutical composition comprising the Figure 1 glycoform and a pharmaceutically acceptable carrier. In one aspect, the present invention provides a method of treating infectious, hyperproliferative diseases and / or conditions, wherein the method comprises administering to an individual in need a pharmaceutical composition comprising a glycoform having Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2.

[0012] In some embodiments, the antibody is a murine, chimeric, humanized, and / or human MC41 antibody, which comprises the following sequences:

[0013] Table 1-1. Amino acid and nucleotide sequences of anti-SSEA-4 murine MC41.

[0014]

[0015] Table 1-2. Amino acid and nucleotide sequences of the second humanized monoclonal antibody hMC41.

[0016] The second

[0017]

[0018]

[0019] Table 1-3. Amino acid and nucleotide sequences of the third humanized monoclonal antibody hMC41.

[0020] The third

[0021]

[0022]

[0023] In one aspect, the present invention provides an isolated monoclonal antibody or a binding fragment thereof that binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, wherein the antibody or fragment thereof comprises an Fc glycoform for enhancing the binding / effector activity of the monoclonal antibody, and wherein the antibody comprises a glycoform having the following formula:

[0024] Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2

[0025] In one embodiment, the antibody is IgG1 and the binding to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→ is specific binding.

[0026] In one embodiment, the antibody comprises a VH having SEQ ID NO:147 or SEQ ID NO:137 and a VL having SEQ ID NO:148 or SEQ ID NO:138.

[0027] In one embodiment, the isolated antibody or its antigen-binding fragment comprises H-CDR1, H-CDR2, and H-CDR3 selected from (i)-(iii) respectively:

[0028] (i) H-CDR1 selected from SEQ ID NO:152 (GFSLTSYG);

[0029] (ii) H-CDR2 selected from SEQ ID NO:153 (IWGEGST);

[0030] (iii) H-CDR3 selected from SEQ ID NO:154 (AMTGTAY);

[0031] and comprising L-CDR1, L-CDR2 and L-CDR3 respectively selected from (iv) to (vi):

[0032] (iv) L-CDR1 selected from SEQ ID NO:149 (SSVSY);

[0033] (v) L-CDR2 selected from SEQ ID NO:150 (DTS); and

[0034] (vi) L-CDR3 selected from SEQ ID NO:151 (HQWSSSPHT).

[0035] In one embodiment, the isolated antibody or antigen-binding fragment further comprises H-FR1, H-FR2, H-FR3 and HFR4 respectively selected from (i)-(iv):

[0036] (i) H-FR1 selected from SEQ ID NO:159 (QVQLKESGPGLVAPSQSLSITCTVS);

[0037] (ii) H-FR2 selected from SEQ ID NO:160 (VSWIRQPPGKGLEWIGV);

[0038] (iii) H-FR3 selected from SEQ ID NO:161 (NYHSVLISRLTISKDNSKSQVFLKLNSLQTDDTATYYC);

[0039] (iv) H-FR4 selected from SEQ ID NO:162 (WGQGTLVTVSS);

[0040] and comprising L-FR1, L-FR2, L-FR3 and L-FR4 respectively selected from (v) to (viii):

[0041] (v) L-FR1 selected from SEQ ID NO:155 (QIVLTQSPAIMSASPGEKVTMTCSAS);

[0042] (vi) L-FR2 selected from SEQ ID NO:156 (MHWYQQKSGTSPKRWIY);

[0043] (vii) L-FR3 selected from SEQ ID NO:157 (KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAATYYC);

[0044] (viii) L-FR4 selected from SEQ ID NO:158 (FGGGTKVEIKR).

[0045] In one embodiment, the antibody is a human antibody.

[0046] In one embodiment, the antibody is a humanized antibody.

[0047] In one embodiment, the antibody comprises a VH having SEQ ID NO:200, SEQ ID No.210 or SEQ ID NO:137 and a VL having SEQ ID No:201, SEQ ID No.211 or SEQ ID No:221.

[0048] In one embodiment, the isolated antibody or its antigen-binding fragment comprises H-CDR1, H-CDR2 and H-CDR3 respectively selected from (i)-(iii):

[0049] (i) H-CDR1 selected from SEQ ID NO:207, SEQ ID NO:217, SEQ ID NO:227;

[0050] (ii) H-CDR2 selected from SEQ ID NO:208, SEQ ID NO:218, SEQ ID NO:228;

[0051] (iii) H-CDR3 selected from SEQ ID NO:209, SEQ ID NO:219, SEQ ID NO:229;

[0052] and comprises L-CDR1, L-CDR2 and L-CDR3 respectively selected from (iv) to (vi):

[0053] (iv) L-CDR1 selected from SEQ ID NO:204, SEQ ID NO:214 and SEQ ID NO:224;

[0054] (v) L-CDR2 selected from SEQ ID NO:205, SEQ ID NO:215 and SEQ ID NO:225;

[0055] (vi) L-CDR3 selected from SEQ ID NO:206, SEQ ID NO:216 and SEQ ID NO:226.

[0056] In one embodiment, the antibody of claim 9, wherein the antibody is a human antibody.

[0057] In one embodiment, the antibody of claim 9, wherein the antibody is a humanized antibody.

[0058] In one embodiment, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment or a single-chain Fv fragment.

[0059] In one aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or a binding fragment thereof as claimed in any one of claims 6, 7, 10 or 11 and a pharmaceutically acceptable carrier.

[0060] In one embodiment, the pharmaceutical composition is suitable for treating hyperproliferative diseases.

[0061] In one aspect, the present invention provides a method of treating cancer in an individual in need thereof, wherein the method comprises administering to the individual a therapeutically effective amount of a pharmaceutical composition as claimed in claim 13, wherein the administered antibody enhances the ADCC activity of the individual.

[0062] In one embodiment, the cancer targeted by the treatment method is selected from the group consisting of: brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, bone cancer, skin cancer, cervical cancer, ovarian cancer and prostate cancer.

[0063] In one embodiment, the method comprises co-administering, as appropriate, the pharmaceutical formulation with at least one other chemotherapeutic agent.

[0064] In another aspect, the present invention also provides a method of preparing a homogeneous antibody population, the method comprising:

[0065] (a) contacting a monoclonal antibody with α-trehalosidase and at least one endoglycosidase;

[0066] (b) generating a detrehalosylated antibody having a single N-acetylglucosamine (GlcNAc); and

[0067] (c) adding a universal polysaccharide to the GlcNAc of the Fc region of the antibody to form a homogeneous antibody having said glycoform.

[0068] In one embodiment, the antibody or its binding fragment comprises an antibody or its binding fragment that specifically binds to one or more antigens selected from the group consisting of Globo H, SSEA-3 and SSEA-4.

[0069] Another aspect of the present invention provides a modified humanized glycoantibody based on MC48. Examples and their amino acid and nucleic acid structures / sequences are provided below:

[0070] Table 17-0. Amino acid and nucleotide sequences of murine monoclonal antibody MC48.

[0071]

[0072] Table 17-1. Amino Acid and Nucleotide Sequences of Humanized Monoclonal Antibody MC48 (the 1st kind)

[0073]

[0074]

[0075] Table 17-2. Amino Acid and Nucleotide Sequences of Humanized Monoclonal Antibody MC48 (the 2nd kind)

[0076]

[0077]

[0078] Table 17-3. Amino Acid and Nucleotide Sequences of Humanized Monoclonal Antibody MC48 (the 3rd kind)

[0079]

[0080]

[0081] Table 17-4. Amino Acid and Nucleotide Sequences of Humanized Monoclonal Antibody MC48 (the 4th kind)

[0082]

[0083]

[0084] Antibodies Specific for SSEA4 and Its Fragments

[0085] One aspect of the present invention provides novel antibodies that bind to SSEA-4 and its fragments. The anti-SSEA-4 antibodies bind to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide) and Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1 (fragment of SSEA-4 hexasaccharide). In some instances, the antibodies are capable of binding to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galβ1. In some instances, the antibodies are capable of binding to Neu5Gcα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (analogue of SSEA-4 hexasaccharide).

[0086] In some embodiments, the method enhances ADCC.

[0087] In one embodiment, the pharmaceutical composition comprises an antibody or a binding fragment thereof having a universal biantennary N-glycan, the terminal of the universal biantennary N-glycan being sialic acid in an α-2,6 linkage.

[0088] In another aspect, the present invention provides a method for treating and / or reducing the risk of cancer in an individual, comprising administering to the individual in need thereof a therapeutically effective amount of a composition as described herein.

[0089] Treatment can reduce tumor size, eliminate malignant cells, prevent cancer metastasis, prevent recurrence, reduce or kill disseminated cancer, extend survival and / or extend the time to cancer progression in tumors.

[0090] In some embodiments, the compositions described herein are formulated as injectables. In some embodiments, the composition is administered subcutaneously.

[0091] Details of certain embodiments of the present invention are set forth herein. Other features, objects, and advantages of the present invention will be apparent from the embodiments, drawings, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 . Structure of optimized universal Fc glycan in therapeutic antibodies.

[0093] Figure 2 . General strategy for preparing homogeneous antibodies, where the optimized universal glycan is located in the Fc region to enhance its therapeutic activity.

[0094] Figure 3 . Demonstration of improved antiviral antibody-dependent cell-mediated cytotoxicity (ADCC) results of anti-influenza virus antibodies.

[0095] Figure 4 . Table listing exemplary increased ADCC activity of anti-CD20 GAb compared to rituximab.

[0096] Figure 5 . Six anti-CD20 GAbs.

[0097] Figure 6A and 6B . Figure 6A At the top of the table, Figure 6BLocated at the bottom of the table. The table lists exemplary FcγRIIIA binding of anti-CD20 GAb and rituximab. Analysis known in the art can be used to measure FcγRIIIA binding. Exemplary assays are described in the examples. Fc receptor binding can be determined as the relative ratio of anti-CD20 GAb to rituximab. In an exemplary embodiment, Fc receptor binding is increased by at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or higher.

[0098] Figure 7 . Binding activities of different homogeneous antibody pairs to different cells having CD20. Figure 7 Show the CDC effects of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc on Ramos cells.

[0099] Figure 8 . Binding activities of different homogeneous antibody pairs to different cells having CD20. Figure 8 Show the CDC effects of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc on Raji cells.

[0100] Figure 9 . Binding activities of different homogeneous antibody pairs to different cells having CD20. Figure 9 Show the CDC effects of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc on SU-DHL-4 cells.

[0101] Figure 10 . Depletion of human SU-DHL-4B cells, as analyzed by FACS. Cells were cultured with different concentrations of anti-CD20 Gab Rituxan-SCT, Rituxan-GlcNAc, and rituximab in the absence or presence of 15% autologous plasma. After washing, the cells were stained with anti-CD2-PE and anti-CD19-FITC. B cell depletion was analyzed on FACS based on CD19+CD2− B cells ( Figure 13 ).

[0102] Figure 11 . Depletion of human Ramos B cells, as analyzed by FACS. Cells were cultured with different concentrations of anti-CD20 Gab Rituxan-SCT, Rituxan-GlcNAc, and rituximab in the absence or presence of 15% autologous plasma. After washing, the cells were stained with anti-CD2-PE and anti-CD19-FITC. B cell depletion was analyzed on FACS based on CD19+CD2− B cells ( Figure 13 ).

[0103] Figure 12 . Depletion of human Raji B cells, as analyzed by FACS. Cells were cultured with different concentrations of anti-CD20 Gab Rituxan-SCT, Rituxan-GlcNAc, and rituximab in the absence or presence of 15% autologous plasma. After washing, the cells were stained with anti-CD2-PE and anti-CD19-FITC. On FACS, B cell depletion was analyzed based on CD19+CD2- B cells ( Figure 13 ).

[0104] Figure 13 . Depletion of human B cells by different isotype antibodies.

[0105] Figure 14 . Table listing exemplary ADCC activities enhanced by anti-HER2 GAb compared to trastuzumab.

[0106] Figure 15 . Table listing exemplary FcγRIIIA binding of anti-HER2 GAb and rituximab.

[0107] Figure 16A . Solid-based ELISA coated with SSEA-4 to determine the binding activity of humanized MC41 phage clones.

[0108] Figure 16B . Solid-based ELISA coated with BSA to determine the binding activity of humanized MC41 phage clones.

[0109] Figure 17A . To evaluate the binding activity of intact humanized MC41 IgG, intact IgGs of humanized MC41 1, 2, 3, and chimeric MC41 (chMC41) were constructed. ELISA results showed that humanized MC41 2 and 3 could react with SSEA-4 in a dose-dependent manner ( Figure 17A ), but did not react with BSA ( Figure 17B ), and the same results were observed for chMC41.

[0110] Figure 17B . To evaluate the binding activity of intact humanized MC41 IgG, intact IgGs of humanized MC41 1, 2, 3, and chimeric MC41 (chMC41) were constructed. ELISA results showed that humanized MC41 2 and 3 could react with SSEA-4 in a dose-dependent manner ( Figure 17A ), but did not react with BSA ( Figure 17B ), and the same results were observed for chMC41.

[0111] Figure 18A and Figure 18B . Figure 18A display Figure 18B the legend of the bar graph. To determine the binding specificities of chMC41 and hMC41, a glycan array was constructed. The results are shown in Figure 18B . Chimeric and humanized MC41 showed greater specific binding than the commercially available SSEA4 antibody (MC813), which only recognizes SSEA4 or SSEA4 modified with hydroxyacetyl groups.

[0112] Figure 19A and 19B . Figure 19A display Figure 19B the legend of the bar graph. To determine the binding specificities of chMC41 and hMC41, a glycan array was constructed. The results are shown in Figure 19B . Chimeric and humanized MC41 showed greater specific binding than the commercially available SSEA4 antibody (MC813), which only recognizes SSEA4 or SSEA4 modified with hydroxyacetyl groups.

[0113] Figure 20A . To study the effector functions of chMC41, an ADCC assay was performed. The HPAC pancreatic cancer cell line was used to evaluate the ADCC and CDC activities of chMC41, the positive control MC813, or the negative control NHIgG.

[0114] Figure 20B . To study the effector functions of chMC41, a CDC assay was performed. The HPAC pancreatic cancer cell line was used to evaluate the ADCC and CDC activities of chMC41, the positive control MC813, or the negative control NHIgG.

[0115] Figure 21A and Figure 21B . To study the effector functions of chMC41 and hMC41, ADCC and CDC assays were performed. The HPAC pancreatic cancer cell line was used to evaluate the ADCC and CDC activities of chMC41, hMC41, the positive control MC813, or the negative controls NHIgG and NMIgG. Figure 21A shows the cancer cell killing activity occurring via ADCC. Figure 21B shows the cancer cell killing activity occurring via CDC.

[0116] Figure 22A . To identify the antibody that binds to SSEA-4, the inventors used a solution containing 2×10 10A phage display human naïve scFv library of [number] members was presented, which was established as described in the inventors' previous report (Lu et al., 2011). First, this library was removed by phage conjugated with Dynabeads, and then phages binding to SSEA-4 were selected by SSEA-4-PEG conjugated Dynabeads. During biopanning, the inventors used two buffer systems: PBS and PBS containing 0.01% Tween 20 (PBST0.01). After five rounds of affinity selection, the phage recovery rate in the fifth round increased, being approximately 55-fold and 80-fold that of the first round in the PBS and PBST0.01 systems, respectively.

[0117] Figure 22B . To identify the antibodies binding to SSEA-4, the inventors used a phage display human naïve scFv library of [number] members, which was established as described in the inventors' previous report (Lu et al., 2011). First, this library was removed by phage conjugated with Dynabeads, and then phages binding to SSEA-4 were selected by SSEA-4-PEG conjugated Dynabeads. During biopanning, the inventors used two buffer systems: PBS and PBS containing 0.01% Tween 20 (PBST0.01). After five rounds of affinity selection, the phage recovery rate in the fifth round increased, being approximately 55-fold and 80-fold that of the first round in the PBS and PBST0.01 systems, respectively. 10 A phage display human naïve scFv library of [number] members was presented, which was established as described in the inventors' previous report (Lu et al., 2011). First, this library was removed by phage conjugated with Dynabeads, and then phages binding to SSEA-4 were selected by SSEA-4-PEG conjugated Dynabeads. During biopanning, the inventors used two buffer systems: PBS and PBS containing 0.01% Tween 20 (PBST0.01). After five rounds of affinity selection, the phage recovery rate in the fifth round increased, being approximately 55-fold and 80-fold that of the first round in the PBS and PBST0.01 systems, respectively.

[0118] Figure 23A . Phage clones were randomly selected and their binding to SSEA-4 was tested by ELISA.

[0119] Figure 23B . Phage clones were randomly selected and their binding to SSEA-4 was tested by ELISA.

[0120] Figure 23C . Phage clones were randomly selected and their binding to SSEA-4 was tested by ELISA.

[0121] Figure 23D . Phage clones were randomly selected and their binding to SSEA-4 was tested by ELISA.

[0122] Figure 24 . To examine the specificity and binding affinity of two phage clones, the inventors performed comparative ELISA using the same phage titer against globo-series glycoconjugates including SSEA-4-BSA, Globo H-BSA, and SSEA-3-BSA.

[0123] Figure 25A.To establish fully human antibodies (hAbs) against SSEA-4, the inventors molecularly engineered the VH and VL coding sequences of p2-78scFv and incorporated them into the human IgG1 backbone. The anti-SSEA-4 p2-78 hAb was generated using the FreeStyle 293 expression system and then purified via a Protein G agarose column. The inventors used Coomassie blue staining to examine the antibody purity by SDS-PAGE analysis.

[0124] Figure 25B .ELISA was performed to study the binding activity of the p2-78 hAb to globo-series glycans.

[0125] Figure 26A .The positive control MC631 of a commercially available IgM antibody. A glycan array containing 203 different glycans was used to further confirm the specificity of the p2-78 hAb.

[0126] Figure 26B .The glycans recognized by the p2-78 hAb.

[0127] Figure 26C .A glycan array containing 203 different glycans was used to further confirm the specificity of the p2-78 hAb.

[0128] Figure 27A .After aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activities of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activities to SSEA-4. The data showed that the binding affinities of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones were maintained compared to the binding affinities of the murine mAb MC48 or MC41.

[0129] Figure 27BAfter aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activities of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activities to SSEA-4. The data showed that the binding affinities of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones were maintained compared to the binding affinities of the murine mAbs MC48 or MC41.

[0130] Figure 28A After aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activities of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activities to SSEA-4. The data showed that the binding affinities of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones were maintained compared to the binding affinities of the murine mAbs MC48 or MC41.

[0131] Figure 28BAfter aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activities of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activities to SSEA-4. The data showed that the binding affinities of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones were maintained compared to the binding affinities of the murine mAb MC48 or MC41.

[0132] Figure 29A and Figure 29B After aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activities of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activities to SSEA-4. The data showed that the binding affinities of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones were maintained compared to the binding affinities of the murine mAb MC48 or MC41.

[0133] Figure 29BAfter aligning the VH and VL variable regions of MC48 and MC41 with the NCBI IgBLAST or IMGT databases, the inventors generated the 1st, 2nd, 3rd, and 4th humanized MC48 sequences and the 1st, 2nd, and 3rd humanized MC41 sequences. The inventors then constructed and generated phage-displayed scFv forms based on these humanized MC48 and MC41 sequences. To determine the binding activity of the humanized MC48 and MC41 phage clones, the inventors performed a solid-based ELISA coated with SSEA-4-BSA. The inventors found that the 3rd and 4th humanized MC48 and the 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4 in a dose-dependent manner, while the 1st and 2nd humanized MC48 and the 1st MC41 scFv lost their binding activity to SSEA-4. The data showed that the binding affinity of the 4th humanized MC48 and the 3rd humanized MC41 scFv phage clones was maintained compared to the binding affinity of the murine mAb MC48 or MC41.

[0134] Figure 30A and 30B . To evaluate the binding activity of the full-length humanized MC41 IgG, the inventors constructed the full-length IgG of the 1st, 2nd, 3rd humanized MC41 and chimeric MC41 (chMC41). The ELISA results showed that the 2nd and 3rd humanized MC41 could react with SSEA-4 in a dose-dependent manner ( Figure 30A ), but not with BSA ( Figure 30B ), and the same results were observed for chMC41.

[0135] Figure 31A and Figure 31B . To determine the binding specificity of chMC41 and hMC41, a glycan array was constructed. The chimeric and humanized MC41 showed greater specific binding than the commercially available SSEA4 antibody (MC813). It only recognized SSEA4 or SSEA4 modified with hydroxyacetyl groups. Figure 31A Show the glycans recognized and Figure 31B Show the array results.

[0136] Figure 32A and Figure 3 . To determine the binding specificity of chMC41 and hMC41, a glycan array was constructed. The chimeric and humanized MC41 showed greater specific binding than the commercially available SSEA4 antibody (MC813). It only recognized SSEA4 or SSEA4 modified with hydroxyacetyl groups. ​ Show the glycans recognized and ​ Show the array results.

[0137] ​ and ​.To study the effector functions of hMC48, chMC41 and hMC41, ADCC and CDC assays were performed. Using HPAC, BxPC3 and PL45 pancreatic cancer cell lines, the ADCC and CDC activities of hMC48 or NHIgG at a concentration of 10 μg / ml were evaluated.

[0138] ​ .HPAC cells were treated with chMC41, hMC41, positive control MC813 or negative control NHIgG.

[0139] ​ .HPAC cells were treated with chMC41, hMC41, positive control MC813 or negative control NHIgG.

[0140] ​ and 35B .Data showed that the effector functions of hMC41 and chMC41 were superior to those of hMC48. Interestingly, humanized MC41 not only maintained its original activity, but also showed stronger cancer cell killing activity via ADCC and CDC than MC813.

[0141] ​ .The binding ability of hMC41 and hMC48 to SSEA-4 was examined by ELISA. The results showed that the binding of hMC41 to SSEA-4 was much better than that of hMC48. The maximum binding value of humanized MC41 was higher and the Kd value was smaller compared to hMC48 (0.2 μg / ml and 4.6 μg / ml for hMC41 and hMC48, respectively). Detailed implementation

[0142] Chemical definition

[0143] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements (CAS version) on the inside front cover of the 75th edition of the Handbook of Chemistry and Physics, and specific functional groups are generally defined as described therein. Additionally, the general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987. Furthermore, exemplary polysaccharide and antibody methods are described in US 20100136042, US 20090317837, and US 20140051127 to Wong et al., the disclosures of each of which are incorporated herein by reference.

[0144] The compounds described herein may contain one or more asymmetric centers and may thus exist in various isomeric forms (e.g., enantiomers and / or diastereomers). For example, the compounds described herein may exist as individual enantiomers, diastereomers, or geometric isomers, or may exist as mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions at page 268 (ed. E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention additionally encompasses the compounds described herein in individual isomeric forms substantially free of other isomers and alternatively in mixtures of various isomers.

[0145] When a numerical range is recited, it is intended to encompass every numerical value and sub-range within that range. For example, " 1-6 C 1-6 " is intended to encompass C1, C2, C3, C4, C5, C6, C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 C 5-6 and C

[0146] Unless otherwise specified, well-known techniques in molecular biology, microbiology, recombinant DNA, and immunology within the skill of the art will be employed to practice the present invention. Such techniques are well described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Edition, Sambrook, Fritsch and Maniatis eds. (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Volumes 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, Harlow and Lane s eds. (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell eds., 1986).

[0147] As used herein, the term "polysaccharide" refers to a polysaccharide or an oligosaccharide. Polysaccharides are also used herein to refer to the carbohydrate portion of the following glycoconjugates, which are glycoproteins, glycolipids, glycopeptides, glycoproteomes, peptidoglycans, lipopolysaccharides, or proteoglycans. Polysaccharides generally consist only of O-glycosidic linkages between monosaccharides. For example, cellulose is a polysaccharide (or more specifically, a glucan) composed of β-1,4-linked D-glucose, and chitin is a polysaccharide composed of β-1,4-linked N-acetyl-D-glucosamine. Polysaccharides can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Polysaccharides can be found linked to proteins, such as glycoproteins and proteoglycans. They are typically found on the extracellular surface. O-linked and N-linked polysaccharides are common in eukaryotes and can also be found in prokaryotes (although less commonly). N-linked polysaccharides are found linked to the R-group nitrogen (N) of asparagine in the sequon. The sequon is the Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid other than proline.

[0148] As used herein, the term "epitope" is defined as a portion of an antigen molecule that contacts the antigen-binding site of an antibody or a T cell receptor.

[0149] As used herein, the term "flow cytometry" or "FACS" means a technique that examines the physical and chemical characteristics of particles or cells suspended in a liquid stream via optical and electronic detection devices.

[0150] A non-naturally occurring antibody or an "isolated" antibody is an antibody that has been identified and separated and / or recovered from the components of its native environment. The components of its native environment are substances that interfere with the research, diagnostic, or therapeutic use of the antibody and can include enzymes, hormones, and other proteins or non-protein solutes. In one embodiment, the antibody is purified (1) to greater than 95 wt% of the antibody, as determined, for example, by the Lowry method, and in some embodiments greater than 99 wt%; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using, for example, a spinning cup sequencer; or (3) to homogeneity, which is achieved by using, for example, Coomassie blue or silver stain and performing SDS-PAGE under reducing or non-reducing conditions. Isolated antibodies include antibodies that are located in situ within recombinant cells because at least one component of the antibody's native environment will not be present. However, isolated antibodies will generally be prepared by at least one purification step.

[0151] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the name of the Fab' in which the cysteine residue of the constant domain bears a free thiol group. The F(ab')2 antibody fragment was initially produced as a pair of Fab' fragments with hinge cysteines therebetween. Other chemical conjugations of antibody fragments are also known.

[0152] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be classified into one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain.

[0153] Depending on the amino acid sequence of the heavy chain constant domain, antibodies (immunoglobulins) can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). An antibody can be part of a larger fusion molecule formed by covalently or non-covalently binding the antibody to one or more other proteins or peptides.

[0154] An "antibody fragment" contains only a part of a complete antibody, where the part retains at least one of the functions that are usually associated with that part when present in the complete antibody, and up to most or all of the functions. In one embodiment, the antibody fragment contains the antigen-binding site of the complete antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment (such as an antibody fragment containing an Fc region) retains at least one biological function that is usually associated with the Fc region when present in the complete antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody with an in vivo half-life substantially similar to that of the complete antibody. For example, such an antibody fragment can contain an antigen-binding arm linked to an Fc sequence that can confer in vivo stability to the fragment.

[0155] The identity or homology of a particular amino acid sequence of the present invention is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to a particular residue after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of homology and without considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions in a particular sequence should not be construed as affecting homology. All sequence alignments referred to in the present invention are such maximum homology alignments. In general, the nucleic acid sequence homology between the polynucleotides, oligonucleotides and fragments of the present invention and a nucleic acid sequence of interest is at least 80%, and more typically, the homology preferably increases to at least 85%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and / or 100%. Two amino acid sequences are homologous if they are partially or completely identical.

[0156] The term "hedgehog-related disorder" refers to or describes a disorder that is typically characterized by, or results in, an abnormal hedgehog pathway function or presentation. Examples of such disorders include, but are not limited to, hyperproliferative diseases, including cancer.

[0157] As used herein, "treatment" refers to a clinical intervention that attempts to alter the natural course of an individual or cell to be treated and may be performed for prophylaxis or during the course of a clinical pathology. Desirable treatment effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing or reducing inflammation and / or tissue / organ damage, reducing the rate of disease progression, improving or alleviating the disease state and alleviating or improving the prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of a disease or disorder.

[0158] Unless otherwise indicated, well-known techniques in the fields of molecular biology, microbiology, recombinant DNA, and immunology within the scope of this technology will be used to implement the present invention. Such techniques are fully described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (edited by D.N. Glover, 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (edited by J.H. Miller and M.P. Calos, 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Volumes 154 and 155 (edited by Wu et al.), Immunochemical Methods In Cell And Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Antibodies: A Laboratory Manual, Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (edited by D.M. Weir and C.C. Blackwell, 1986).

[0159] As used herein, the term "polysaccharide" refers to a polysaccharide or an oligosaccharide. Polysaccharides are also used herein to refer to the carbohydrate portion of a glycoconjugate, which is a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan. Polysaccharides typically consist only of O-glycosidic linkages between monosaccharides. For example, cellulose is a polysaccharide (or more specifically, a glucan) composed of β-1,4-linked D-glucose, while chitin is a polysaccharide composed of β-1,4-linked N-acetyl-D-glucosamine. Polysaccharides can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Polysaccharides can be found linked to proteins, such as glycoproteins and proteoglycans. They are typically found on the outer surface of cells. O-linked and N-linked polysaccharides are common in eukaryotes and can also be found in prokaryotes (although less commonly). N-linked polysaccharides are found linked to the R-group nitrogen (N) of asparagine in a sequon. A sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid other than proline.

[0160] As used herein, the term "antigen" is defined as any substance capable of eliciting an immune response.

[0161] As used herein, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0162] As used herein, the term "CD1d" refers to a member of the CD1 (cluster of differentiation 1) family of glycoproteins, expressed on the surface of various human antigen-presenting cells. Lipid antigens presented by CD1d activate natural killer T cells. CD1d has a deep antigen-binding groove in which glycolipid antigens bind. CD1d molecules expressed on dendritic cells can bind and present glycolipids, including α-GalCer analogs such as C34.

[0163] As used herein, the term "epitope" is defined as a part of an antigen molecule that contacts the antigen-binding site of an antibody or a T cell receptor.

[0164] As used herein, the term "vaccine" refers to a preparation containing an antigen, which is composed of whole pathogenic organisms (killed or attenuated) or components of these organisms (such as proteins, peptides, or polysaccharides), and is used to confer immunity against diseases caused by these organisms. Vaccine preparations can be natural, synthetic, or obtained by recombinant DNA technology.

[0165] As used herein, the term "antigen specificity" refers to a property of a cell population such that the provision of a specific antigen or antigen fragment causes the proliferation of specific cells.

[0166] As used herein, the term "specifically binds" refers to the interaction between a binding pair (e.g., an antibody and an antigen). In various instances, the specific expression of specific binding can be an affinity constant of about 10 -6 moles / liter, about 10 -7 moles / liter or about 10 -8 moles / liter or less.

[0167] An "isolated" antibody is one that has been identified and separated from and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that interfere with the study, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In one embodiment, the antibody is purified (1) to greater than 95 wt% of the antibody, as determined, for example, by the Lowry method, and in some embodiments more than 99 wt%; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use, for example, of a spinning cup sequenator; or (3) to homogeneity, which is achieved by use, for example, of Coomassie blue or silver stain and SDS-PAGE under reducing or non-reducing conditions. Isolated antibodies include antibodies that are in situ within recombinant cells, since there will be at least one component of the antibody's natural environment that is not present. However, isolated antibodies are generally prepared by at least one purification step.

[0168] As used herein, the phrases "substantially similar", "substantially identical", "equivalent", or "substantially equivalent" indicate that the degree of similarity between two values (e.g., one associated with a molecule and the other associated with a reference / comparison molecule) is great enough such that, in the context of the biological characteristics measured in accordance with these values (e.g., Kd values, antiviral activity, etc.), one of ordinary skill in the art would consider the difference between the two values to be minimal or without biological and / or statistical significance. As a function of the value of the reference / comparison molecule, the difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%.

[0169] As used herein, the phrases "substantially reduced" or "substantially different" indicate that the degree of difference between two values (generally, one associated with a molecule and the other associated with a reference / comparison molecule) is great enough such that, in the context of the biological characteristics measured in accordance with these values (e.g., Kd values), one of ordinary skill in the art would consider the difference between the two values to be statistically significant. As a function of the value of the reference / comparison molecule, the difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0170] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (such as an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigen more rapidly and tend to remain bound for longer. A variety of methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

[0171] In one embodiment, the "Kd" or "Kd value" according to the present invention is measured by a radioimmunoassay (RIA) using the Fab form of the antibody of interest and its antigen, as described in the following assay. The solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of radiolabeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with a plate coated with an anti-Fab antibody (Chen et al., (1999) J. Mol. Biol. 293:865-881). To establish the assay conditions, microtiter plates (Dynex) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Lab) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (about 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM 125 I) radiolabeled antigen is used. 125The I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., in accordance with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Subsequently, the Fab of interest is incubated overnight; however, the incubation can be continued for a longer period (e.g., 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). Subsequently, the solution is removed and the plate is washed 8 times with 0.1% Tween-20 in PBS. When the plate is dry, 150 μl / well of scintillant (MicroScint-20; Packard) is added, and the plate is counted for ten minutes using a Topcount gamma counter (Packard). The concentration of each Fab that provides less than or equal to 20% of the maximum binding is selected for the competitive binding assay. According to another embodiment, the Kd or Kd value is measured by surface plasmon resonance analysis using a BIAcore CM5 chip immobilized with the antigen having approximately 10 response units (RU) at 25 °C TM -2000 or BIAcore TM -3000 (BIAcore, Inc., Piscataway, N.J.). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8), and then injected at a flow rate of 5 μl / min to couple the protein to approximately 10 response units (RU). Following the injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. In each experiment, the activation spots and ethanolamine blocking without immobilization are performed for the protein to be used in the reference subtraction. For kinetic measurements, at 25 °C, serial two-fold dilutions (0.78 nM to 500 nM) of the Fab in PBS containing 0.05% Tween 20 (PBST) are injected at a flow rate of approximately 25 μl / min. Using a simple one-to-one Langmuir binding model ( Evaluation Software version 3.2), the association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, e.g., Chen, Y. et al., (1999) J. Mol Biol 293:865-881. If the binding rate obtained by the above surface plasmon resonance analysis exceeds 10 6 M -1 s-1 The association rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antigen-binding antibody (Fab form) in PBS (pH 7.2) at 25 °C as the antigen concentration is increased, as measured using a spectrofluorometer (such as a stopped-flow equipped spectrofluorometer with stirred cuvettes (Aviv Instruments) or an 8000 series SLM-Aminco spectrofluorometer (ThermoSpectronic)).

[0172] The "association rate" or "rate of association" or "association rate" or "kon" according to the present invention can also use the same surface plasmon resonance technique, using a BIAcore with a CM5 chip immobilized with antigen having about 10 response units (RU) at 25 °C TM -2000 or BIAcore TM -3000 (BIAcore, Inc., Piscataway, NJ) to determine. Briefly, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), and then injected at a flow rate of 5 μl / min to couple the protein to reach about 10 response units (RU). After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, at 25 °C, two-fold serial dilutions (from 0.78 nM to 500 nM) of Fab in PBS (PBST) containing 0.05% Tween 20 are injected at a flow rate of about 25 μl / min. Using a simple one-to-one Langmuir binding model ( evaluation software version 3.2), the association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, for example, Chen, Y. et al., (1999) J. Mol Biol 293:865-881. However, if the association rate obtained by the above surface plasmon resonance analysis exceeds 10 6 M -1 s -1The association rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antigen - antibody (Fab form) in PBS (pH 7.2) at 25 °C with increasing antigen concentration, as measured using a spectrofluorometer (such as a stopped - flow equipped spectrofluorometer (Aviv Instruments) with stirred cuvettes or an 8000 series SLM - Aminco spectrofluorometer (ThermoSpectronic)).

[0173] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One class of vectors is "plasmids", which refers to a circular double - stranded DNA loop to which other DNA segments can be ligated. Another class of vectors is bacteriophage vectors. Another type of vector is viral vectors, into which other DNA segments can be ligated to the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non - episomal mammalian vectors) can integrate into the host cell genome after introduction into the host cell and thus replicate with the host genome. In addition, some vectors are capable of directing the expression of genes operably linked to them. These vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors suitable for recombinant DNA techniques are usually in the form of plasmids. In this specification, since plasmids are the most commonly used vector form, "plasmid" and "vector" are used interchangeably.

[0174] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substance that can be incorporated into the polymer by DNA or RNA polymerase or synthetic reactions. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. If modified, the nucleotide structure can be modified before or after assembly of the polymer. The nucleotide sequence can be interspersed with non-nucleotide components. Polynucleotides can be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, substitution of one or more natural nucleotides with analogs, i.e., "caps"; internucleotide modifications such as those using uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those using charged linkages (e.g., phosphorothioates, dithiophosphates, etc.), those containing pendant moieties (such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those using intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those using modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl group normally present in the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group or activated to prepare other linkages with other nucleotides; or can be bound to a solid or semi-solid support. The 5' and 3' terminal OHs can be phosphorylated or replaced by an amine or an organic capping group having 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized relative to standard protecting groups. Polynucleotides can also contain ribose or deoxyribose analog forms commonly known in the art, including, for example, 2'-O-methyl-ribose, 2'-O-allyl ribose, 2'-fluoro-ribose or 2'-azido-ribose, cyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxoses), pyranoses, furanoses, sedoheptuloses, acyclic analogs and basic nucleoside analogs such as methyl ribonucleosides. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include (but are not limited to) examples where the phosphate group is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("acetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aryl aldehyde group optionally containing an ether (-O-) linkage.All linkages in the polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0175] As used herein, the term "oligonucleotide" generally refers to short polynucleotides, usually single-stranded and usually synthetic, that are typically (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The foregoing description of polynucleotides applies equally and fully to oligonucleotides.

[0176] "Antibody" (Ab) and "immunoglobulin" (Ig) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that typically lack antigen specificity. For example, the latter type of polypeptide is produced in small amounts by the lymphatic system and in large amounts by myelomas.

[0177] The terms "antibody" and "immunoglobulin" are used interchangeably in a broad sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). Antibodies can be chimeric antibodies, human antibodies, humanized antibodies, and / or affinity matured antibodies.

[0178] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. These domains are usually the most variable parts of the antibody and contain the antigen-binding sites.

[0179] The term "variable" means that certain portions of the variable domains among antibodies vary extensively in sequence and these portions are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable domain. It is concentrated in three segments of the variable domains of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The relatively conserved parts of the variable domains are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FR regions, which are connected by three CDRs (forming loop connections) and mainly adopt a β-sheet conformation (forming part of a β-sheet structure in some cases). The CDRs in each chain are held closely together by the FR regions and together with the CDRs from other chains participate in the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as enabling the antibody to participate in antibody-dependent cell cytotoxicity.

[0180] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment yields F(ab')2 fragments, which have two antigen-binding sites and are still capable of cross-linking antigens.

[0181] "Fv" is the smallest antibody fragment that contains a complete antigen-recognition site and antigen-binding site. In the double-chain Fv species, this region is composed of a dimer of a heavy-chain variable domain non-covalently and tightly associated with a light-chain variable domain. In the single-chain Fv species, a heavy-chain variable domain and a light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in the double-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Overall, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv, containing only three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but with a lower affinity than the entire binding site.

[0182] Fab fragments also contain the light-chain constant domain and the first heavy-chain constant domain (CH1). Fab' fragments differ from Fab fragments in that several residues are added at the carboxyl terminus of the heavy-chain CH1 domain that includes one or more cysteines from the antibody hinge region. Fab'-SH refers herein to a Fab' in which the cysteine residue of the constant domain bears a free thiol group. F(ab')2 antibody fragments are initially produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0183] Based on the amino acid sequence of the constant domain, the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types (called κ and λ).

[0184] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0185] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody in a substantially intact form, as opposed to antibody fragments as defined below. These terms particularly refer to antibodies having heavy chains that contain an Fc region.

[0186] An "antibody fragment" contains only a portion of a full-length antibody, where the portion retains at least one, and most or all, of the functions that are usually associated with that portion when present in the full-length antibody. In one embodiment, an antibody fragment contains the antigen-binding site of a full-length antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment (e.g., one that contains an Fc region) retains at least one biological function that is usually associated with the Fc region when present in the full-length antibody, such as FcRn binding, antibody half-life modulation, ADCC function, and complement binding. In one embodiment, an antibody fragment is a monovalent antibody having a substantially similar in vivo half-life to the full-length antibody. For example, the antibody fragment can contain an antigen-binding arm linked to an Fc sequence that is capable of conferring in vivo stability to the fragment.

[0187] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts or only contain homogeneous glycoform profiles (the glycoantibodies in the population have only a single polysaccharide or a single polysaccharide profile). Examples of homogeneous antibody compositions that enhance effector function by using 2,3- and 2,6-sialyl and desialylated complex biantennary polysaccharides at the Fc-297 position are described in US12 / 959,351. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a discrete mixture of antibodies. The monoclonal antibodies typically include antibodies that comprise a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a method that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method can be selecting a unique clone from a plurality of clones (such as a fusionoma clone library, a phage clone library, or a recombinant DNA clone library). It should be understood that the selected targeting binding sequence can be further modified, e.g., to enhance affinity for the target, humanize the targeting binding sequence, enhance its yield in cell culture, reduce its in vivo immunogenicity, form multispecific antibodies, etc., and antibodies that comprise the modified targeting binding sequence are also monoclonal antibodies of the present invention. Compared to polyclonal antibody preparations that typically include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to specificity, an advantage of monoclonal antibody preparations is also that they are generally not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a population of substantially homogeneous antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, the monoclonal antibodies used according to the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)); recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567); phage display techniques (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol.340(5):1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004), and techniques for generating human or human-like antibodies in animals having a part or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (see, e.g., WO98 / 24893; WO96 / 34096; WO96 / 33735; WO91 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993); Jakobovits et al., Nature 362:255-258(1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016; Marks et al., Bio.Technology 10:779-783(1992); Lonberg et al., Nature 368:856-859(1994); Morrison, Nature 368:812-813(1994); Fishwild et al., Nature Biotechnol. 14:845-851(1996); Neuberger, Nature Biotechnol. 14:826(1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93(1995).

[0188] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is the same or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is the same or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of these antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)).

[0189] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains the minimal sequence obtained from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the recipient hypervariable regions are replaced with residues from the hypervariable regions of a non-human species (donor antibody) having the desired specificity, affinity, and / or capacity (such as a mouse, rat, rabbit, or non-human primate). In some instances, the framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. In addition, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody efficacy. In general, a humanized antibody will contain substantially all of at least one and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are the FRs of a human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc) (usually a human immunoglobulin constant region). For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0190] The terms "hypervariable region", "HVR", or "HV", when used herein, refer to regions within the variable domain of an antibody that are highly variable in sequence and / or form structurally defined loops. Generally, an antibody contains six hypervariable regions; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). There are numerous depictions of hypervariable regions used herein. Kabat complementarity determining regions (CDRs) are sequence-based and most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the positions of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" hypervariable regions are analyzed based on available complex crystal structures. Residues from each of these hypervariable regions are indicated below.

[0191] Loop Kabat AbM Chothia Contact

[0192] L1 L24-L34 L24-L34 L26-L32 L30-L36

[0193] L2 L50-L56 L50-L56 L50-L52 L46-L55

[0194] L3 L89-L97 L89-L97 L91-L96 L89-L96

[0195] H1 H31-H35B H26-H35B H26-H32 H30-H35B

[0196] (Kabat numbering)

[0197] H1 H31-H35 H26-H35 H26-H32 H30-H35

[0198] (Chothia numbering)

[0199] H2 H50-H65 H50-H58 H53-H55 H47-H58

[0200] H3 H95-H102 H95-H102 H96-H101 H93-H101

[0201] The hypervariable regions may include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al. supra.

[0202] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0203] The terms "Kabat numbering of variable domain residues" or "Kabat numbering of amino acid positions" and variations thereof refer to the numbering system used in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991) for compiling the variable domains of the heavy or light chains of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or HVR of the variable domain. For example, the heavy chain variable domain may include an insertion of a single amino acid after residue 52 of H2 (residue 52a according to Kabat) and insertion residues after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues in a given antibody can be determined by aligning the antibody sequence with the homologous regions of the "standard" Kabat numbered sequence.

[0204] "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains enabling the scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0205] The term "bispecific antibody" refers to small antibody fragments having two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) joined to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domain of another chain and create two antigen-binding sites. Bispecific antibodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0206] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or made by any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0207] An "affinity matured" antibody is an antibody in which one or more changes have been made in one or more of the HVRs such that the affinity of the antibody for the antigen is increased (compared to the parent antibody that does not have those changes). In one embodiment, the affinity matured antibody has a nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be prepared by procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Induction of random mutations of CDR and / or framework residues is described below: Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0208] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Some blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0209] As used herein, an "agonist antibody" is an antibody that mimics at least one functional activity of a polypeptide of interest.

[0210] "Disease condition" refers to any medical condition that would benefit from antibody treatment of the invention. This includes chronic and acute conditions or diseases, including those pathological conditions that render a mammal susceptible to the disease condition. Non-limiting examples of disease conditions to be treated herein include cancer.

[0211] The terms "proliferative disease condition" and "hyperproliferative disease condition" refer to disease conditions associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disease condition is cancer.

[0212] As used herein, "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign), and all precancerous and cancerous cells and tissues. As recited herein, the terms "cancer", "cancerous", "proliferative disease condition", "hyperproliferative disease condition", and "tumor" are not mutually exclusive.

[0213] The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal, typically characterized by unregulated cell growth / proliferation. Examples of cancer include (but are not limited to) carcinomas, lymphomas (such as Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. More specific examples of these cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0214] As used herein, "treatment" refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and can be for prophylactic purposes or during a clinical pathological process. Desirable treatment outcomes include preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing or slowing inflammation and / or tissue / organ damage, reducing the rate of disease progression, improving or slowing the disease state, and symptom remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or disorder.

[0215] "Individual" or "subject" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include (but are not limited to) farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the vertebrate is a human.

[0216] A "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo animals, sport animals or pet animals such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is a human.

[0217] An "effective amount" refers to the amount of dosage and time necessary to effectively achieve the desired therapeutic or prophylactic outcome.

[0218] The "therapeutically effective amount" of the substance / molecule of the present invention may vary depending on factors such as the disease state, age, gender and weight of the individual, as well as the ability of the substance / molecule to induce the desired response in the individual. The therapeutically effective amount is also an amount where the therapeutic beneficial effect exceeds any toxic or harmful effects of the substance / molecule. A "prophylactically effective amount" refers to the amount of dosage and time necessary to effectively achieve the desired prophylactic outcome. Since the prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount is usually (but not necessarily) less than the therapeutically effective amount.

[0219] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., radioactive isotopes of At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 and Lu); chemotherapeutic agents such as methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and their fragments (such as nucleases, antibiotics and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including their fragments and / or variants), and various anti-tumor or anti-cancer agents disclosed hereinafter. Other cytotoxic agents are described hereinafter. Tumoricidal agents can destroy tumor cells.

[0220] A "chemotherapeutic agent" is a compound that can be used to treat cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); Δ-9-tetrahydrocannabinol (dronabinol, )); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan) CPT-11 (irinotecan; ) Acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheamicin ω1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186(1994)); dynemicin, including dynemicin A; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine,. Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone;Anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Cytarabine (「Ara-C」); Thiotepa; Taxoids, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.); Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel, ABRAXANE TM (American Pharmaceutical Partners, Schaumberg, Ill.) and Docetaxel ( Rorer, Antony, France); Chlorambucil; Gemcitabine 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin; Vinblastine Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine Oxaliplatin; Leucovovin; Vinorelbine Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid; Capecitabine Pharmaceutically acceptable salts, acids or derivatives of any of the above therapeutic agents; and combinations of two or more of the above therapeutic agents, such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone) and FOLFOX (oxaliplatin (ELOXATINTM ) (Abbreviation of the treatment regimen in combination with 5-FU and lucovorin).

[0221] Pharmaceutical formulation

[0222] The pharmaceutical composition is administered in a therapeutically effective, protective, and therapeutic amount in a manner compatible with the dosage formulation. The exact amount of the active ingredient required for administration depends on the judgment of the practitioner. However, those skilled in the art can readily determine a suitable dosage range. The suitable regimen for the initial and booster doses is also variable, but may include an initial administration followed by subsequent administrations. The vaccine dosage may also vary depending on the route of administration and the body size of the host.

[0223] Methods for generating monoclonal and polyclonal antibodies and their fragments in animals (such as mice, rabbits, goats, sheep, or horses) are well known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact immunoglobulin molecules as well as their fragments, such as Fab, F(ab')2, Fv, scFv (single-chain antibody), and dAb (domain antibody; Ward et al., (1989) Nature, 341, 544).

[0224] The compositions disclosed herein can be included in pharmaceutical compositions together with other active agents, carriers, vehicles, excipients, or adjuvants that can be recognized by those skilled in the art upon reading the present invention.

[0225] The pharmaceutical composition preferably comprises at least one pharmaceutically acceptable carrier. In these pharmaceutical compositions, the compositions disclosed herein form "active compounds", also referred to as "active agents". As used herein, the phrase "pharmaceutically acceptable carrier" includes solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and tonicity regulators, such as sodium chloride or dextrose. The pH may be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0226] Clinical applications

[0227] The present invention provides selected and directed optimized glycoantibodies that are suitable for treating proliferative diseases in an individual, such as cancer (e.g., lung cancer, colorectal cancer, pancreatic cancer, biliary tract cancer, or endometrial cancer), benign neoplasms, or angiogenesis.

[0228] The compositions described herein may also be used for cancer treatment and diagnosis. Methods for generating monoclonal and polyclonal antibodies and their fragments in humans and / or animals (e.g., mice, rabbits, goats, sheep, or horses) are well known in the art. See, e.g., Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact immunoglobulin molecules as well as fragments thereof, such as Fab, F(ab)2, Fv, scFv (single-chain antibody), and dAb (domain antibody; Ward et al., (1989) Nature, 341, 544).

[0229] These compositions may further comprise suitable carriers well known in the art, such as pharmaceutically acceptable excipients, including buffers.

[0230] Also provided are non-naturally occurring and / or isolated antibodies and polynucleotides. In certain embodiments, the isolated antibodies and polynucleotides are substantially pure.

[0231] The antigen-binding domain of an antibody is formed by two variable (V) regions of approximately 110 amino acids, one from the light chain (VL) and the heavy chain (VH), both of which have three hypervariable loops or complementarity-determining regions (CDRs). The variable domains can be functionally presented on phage as single-chain Fv (scFv) fragments (where VH and VL are covalently linked via a short flexible peptide); or as Fab fragments (where they are each fused to a constant domain and interact non-covalently), as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are referred to as "Fv phage clones" or "Fv clones".

[0232] The repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then a search can be made for antigen-binding clones, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). An immune-derived library provides antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, native repertoires can be cloned without any immunization, providing a source of single human antibodies against a wide range of non-self and self antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, native libraries can also be prepared synthetically as follows: unrearranged V gene segments are cloned from stem cells, and PCR primers containing random sequences are used to encode the highly variable CDR3 region and effect in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0233] Antibody fragments are presented using filamentous phage by fusion to the minor coat protein pIII. The antibody fragments can be presented as single-chain Fv fragments, where the VH and VL domains are linked on the same polypeptide chain via a flexible polypeptide spacer, for example as described by Marks et al., J. Mol. Biol., 222:581-597 (1991); or as Fab fragments, where one chain is fused to pIII and the other chain is secreted into the periplasm of the bacterial host cell, where an assembly presenting the Fab-coat protein structure is formed on the phage surface by displacing some of the wild-type coat protein, for example as described by Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

[0234] Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained as follows: genomic DNA or mRNA is isolated from lymphocytes and then polymerase chain reaction (PCR) is performed with primers that match the 5' and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), thereby preparing a diverse V gene repertoire for expression. The V genes can be amplified from cDNA and genomic DNA, where the reverse primer is located at the 5' end of the exon encoding the mature V domain and the forward primer is primarily located within the J segment, as described in Orlandi et al., (1989) and Ward et al., Nature, 341:544-546 (1989). However, when amplifying from cDNA, the reverse primer can also be located in the leader exon, as described in Jones et al., Biotechnol., 9:88-89 (1991), and the forward primer is located within the constant region, as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers, as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized as follows: PCR primers targeting each V gene family are used to amplify all available VH and VL arrangements present in an immune cell nucleic acid sample, for example as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). To clone the amplified DNA into an expression vector, a rare restriction site can be introduced in the form of a tag into the PCR primer at one end, as described in Orlandi et al. (1989); or PCR amplification can be further performed with tagged primers, as described in Clackson et al., Nature, 352:624-628 (1991).

[0235] The repertoire of synthetically rearranged V genes can be derived in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)) and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993)); these cloned segments (including all major conformations of the H1 and H2 loops) can be used to generate a diverse repertoire of VH genes using PCR primers encoding H3 loops of diverse sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be prepared in which all sequence diversity is concentrated in a long H3 loop of a single length, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-1461 (1993)) and can be used to prepare synthetic light chain repertoires. Based on a range of VH and VL folds and L3 and H3 lengths, synthetic V gene repertoires will encode antibodies with a considerable amount of structural diversity. After amplification of the DNA encoding the V genes, germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0236] Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires in several ways. Each repertoire can be generated in a different vector and the recombinant vectors can be recombined in vitro (e.g., as described in Hogrefe et al., Gene, 128:119-126 (1993)) or in vivo by co-infection, such as the loxP system described in Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). The in vivo recombination method takes advantage of the double-stranded nature of Fab fragments to overcome the limitations imposed on library size by the transformation efficiency of Escherichia coli. The native VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of bacteria containing the phagemid, such that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals such that the VH and VL genes are recombined on a single replicon and co-packaged into phage virions. These huge libraries provide a large number of diverse antibodies with good affinities (about 10 -8 Kd of M -1 )

[0237] Alternatively, the repertoires can be sequentially cloned into the same vector, for example as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or assembled together by PCR and then cloned, for example as described by Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be utilized to ligate VH and VL DNA using DNA encoding a flexible peptide spacer to form single-chain Fv (scFv) repertoires. In yet another technique, "intracellular PCR assembly" is used to combine VH and VL genes within lymphocytes by PCR, and then clonal repertoires of the ligated genes are selected, as described by Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

[0238] The library can be screened by any technique known in the art. The target can be used to coat the wells of a capture plate, expressed on host cells attached to the capture plate or used for cell sorting, or conjugated to biotin for capture by streptavidin-coated beads, or used for panning a phage display library in any other method known in the art.

[0239] Wash the phage bound to the solid phase and then elute by acid, for example as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or by base, for example as described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or by SSEA-3 / SSEA-4 / GLOBO H antigen competition elution, for example using a procedure similar to the antigen competition method of Clackson et al., Nature, 352:624-628 (1991). The phage can be enriched 20-1,000-fold in a single round of selection. In addition, the enriched phage can be grown in a bacterial culture and further subjected to multiple rounds of selection.

[0240] The efficiency of selection depends on a variety of factors, including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously engage with the antigen. Antibodies with fast dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage display, and a high coating density of the antigen in the solid phase. High density not only stabilizes the phage through multivalent interactions, but also facilitates the reassociation of dissociated phage. The selection of antibodies with slow dissociation kinetics (and good binding affinity) can be promoted by using long washes and monovalent phage display (as described in Bass et al., Proteins, 8:309-314 (1990) and WO 92 / 09690) and a low coating density of the antigen (as described in Marks et al., Biotechnol., 10:779-783 (1992)).

[0241] However, random mutagenesis of selected antibodies (e.g., as performed in some affinity maturation techniques described above) can generate numerous mutants, most of which bind to the antigen, while a few have higher affinity. In the presence of limited SSEA-3 / SSEA-4 / GLOBO H, a few high-affinity phage may prevail. To retain all higher-affinity mutants, phage can be incubated with an excess of biotinylated SSEA-3 / SSEA-4 / GLOBO H, but at a molar concentration lower than the target molar affinity constant for SSEA-3 / SSEA-4 / GLOBO H. High-affinity binding phage can then be captured using streptavidin-coated paramagnetic beads. This type of "equilibrium capture" allows selection of antibodies based on binding affinity, with sensitivity allowing isolation of mutant clones with at least twofold higher affinity from a large excess of phage with lower affinity. Conditions used to wash phage bound to the solid phase can also be manipulated to allow for differentiation based on the dissociation kinetics.

[0242] DNA encoding the Fv clones of the present invention is readily isolated and sequenced using known procedures (e.g., using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA templates). After isolation, the DNA can be placed into expression vectors and then transfected into host cells that do not otherwise produce immunoglobulins (e.g., E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to synthesize the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol. Revs, 130:151 (1992).

[0243] DNA encoding the Fv germlines of the present invention can be combined with known DNA sequences encoding the heavy chain and / or light chain constant regions (e.g., appropriate DNA sequences can be obtained from Kabat et al., supra) to form germlines encoding full-length or partial-length heavy and / or light chains. It should be understood that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. The Fv germlines are derived from the variable domain DNA of an animal (such as a human) species and are then fused with the constant region DNA of another animal species to form a coding sequence for "hybrid" coding sequences as used herein, and the definitions of "chimeric" and "hybrid" antibodies include full-length heavy and / or light chains. In one embodiment, the Fv germlines derived from human variable DNA are fused with human constant region DNA to form coding sequences for all-human full-length or partial-length heavy and / or light chains.

[0244] Antibodies made from a naive library (natural or synthetic) can have moderate affinity (about 10 6 to 10 7 M -1 for the Kd -1 ), but affinity maturation can also be carried out in vitro by constructing a second library and reselecting from the second library, as described by Winter et al. (1994) (supra). For example, random mutations can be introduced in vitro by using error-prone polymerases (reported in Leung et al., Technique 1:11-15 (1989)) in the methods of Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or Gram et al., Proc. Natl. Acad. Sci USA, 89:3576-3580 (1992). Alternatively, affinity maturation can be carried out as follows: random mutations are introduced into one or more CDRs in the selected individual Fv germlines (e.g., using PCR with primers carrying random sequences spanning the CDRs of interest) and high-affinity germlines are screened. WO 9607754 (published March 14, 1996) describes a method for inducing mutations in the complementarity-determining regions of immunoglobulin light chains to generate a light chain gene library. Another effective method is to recombine the VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants from unimmunized donors and screen for higher affinity in several rounds of chain shuffling, as described by Marks et al., Biotechnol., 10:779-783 (1992). This technique can generate antibodies and antibody fragments with affinities in the range of 10 -9 M.

[0245] Other methods for generating and evaluating antibody affinity are well known in the art and are described, for example, in Kohler et al., Nature 256:495 (1975); U.S. Patent No. 4,816,567; Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); Munson et al., Anal. Biochem., 107:220 (1980); Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989); Abrahmsen et al., EMBO J., 4:3901 (1985); Methods in Enzymology, Volume 44 (1976); Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).

[0246] General methods

[0247] Antibodies can be generated using routine skills in the art, including the techniques described herein, such as hybridoma technology and screening phage display libraries for binding molecules. These methods are well established in the art.

[0248] Briefly, the antibodies of the present invention can be generated by using combinatorial libraries to screen for synthetic antibody clones having the desired activity. In principle, synthetic antibody clones are selected by screening phage libraries containing phages presenting various fragments of antibody variable regions (Fvs) fused to the phage coat protein. These phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen adsorb to the antigen and are thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any of the antibodies of the present invention can be obtained by designing a suitable antigen screening procedure to select the phage clones of interest, followed by constructing full-length antibody clones using the Fv sequences derived from the phage clones of interest and suitable constant region (Fc) sequences, which are described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda Md. (1991), Vols. 1-3.

[0249] Monoclonal antibodies can be obtained from substantially homogeneous populations of antibodies, i.e., each individual antibody constituting the population is identical, but may contain minor amounts of possible naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0250] The monoclonal antibodies of the present invention can be prepared by a variety of methods known in the art, including the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or they can be prepared by recombinant DNA methods (e.g., U.S. Patent No. 4,816,567).

[0251] Vectors, host cells, and recombinant methods

[0252] To produce the antibodies of the present invention recombinantly, the nucleic acid encoding them is isolated and inserted into a replicable vector for further propagation (DNA amplification) or expression. The DNA encoding the antibody is readily isolated and sequenced using known procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the antibody heavy and light chains). A variety of vectors are available. The choice of vector depends in part on the host cell to be used. Host cells include (but are not limited to) cells of prokaryotic or eukaryotic (usually mammalian) origin. It should be understood that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species.

[0253] Production of antibodies using prokaryotic host cells

[0254] Vector construction

[0255] Polynucleotide sequences encoding the polypeptide components of the antibodies of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of the appropriate vector will mainly depend on the size of the nucleic acid inserted into the vector and the particular host cell to be transformed by the vector. Each vector contains different components, depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it is present. Vector components generally include, but are not limited to: an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, an inserted sequence of heterologous nucleic acid, and a transcription termination sequence.

[0256] Generally, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in conjunction with these hosts. The vector usually carries a replication site and a marker sequence capable of providing phenotypic selection in the transformed cell. For example, Escherichia coli is typically transformed using pBR322 (a plasmid derived from the Escherichia coli species). pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and thus provides an easy way to identify transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain or be modified to contain promoters that can be used by the microorganism to express endogenous proteins. Examples of pBR322 derivatives for expressing specific antibodies are described in detail in U.S. Patent No. 5,648,237 to Carter et al.

[0257] In addition, bacteriophage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in conjunction with these hosts. For example, bacteriophages such as λGEM TM -11 can be used to prepare recombinant vectors that can be used to transform sensitive host cells such as Escherichia coli LE392.

[0258] The expression vectors of the present invention can contain two or more promoter-cistron pairs encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates the expression of the cistron. Prokaryotic promoters are typically divided into two categories: inducible promoters and constitutive promoters. An inducible promoter is a promoter under whose control the transcriptional level of the cistron begins to increase in response to changes in culture conditions (such as the presence or absence of nutrients or temperature changes).

[0259] Many promoters that can be recognized by a variety of potential host cells are well known. The selected promoter is operably linked to the cistronic DNA encoding the light or heavy chain, which is achieved by restriction enzyme digestion to remove the promoter in the source DNA and insert the isolated promoter sequence into the vector of the present invention. Both native promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are used because they generally allow increased transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0260] Promoters suitable for prokaryotic hosts include the PhoA promoter, the β-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, enabling those skilled in the art to operably join these nucleotide sequences to the cistrons encoding the target light and heavy chains using linkers or adaptors that provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0261] In one aspect of the present invention, each cistron within the recombinant vector contains a secretion signal sequence component that directs the translocation of the expressed polypeptide across the membrane. Generally, the signal sequence can be a component of the vector, or it can be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or the heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequence used in the two cistrons of the expression system is the STII signal sequence or a variant thereof.

[0262] In another aspect, the production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell, and thus a secretion signal sequence is not required within each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded, and assembled in the cytoplasm to form a functional immunoglobulin. Certain host strains (such as the Escherichia coli trxB- strain) provide cytoplasmic conditions that are favorable for disulfide bond formation, allowing the correct folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0263] The antibodies of the present invention can also be produced using an expression system in which the quantitative ratio of the expressed polypeptide components can be adjusted to maximize the yield of the secreted and correctly assembled antibodies of the present invention. Such adjustment is accomplished at least in part by simultaneously adjusting the translational strength of the polypeptide components.

[0264] A technique for adjusting translational strength is disclosed in U.S. Patent No. 5,840,523 to Simmons et al. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants with a range of translational strengths can be generated, thereby providing a suitable means to adjust this factor according to the desired amount of expression of a particular strand. TIR variants can be generated by known mutagenesis techniques that produce codon changes that alter the amino acid sequence. In some embodiments, the nucleotide sequence changes are silent. TIR changes can include, for example, changes in the number or spacing of the Shine-Dalgarno sequence, as well as changes in the signal sequence. One method for generating mutant signal sequences is to generate a "codon library" (i.e., changes that are silent) at the start of the coding sequence that does not change the amino acid sequence of the signal sequence. This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids (such as leucine, serine, and arginine) have multiple first and second positions that can be used to increase the complexity when preparing the library. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0265] In one embodiment, a set of vectors is generated that have a series of TIR strengths for each cistron. This limited set compares the expression levels of the different strands and the yields of the desired antibody product at different combinations of TIR strengths. The TIR strength can be determined by quantifying the expression level of a reporter gene, as described in detail in U.S. Patent No. 5,840,523 to Simmons et al. Based on the translational strength comparison, the desired individual TIRs to be combined in the expression vector constructs of the present invention are selected.

[0266] Prokaryotic host cells suitable for expressing the antibodies of the present invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas species (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, Escherichia coli cells are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219); ATCC deposit number 27,325) and its derivatives, including strain 33D3 having the genotype W3110ΔfhuA(ΔtonA)ptr3 lacIq lacL8ΔompTΔ(nmpc-fepE)degP41kanR (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select an appropriate bacterium taking into account the replicability of the replicon in the bacterial cell. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to supply the replicon, it is advisable to use Escherichia, Serratia, or Salmonella species as the host. Typically, the host cells should secrete a minimal amount of proteolytic enzymes, and other protease inhibitors should be incorporated into the cell culture.

[0267] Antibody production

[0268] The host cells are transformed with the above expression vectors and cultured in a known nutrient medium that is adjusted as appropriate to induce the promoter, select transformants, or amplify the gene encoding the desired sequence.

[0269] Transformation means introducing DNA into a prokaryotic host such that the DNA can replicate as an extrachromosomal element or as a chromosomal integrant. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. The calcium treatment method using calcium chloride is commonly used for bacterial cells with a substantial cell wall barrier. Another transformation method is the use of polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0270] Prokaryotic cells for producing the polypeptides of the present invention are grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria broth (LB) and essential nutrient supplements. In some embodiments, the medium also contains a selection agent chosen based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing the ampicillin resistance gene.

[0271] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may be included at appropriate concentrations, introduced individually or in combination with other supplements or media such as complex nitrogen sources. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteamine, cystamine, thioglycolate, dithiothreitol, and dithioerythritol.

[0272] The prokaryotic host cells are cultured at a suitable temperature. For the growth of Escherichia coli, for example, growth occurs in a temperature range including (but not limited to) about 20°C to about 39°C, about 25°C to about 37°C, and about 30°C. The pH of the medium can be any pH within the range of about 5 to about 9, depending primarily on the host organism. For Escherichia coli, the pH can be about 6.8 to about 7.4, or about 7.0.

[0273] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the present invention, the PhoA promoter is used to control polypeptide transcription. Accordingly, the transformed host cells are cultured in a phosphate-limited medium for induction. In one embodiment, the phosphate-limited medium is the C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133 - 147). Depending on the vector construct used, a variety of other inducers can be used, as known in the art.

[0274] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered from the periplasm of the host cell. Protein recovery typically involves disrupting the microorganism, usually by osmotic shock, sonication, or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

[0275] In one aspect of the invention, antibodies are produced in large quantities by fermentation methods. Various large-scale batch-fed fermentation procedures can be utilized to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, for example, a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to disperse oxygen and nutrients, especially glucose (a common carbon / energy source). Small-scale fermentations generally refer to fermentations carried out in fermenters with a volume capacity of no more than about 100 liters and ranging from about 1 liter to about 100 liters.

[0276] In the fermentation method, protein expression is typically initiated after the cells have grown to the desired density under suitable conditions (e.g., an OD550 of about 180 - 220), at which stage the cells are in the early stationary phase. Depending on the vector construct used, a variety of other inducers can be used, as known in the art and described above. The cells can be grown for a shorter period before induction. The cells are typically induced for about 12 - 50 hours, although longer or shorter induction times can be used.

[0277] To enhance the yield and quality of the polypeptides of the present invention, various fermentation conditions can be modified. For example, to enhance the correct assembly and folding of the secreted antibody polypeptides, other vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl-prolyl cis,trans isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. Chaperone proteins have been shown to promote the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J.Biol.Chem. 275:17100-17105; Ramm and Pluckthun (2000) J.Biol.Chem. 275:17106-17113; Arie et al., (2001) Mol.Microbiol. 39:199-210.

[0278] To minimize proteolysis of the expressed heterologous proteins (especially proteolysis-sensitive proteins), the present invention can use certain host strains lacking proteolytic enzymes. For example, the host cell strain can be modified to create gene mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some Escherichia coli protease-deficient strains are available and are described, for example, in Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0279] In one embodiment, an Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as the host cell in the expression system of the present invention.

[0280] Antibody Purification

[0281] In one embodiment, the antibody proteins produced herein are further purified to obtain a substantially homogeneous preparation for further analysis and use. Standard protein purification methods known in the art can be used. The following procedures illustrate suitable purification procedures: separation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, or cation exchange resin chromatography (such as DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0282] In one aspect, the antibody products of the invention are purified by immunoaffinity using Protein A immobilized on a solid phase. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the Fc region of antibodies with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a column containing a glass or silica surface, or a controlled pore glass column or a silica column. In some applications, the column is coated with a reagent such as glycerol to prevent non-specific attachment of contaminants as much as possible.

[0283] As a first step in purification, a preparation derived from cell culture as described above can be applied to the solid phase to which Protein A is immobilized to allow the antibody of interest to specifically bind to Protein A. The solid phase is then washed to remove contaminants that non-specifically bind to the solid phase. Finally, the antibody of interest is recovered by elution from the solid phase.

[0284] Production of antibodies using eukaryotic host cells

[0285] Vector components generally include (but are not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0286] (i) Signal sequence component

[0287] Vectors for use in eukaryotic host cells may also contain a signal sequence, or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is a signal sequence that will be recognized and processed by the host cell (i.e., cleaved by signal peptidase). When expressed in mammalian cells, mammalian signal sequences as well as viral secretory leader sequences, such as the herpes simplex virus gD signal, can be utilized.

[0288] The DNA of this precursor region is ligated in-frame to the DNA encoding the antibody.

[0289] (ii) Origin of replication

[0290] Generally speaking, mammalian expression vectors do not require origin-of-replication components. For example, the SV40 origin is commonly used only because it contains an early promoter.

[0291] (iii) Selectable gene components

[0292] Expression vectors and cloning vectors may contain selectable genes, also known as selectable markers. Typical selectable genes encode the following proteins: (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate, or tetracycline); (b) complement auxotrophies (if necessary); or (c) supply key nutrients not available from complex media.

[0293] One example of a selection scheme is to use a drug to block host cell growth. Those cells successfully transformed with a heterologous gene produce a protein that confers resistance to the drug and thus survive the selection scheme. Examples of such dominant selections are the use of the drugs neomycin, mycophenolic acid, and hygromycin.

[0294] Another example of selectable markers suitable for mammalian cells are those markers that can identify cells capable of taking up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and metallothionein-II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc.

[0295] For example, cells transformed with a DHFR selectable gene can first be identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR, suitable host cells include, for example, Chinese hamster ovary (CHO) cell lines lacking DHFR activity (such as ATCC CRL-9096).

[0296] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an antibody, wild-type DHFR protein, and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, for example kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0297] (iv) Promoter components

[0298] Expression vectors and cloning vectors usually contain a promoter, which is recognized by the host organism and operably linked to a nucleic acid encoding a polypeptide of interest (such as an antibody). Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription start of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal for adding a poly-A tail to the 3' end of the coding sequence. All of these sequences are preferably inserted into eukaryotic expression vectors.

[0299] In mammalian host cells, transcription of antibody polypeptides from vectors can be controlled by, for example, the following promoters: promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40); heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoter; or heat shock promoters, provided that such promoters are compatible with the host cell system.

[0300] The early and late promoters of the SV40 virus are preferably obtained in the form of an SV40 restriction fragment that also contains the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is preferably obtained in the form of a HindIII E restriction fragment. A system for expressing DNA using bovine papillomavirus as a vector in mammalian hosts is disclosed in U.S. Patent No. 4,419,446. Variations of this system are described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598 - 601 (1982), which is about the expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter obtained from herpes simplex virus. Alternatively, the long terminal repeat sequence of Rous Sarcoma Virus can be used as a promoter.

[0301] (v) Enhancer component constituents

[0302] Enhancement of DNA encoding the antibody polypeptides of the present invention for transcription in higher eukaryotes is usually achieved by inserting enhancer sequences into the vector. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, typically, enhancers derived from eukaryotic cell viruses will be used. Examples include the SV40 enhancer (bp 100-270) located downstream of the origin of replication, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located downstream of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982), which is about enhancer elements for activating eukaryotic promoters. The enhancer can be spliced into the expression vector at the 5' or 3' position of the antibody polypeptide coding sequence, but is usually located at the 5' site relative to the promoter.

[0303] (vi) Transcription termination components

[0304] Expression vectors used in eukaryotic host cells typically will also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are usually obtained from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed in the untranslated portion of the mRNA encoding the antibody as polyadenylation fragments. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0305] (vii) Selection and transformation of host cells

[0306] The host cells suitable for the DNA in the cloning or expression vectors in this article include the higher eukaryotic cells described in this article, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are the simian kidney CV1 line transformed with SV40 (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and the human liver tumor line (Hep G2).

[0307] The host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in a known nutrient medium that is appropriately conditioned to induce the promoter, select transformants, or amplify the gene encoding the desired sequence.

[0308] (viii) Culturing the host cells

[0309] Host cells for producing the antibodies of the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in the following references can be used as a host cell medium: Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; or No. 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Re. 30,985. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are usually present at final concentrations in the micromolar concentration range), and glucose or an equivalent energy source. Appropriate concentrations known to those skilled in the art can include any other necessary supplements. Culture conditions such as temperature, pH, and similar conditions are those previously selected for the host cells used for expression and will be apparent to those of ordinary skill in the art.

[0310] (ix) Purification of the antibody

[0311] When using recombinant techniques, the antibody can be produced intracellularly or secreted directly into the medium. If the antibody is produced intracellularly, the first step usually involves removing particulate debris (host cells or lysed fragments) by, for example, centrifugation or ultrafiltration. In the case where the antibody is secreted into the medium, the supernatant of such an expression system is usually first concentrated using a commercially available protein concentration filter (such as an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0312] Antibody compositions prepared from leukocytes can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, where affinity chromatography is a commonly acceptable purification technique. The suitability of an affinity reagent, such as Protein A, as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). All murine isotypes and human γ3 are recommended to use Protein G (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices can also be utilized. Compared to the flow rate and processing time achievable with agarose, a mechanically stable matrix, such as controlled pore glass or poly(styrene-divinyl)benzene, can enable a faster flow rate and shorter processing time. In the case where the antibody contains a CH3 domain, Bakerbond ABX TM resin (J.T. Baker, Phillipsburg, N.J.) is suitable for purification. Other protein purification techniques can also be utilized, such as separation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSE TM chromatography, anion or cation exchange resins (such as polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

[0313] After any preliminary purification step, further purification steps can be performed on the mixture containing the antibody of interest and contaminants, if desired, such as low pH hydrophobic interaction chromatography using an elution buffer with a pH of about 2.5 - 4.5, which is typically performed at a low salt concentration (e.g., about 0 - 0.25 M salt).

[0314] It should be noted that, in general, techniques and methods for preparing antibodies for use in research, testing, and clinical applications have been well established in this art, which are consistent with the above and / or considered suitable by those skilled in the art for the specific antibody of interest.

[0315] Activity assays

[0316] The physical / chemical properties and biological functions of the antibodies of the present invention can be characterized by a variety of assays known in the art.

[0317] The purified antibodies can be further characterized by a series of assays, including (but not limited to) N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0318] When needed, the biological activity of the antibody is analyzed. In some embodiments, the antigen-binding activity of the antibodies of the invention is tested. Antigen-binding assays known in the art and usable herein include, without limitation, any direct or competitive binding assay using techniques such as the following: Western blot, radioimmunoassay, enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, and protein A immunoassay.

[0319] In one embodiment, the invention encompasses antibodies with altered effector functions, some but not all of which are critical for in vivo antibody half-life, making them desirable candidates for many applications where certain effector functions, such as complement and ADCC, are unnecessary or detrimental. In certain embodiments, the Fc activity of the antibody is measured to ensure that only the desired properties are maintained. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / elimination of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody does not have FcγR binding capacity (and thus may not have ADCC activity), but retains FcRn binding capacity. The primary cells used to mediate ADCC, i.e., NK cells, only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is outlined in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in vitro assay for evaluating the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo (e.g., in an animal model such as the animal model disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998)). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and thus lacks CDC activity. To evaluate complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art.

[0320] Antibody fragments

[0321] The present invention encompasses antibody fragments. In certain instances, the use of antibody fragments has advantages over intact antibodies. The smaller size of the fragments allows for rapid clearance and enhanced access to solid tumors.

[0322] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992)); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from Escherichia coli, thus allowing for easy production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with extended in vivo half-lives and containing salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO93 / 16185; U.S. Patent No. 5,571,894; and 5,587,458. Fv and sFv are the only antibody fragments that have a complete binding site but lack a constant region; thus, they are useful for reducing non-specific binding during in vivo use. sFv fusion proteins can be constructed to generate fusions of effector proteins at the amino or carboxyl terminus of the sFv. See Antibody Engineering, Borrebaeck ed., supra. Antibody fragments can also be "linear antibodies," such as those described in U.S. Patent No. 5,641,870. These linear antibody fragments can be monospecific or bispecific.

[0323] Humanized antibodies

[0324] Any of the antibodies described herein can be a full-length antibody or an antigen-binding fragment thereof. In some instances, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment. In some instances, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment. In some instances, the isolated antibody is a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody.

[0325] Any one of the antibodies described herein has one or more of the following characteristics:

[0326] a) is a recombinant antibody, monoclonal antibody, chimeric antibody, humanized antibody, human antibody, antibody fragment, bispecific antibody, monospecific antibody, monovalent antibody, IgG1 antibody, IgG2 antibody, or antibody derivative; b) is a human, murine, humanized or chimeric antibody, antigen-binding fragment, or antibody derivative; c) is a single-chain antibody fragment, multibody, Fab fragment, and / or immunoglobulin IgG, IgM, IgA, IgE, IgD isotype and / or its subclass; d) has one or more of the following characteristics: (i) mediates ADCC and / or CDC of cancer cells; (ii) induces and / or promotes apoptosis of cancer cells; (iii) inhibits the proliferation of target cells of cancer cells; (iv) induces and / or promotes phagocytosis of cancer cells; and / or (v) induces and / or promotes the release of cytotoxic agents; e) specifically binds to a tumor-associated carbohydrate antigen, which is a tumor-specific carbohydrate antigen; f) does not bind to antigens expressed on non-cancer cells, non-tumor cells, benign cancer cells and / or benign tumor cells; and / or g) specifically binds to tumor-associated carbohydrate antigens expressed on cancer stem cells and normal cancer cells.

[0327] Preferably, the antibody binds specifically to its corresponding antigen. The term "specifically" is generally used to refer to a situation where one member of a binding pair does not show any significant binding to molecules other than its specific binding partner, for example, having a cross-reactivity of less than about 30%, preferably 20%, 10%, or 1% with any other molecule other than those specified herein.

[0328] The antibody is suitable for binding to its target antigen determinant with high affinity (low KD value), and the KD is preferably in the range of nanomolar concentration or lower. Affinity can be measured by methods known in the art (such as surface plasmon resonance).

[0329] Exemplary antibody preparation

[0330] Exemplary antibodies capable of binding to the Globo H antigen determinant and SSEA-4 antigen determinant described herein can be generated by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Host animal immunization and hybridoma technology

[0331] Exemplary polyclonal antibodies against anti-Globo H and anti-SSEA-4 antibodies can be prepared by collecting blood from immunized mammals checked by the increase in the desired antibodies in the serum and separating the serum from the blood by any known method. The polyclonal antibodies include sera containing the polyclonal antibodies, and eluates containing the polyclonal antibodies can be separated from the sera.

[0332] Polyclonal antibodies are generally produced in a host animal (such as a rabbit, mouse, horse, or goat) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It is applicable to use bifunctional agents or derivatives (such as maleimidobenzoyl sulfosuccinimide ester (binding via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, etc.) to bind the relevant antigen to a protein immunogenic in the species to be immunized (such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

[0333] Any mammal can be immunized with an antigen to produce the desired antibodies. Generally, rodents, lagomorphs, or primates can be used. Rodents include, for example, mice, rats, and hamsters. Lagomorphs include, for example, rabbits. Primates include, for example, catarrhines (Old World monkeys), such as cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees.

[0334] Methods for immunizing animals with antigens are known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard immunization method for mammals. More specifically, the antigen can be diluted and suspended in an appropriate amount of phosphate-buffered saline (PBS), physiological saline, etc. If necessary, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant (such as Freund's complete adjuvant) to form an emulsion, and then administered to the mammal. The animal is immunized against the antigen, immunogenic conjugate, or derivative by combining 1 mg or 1 μg of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's incomplete adjuvant.

[0335] Animals can be boost - immunized until the titer levels off by administering several times at intervals of 4 to 21 days an antigen mixed with an appropriate amount of Freund's incomplete adjuvant. Animals are boost - immunized by subcutaneous injection at multiple sites with Freund's complete adjuvant containing 1 / 5 to 1 / 10 of the original amount of the peptide or conjugate. The animals are bled 7 to 14 days later and the antibody titers of the sera are analyzed. Immunization can also be carried out using a suitable carrier. After immunization as described above, the increase in the content of the required antibody in the serum is checked by standard methods. Preferably, the animals are boost - immunized with conjugates of the same antigen but conjugated to different proteins and / or conjugated via different cross - linking reagents. The conjugates can also be produced in recombinant cell cultures in the form of protein fusions. Similarly, aggregating agents such as alum are suitable for enhancing the immune response.

[0336] Over the past twenty to thirty years, a variety of methods have been developed for preparing chimeric, humanized or human antibodies for in vivo therapeutic applications in humans. The most widely used and proven method is to prepare murine mAbs using the hybridoma method, followed by humanizing the mAb by replacing the V H and V L domain framework regions and constant domains with the most homologous human framework regions of human V H and V L domains and the constant domains of the desired human γ - immunoglobulin isotype and subclass. Many mAbs used clinically (such as Xolair) are humanized mAbs of the human γ1, κ isotype and subclass and are prepared using this method.

[0337] In some embodiments, antibodies can be produced by well - known hybridoma techniques. Kohler et al., Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal (such as a hamster or rabbit) is immunized as described above to induce lymphocytes to produce or be capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0338] To prepare monoclonal antibodies, immune cells are collected from an antigen - immunized mammal and the increase in the content of the required antibody in the serum is checked as described above, and cell fusion is carried out. The immune cells for cell fusion are preferably obtained from the spleen. Other preferred parental cells to be fused with the above - mentioned immune cells include, for example, myeloma cells of a mammal, and more preferably myeloma cells having an acquired property of being selectable by a drug for the fused cells.

[0339] Preferred myeloma cells are myeloma cells that are efficiently fusogenic, render the selected antibody-producing cells to stably produce antibodies in high amounts, and are sensitive to media such as HAT medium. Among them, preferred myeloma cell lines are murine myeloma cell lines such as myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors, obtained from the Salk Institute Cell Distribution Center (San Diego, Calif., USA); and the SP-2 cells obtained from the American Type Culture Collection (Rockville, Md., USA). Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0340] The above-mentioned immune cells and myeloma cells can be fused according to known methods, such as the method of Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Then, lymphocytes and myeloma cells are fused using a suitable fusogen (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The resulting hybridomas obtained by cell fusion can be selected by culturing them in a standard selection medium such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). The cell culture is typically continued in HAT medium for several days to weeks (a time sufficient to allow all other cells other than the desired hybridomas (unfused cells) to die). Then, standard limiting dilution is performed to screen and clone the hybridoma cells that produce the desired antibody.

[0341] The hybridomas thus prepared are inoculated and grown in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for the hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT.

[0342] The medium in which the hybridoma cells grow is analyzed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays. Enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence absorbance measurement can be used to measure the antigen-binding activity of the antibodies of the present invention. In ELISA, the antibodies of the present invention are immobilized on a plate, the proteins of the present invention are applied to the plate, and then a sample containing the desired antibodies, such as the culture supernatant of antibody-producing cells or a purified antibody, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme (such as alkaline phosphatase) is applied, and the plate is incubated. Then, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate, and the absorbance is measured to evaluate the antigen-binding activity of the sample. Protein fragments, such as C-terminal or N-terminal fragments, can be used in this method. The activity of the antibodies of the present invention can be evaluated using BIAcore (Pharmacia). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0343] Using any known method, including the methods described above, hybridoma cells that produce antibodies that bind to the epitopes described herein can be identified and selected for further characterization.

[0344] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, they can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Media suitable for this purpose include, for example, D-MEM or RPMI-1640 media. The monoclonal antibodies secreted by the subclones are preferably separated from the medium, ascites, or serum by known immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0345] In addition, the hybridoma cells can be grown in vivo in the form of ascitic tumors in an animal. For example, the resulting hybridoma can subsequently be transplanted into the peritoneal cavity of a mouse, and the ascites can be harvested.

[0346] The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, Protein A or Protein G columns, DEAE ion exchange chromatography, or affinity columns conjugated with the proteins of the present invention. The antibodies of the present invention can be used not only for purifying and detecting the proteins of the present invention, but also as candidates for agonists and antagonists of the proteins of the present invention. In addition, such antibodies can be applied to antibody therapy for diseases related to the proteins of the present invention.

[0347] Recombinant technology

[0348] The monoclonal antibodies thus obtained can also be prepared recombinantly using genetic engineering techniques (see, for example, Borrebaeck C.A.K. and Larrick J.W., Therapeutic Monoclonal Antibodies, published by MacMillan Publishers LTD, UK, 1990). DNA encoding the antibodies can be cloned from immune cells such as antibody-producing hybridomas or immunolymphocytes, inserted into an appropriate vector, and introduced into host cells to produce recombinant antibodies. The present invention also provides recombinant antibodies prepared as described above.

[0349] When the obtained antibodies are to be administered to humans (antibody therapy), human antibodies or humanized antibodies are preferably used to reduce immunogenicity. For example, transgenic animals having a human antibody gene repertoire can be immunized with an antigen selected from a protein, a cell expressing the protein, or a lysate thereof. Then, antibody-producing cells are collected from the animals and fused with myeloma cells to obtain hybridomas from which human antibodies against the protein can be prepared. Alternatively, antibody-producing immune cells such as immunolymphocytes can be immortalized by oncogenes and used to prepare monoclonal antibodies.

[0350] DNA encoding monoclonal antibodies can be easily isolated and sequenced using well-known procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding murine heavy and light antibody chains). Hybridoma cells are used as a preferred source of the DNA. After isolation, the DNA can be placed into an expression vector and then transfected into host cells such as Escherichia coli cells that do not originally produce immunoglobulins, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells to synthesize monoclonal antibodies in the recombinant host cells. Discussion articles on the recombinant expression of the DNA encoding the antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev., 130:151-188 (1992).

[0351] DNA encoding the antibody produced by the above hybridoma cells can be genetically modified via conventional techniques to produce a genetically engineered antibody. Genetically engineered antibodies (such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies) can be produced via, for example, well-known recombinant techniques. The DNA can then be modified, for example, by replacing homologous murine sequences with human heavy and light chain constant domain encoding sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851) or by covalently linking all or part of a non-immunoglobulin polypeptide encoding sequence to an immunoglobulin encoding sequence. In this way, genetically engineered antibodies with binding specificity for a target antigen, such as "chimeric" or "hybrid" antibodies, can be prepared.

[0352] Techniques developed for the preparation of "chimeric antibodies" are well known in the art. See, for example, Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al., (1984) Nature 312, 604; and Takeda et al., (1984) Nature 314:452.

[0353] Typically, these non-immunoglobulin polypeptides are used to replace the antibody constant domain or the variable domain of the antibody that has one antigen-binding site to form a chimeric bivalent antibody that contains one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0354] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide bond exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0355] Methods for humanizing non-human antibodies are well known in the art. In general, one or more amino acid residues from non-human sources have been introduced into humanized antibodies. These non-human amino acid residues are usually referred to as "import" residues, which are typically taken from "import" variable domains. Humanization can generally follow the method of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), and is carried out by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, these "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain has been replaced by the corresponding sequences of a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues have been replaced by residues from similar sites of a rodent antibody.

[0356] The selection of human variable domains (light and heavy chains) that can be used to prepare humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the variable domain sequences of a rodent antibody are screened against the entire library of known human variable domain sequences. The human sequence that is most similar to the rodent sequence is then regarded as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework that is derived from the consensus sequence of all human antibodies having a particular light or heavy chain subgroup. Several different humanized antibodies can use the same framework (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0357] More importantly, the antibody is humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, a humanized antibody is prepared by using three-dimensional models of the parental and humanized sequences to analyze the parental sequence and various contemplated humanized products. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and present the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these presentations allows analysis of the possible roles of residues when the candidate immunoglobulin sequence functions, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and donor sequences and combined to obtain the desired antibody characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

[0358] Alternatively, in the absence of endogenous immunoglobulins, transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies upon immunization can now be produced. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (J H ) gene in chimeric and germline mutant mice results in complete suppression of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into these germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

[0359] Any nucleic acid encoding any of the anti-Globo H and anti-SSEA-4 antibodies (including heavy chain, light chain or both) described herein, a vector (such as an expression vector) comprising one or more nucleic acids, and a host cell comprising one or more vectors also fall within the scope of the present invention. In some instances, the vector comprises a nucleic acid that comprises a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-Globo H antibody as described herein. In some instances, the vector comprises a nucleic acid that comprises a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-SSEA-4 antibody as described herein. In other instances, the vector comprises nucleotide sequences encoding a heavy chain variable region and a light chain variable region, the expression of which can be controlled by a single promoter or two separate promoters. Also provided herein are methods for preparing any of the anti-Globo H and anti-SSEA-4 antibodies as described herein, for example, by recombinant techniques described in this section.

[0360] Other techniques for preparing antibodies

[0361] In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins. Transgenic animals designed to produce more desirable (e.g., fully human antibodies) or more robust immune responses can also be used to generate humanized or human antibodies. Examples of such techniques are the Xenomouse of Amgen, Inc. (Fremont, Calif.) RTM and the HuMAb-Mouse of Medarex, Inc. (Princeton, N.J.) RTM and the TC Mouse TM . In another alternative, antibodies can be produced recombinantly by phage display techniques. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, human antibodies and antibody fragments can be generated in vitro from immunoglobulin variable (V) domain gene repertoires of non-immunized donors using phage display techniques (McCafferty et al., (1990) Nature 348:552-553).

[0362] Antigen-binding fragments of intact (full-length) antibodies can be prepared by conventional methods. For example, F(ab')2 fragments can be generated by pepsin digestion of antibody molecules, and Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments.

[0363] Alternatively, the anti-Globo H and anti-SSEA-4 antibodies described herein can be isolated from antibody phage libraries (such as single-chain antibody phage libraries) generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol Biol., 222:581-597 (1991). Subsequent publications describe strategies for generating high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0364] Antibodies obtained as described herein can be purified to homogeneity. For example, the isolation and purification of antibodies can be performed according to the separation and purification methods used for general proteins. For example, antibodies can be isolated and isolated by appropriate selection and combined use of column chromatography (such as affinity chromatography), filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and other methods (Antibodies: A Laboratory Manual. Edited by Harlow and David Lane, Cold Spring Harbor Laboratory, 1988) (but not limited to this). The concentration of the antibodies obtained as above can be determined by absorbance measurement, enzyme-linked immunosorbent assay (ELISA), etc. Exemplary chromatography other than affinity chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and similar methods (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). The chromatography procedure can be performed by liquid chromatography (such as HPLC, FPLC).

[0365] Antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of the antibody-antigen complex, competition analysis, gene fragment expression analysis, and synthetic peptide-based analysis, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies (a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999). In another example, epitope mapping can be used to determine the sequence to which the antibody binds. An epitope can be a linear epitope (i.e., contained within a single stretch of amino acids), or it can be a conformational epitope formed by the three-dimensional interaction of amino acids that may not necessarily be contained within a single stretch (the primary structure linear sequence). Peptides of different lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays for the antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and measuring the binding of the antibody. According to gene fragment expression analysis, the open reading frame encoding the target antigen can be fragmented randomly or according to a specific genetic construct, and the reactivity of the expressed fragments of the antigen with the antibody to be tested is measured. The gene fragments can be generated, for example, by PCR, followed by in vitro transcription and translation into protein in the presence of radioactive amino acids. Subsequently, the binding of the antibody to the radiolabeled antigen fragment is determined by immunoprecipitation and gel electrophoresis. Alternatively, certain epitopes can be identified by using large libraries of random peptide sequences presented on the surface of phage particles (phage libraries). Or, defined libraries of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay.

[0366] In another example, mutagenesis, domain swapping experiments, and alanine scanning mutagenesis of the antigen-binding domain can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using target antigen mutants in which multiple residues in the epitope-binding region of the candidate antibody are replaced (swapped) with the sequence of a closely related but antigenically distinct protein, such as another member of the neurotrophin family. By assessing the binding of the antibody to the mutant target protein, the importance of specific antigen fragments for antibody binding can be evaluated.

[0367] Alternatively, competition analysis can be performed using other antibodies known to bind to the same antigen to determine whether one antibody (e.g., the MC45 antibody described herein) binds to the same epitope as the other antibodies. Competition analysis is well known to those skilled in the art.

[0368] Other aspects of suitable exemplary methods for preparing antibodies

[0369] In this art, methods for generating monoclonal and polyclonal antibodies and their fragments in animals such as mice, rabbits, goats, sheep or horses are well known. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact immunoglobulin molecules as well as fragments thereof such as Fab, F(ab')2, Fv, scFv (single-chain antibody) and dAb (domain antibody; Ward et al., (1989) Nature, 341, 544).

[0370] The compositions disclosed herein can be included in pharmaceutical compositions together with other active agents, carriers, vehicles, excipients or adjuvants that can be recognized by those skilled in the art upon reading the present invention.

[0371] The pharmaceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. In these pharmaceutical compositions, the compositions disclosed herein form the "active compound", also referred to as the "active agent". As used herein, the phrase "pharmaceutically acceptable carrier" includes solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity regulators such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[0372] Provided are compositions comprising at least one anti-SSEA-3 / SSEA-4 / Globo H antibody or at least one polynucleotide comprising a sequence encoding an anti-SSEA-3 / SSEA-4 / Globo H antibody. In certain embodiments, the compositions can be pharmaceutical compositions. As used herein, a composition comprises one or more antibodies that bind to one or more of SSEA-3 / SSEA-4 / Globo H and / or one or more polynucleotides comprising a sequence encoding one or more antibodies that bind to one or more of SSEA-3 / SSEA-4 / Globo H. These compositions can further comprise suitable carriers well known in the art, such as pharmaceutically acceptable excipients, including buffers.

[0373] Also provided are isolated antibodies and polynucleotides. In certain embodiments, the isolated antibodies and polynucleotides are substantially pure.

[0374] In one embodiment, the anti-SSEA-3 / SSEA-4 / Globo H antibody is a monoclonal antibody. In another embodiment, fragments of the anti-SSEA-3 / SSEA-4 / Globo H antibody are provided (e.g., Fab, Fab'-SH, and F(ab')2 fragments). These antibody fragments can be produced by conventional means such as enzymatic digestion, or can be produced by recombinant techniques. These antibody fragments can be chimeric antibody fragments, humanized antibody fragments, or human antibody fragments. These fragments can be used to achieve the diagnostic and therapeutic purposes described below.

[0375] A variety of methods known in the art for generating phage display libraries from which antibodies of interest can be obtained. One method for generating an antibody of interest is via the use of a phage antibody library as described by Lee et al., J. Mol. Biol. (2004), 340(5):1073-93.

[0376] The anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be generated by screening a combinatorial library to isolate synthetic antibody clones with the desired activity. In principle, synthetic antibody clones are selected by screening a phage library containing various fragments of phage presenting antibody variable regions (Fv) fused to the phage coat protein. These phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any anti-SSEA-3 / SSEA-4 / Globo H antibody of the present invention can be obtained by designing a suitable antigen screening procedure to select the phage clones of interest, followed by constructing full-length anti-SSEA-3 / SSEA-4 / Globo H antibody clones using the Fv sequences from the phage clones of interest and suitable constant region (Fc) sequences as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

[0377] The antigen-binding domain of an antibody is formed by two variable (V) regions of approximately 110 amino acids each, one from the light chain (VL) and one from the heavy chain (VH), both of which have three hypervariable loops or complementarity-determining regions (CDRs). The variable domains can be functionally presented on phage as single-chain Fv (scFv) fragments (where VH and VL are covalently linked via a flexible short peptide) or Fab fragments (where VH and VL are each fused to a constant domain and interact non-covalently), as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

[0378] The VH and VL gene lineages can be separately cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then antigen-binding clones can be searched for, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, natural lineages can be cloned to provide a single source of human antibodies for a wide range of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a naive library can also be obtained synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and complete in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0379] Antibody fragments are presented using filamentous phage by fusing them to a small amount of the coat protein pIII. These antibody fragments can be presented as single-chain Fv fragments, where the VH domain and the VL domain are linked on the same polypeptide chain by a flexible polypeptide spacer, for example as described by Marks et al., J. Mol. Biol., 222:581-597 (1991); or as Fab fragments, where one chain is fused to pIII and the other chain is secreted into the periplasm of the bacterial host cell, where a Fab-coat protein structure presented on the phage surface is assembled by displacing a part of the wild-type coat protein, for example as described by Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

[0380] Generally speaking, nucleic acids encoding antibody gene fragments can be obtained from immune cells collected from humans or animals. If a library preferentially enriched for anti-SSEA-3 / SSEA-4 / Globo H clones is desired, individuals are immunized with SSEA-3 / SSEA-4 / Globo H to generate an antibody response, and spleen cells and / or circulating B cells or other peripheral blood lymphocytes (PBLs) are recovered for library construction. In one embodiment, a human antibody gene fragment library preferentially enriched for anti-human SSEA-3 / SSEA-4 / Globo H clones is obtained as follows: an anti-human SSEA-3 / SSEA-4 / Globo H antibody response is generated in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), such that immunization with SSEA-3 / SSEA-4 / Globo H generates B cells that produce human antibodies against SSEA-3 / SSEA-4 / Globo H. The generation of transgenic mice for the production of human antibodies is described below.

[0381] By using screening procedures suitable for isolating B cells expressing SSEA-3 / SSEA-4 / Globo H-specific antibodies, the anti-SSEA-3 / SSEA-4 / Globo H-reactive cell population can be further enriched. Screening procedures include, for example, separating cells by SSEA-3 / SSEA-4 / Globo H affinity chromatography, or adsorbing cells to SSEA-3 / SSEA-4 / Globo H labeled with a fluorescent dye, followed by fluorescence-activated cell sorting (FACS).

[0382] Alternatively, the use of spleen cells and / or B cells or other PBLs from non-immunized donors may more appropriately represent the possible antibody repertoire, and also allows the construction of antibody libraries using any animal (human or non-human) species in which SSEA-3 / SSEA-4 / Globo H is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from an individual to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorph, wolf, dog, cat, pig, cow, horse, and avian species, among others.

[0383] Recover and amplify nucleic acids encoding antibody variable gene segments (including VH and VL segments) from the cells of interest. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes and then performing polymerase chain reaction (PCR) using primers that match the 5' and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), to prepare various V gene repertoires for expression. V genes can be amplified from cDNA and genomic DNA using a reverse primer at the 5' end of the exon encoding the mature V-domain and a forward primer located within the J-segment, as described in Orlandi et al., (1989) and Ward et al., Nature, 341:544-546 (1989). However, when amplifying from cDNA, the reverse primer can also be positioned in the leader exon, as described in Jones et al., Biotechnol., 9:88-89 (1991), and the forward primer can also be positioned within the constant region, as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers, as described in Orlandi et al. (1989) or Sastry et al. (1989). In some embodiments, library diversity is maximized by using PCR primers that target each V-gene family to amplify all available VH and VL arrangements present in an immunocyte nucleic acid sample, for example as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991), or as described in Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). When cloning the amplified DNA into an expression vector, a rare restriction site can be introduced at one end into the PCR primer as a tag, as described in Orlandi et al. (1989); or further PCR amplification can be performed using tagged primers, as described in Clackson et al., Nature, 352:624-628 (1991).

[0384] Synthetically rearranged V gene lineages can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)) and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993)); various VH gene lineages can be generated using these cloned segments (including all major conformations of the H1 and H2 loops) and PCR primers encoding H3 loops of various sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH lineages with all sequence diversity concentrated in a single-length long H3 loop can also be prepared, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). The human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-1461 (1993)) and can be used to prepare synthetic light chain lineages. Based on a range of VH and VL folds and L3 and H3 lengths, synthetic V gene lineages will encode antibodies with a large amount of structural diversity. After amplification of the DNA encoding the V-genes, germline V-gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0385] Antibody fragment lineages can be constructed in several ways by combining VH and VL gene lineages. Various lineages can be established in different vectors and the recombinant vectors in vitro (e.g., as described in Hogrefe et al., Gene, 128:119-126 (1993)), or the recombinant vectors in vivo by co-infection (e.g., the loxP system described in Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). The in vivo recombination method takes advantage of the double-stranded nature of Fab fragments to overcome the limitation imposed by the E. coli transformation efficiency on library size. The native VH and VL lineages are cloned separately, one into a phagemid and the other into a phage vector. Then the two libraries are combined by phage infection of bacteria containing the phagemid, such that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals such that the VH and VL genes are recombined onto a single replicon and co-packaged into phage virions. These huge libraries provide a large number of antibodies with good affinity (about 10 -8 Kd of M -1 ) of various antibodies.

[0386] Alternatively, these lineages can be cloned sequentially into the same vector, for example as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); or assembled together by PCR and then cloned, for example as described by Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to ligate VH and VL DNA with DNA encoding a flexible peptide spacer to form single-chain Fv (scFv) lineages. In yet another technique, "intracellular PCR assembly" is used to combine VH and VL genes by PCR within lymphocytes, followed by cloning of pure lineages of the ligated genes, as described by Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

[0387] The library can be screened by any art known in the art. For example, the SSEA-3 / SSEA-4 / Globo H target can be used to coat the wells of a capture plate, expressed on host cells attached to the capture plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method known in the art for panning phage display libraries.

[0388] A phage library sample is contacted with immobilized SSEA-3 / SSEA-4 / Globo H under conditions suitable for at least a portion of the phage particles to bind to the adsorbent. Generally, physiological conditions are mimicked to select the conditions, including pH, ionic strength, temperature, and the like. The phage bound to the solid phase are washed and then eluted by acid, for example as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); or eluted by base, for example as described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or by SSEA-3 / SSEA-4 / Globo H antigen competition, for example using a procedure similar to the antigen competition method of Clackson et al., Nature, 352:624-628 (1991). The phage can be enriched about 20-fold to about 1,000-fold in a single round of selection. In addition, the enriched phage can be grown in a bacterial culture and subjected to another round of selection.

[0389] The selection efficiency depends on many factors, including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be retained by using short washing times, multivalent phage display, and high coating densities of the antigen on the solid phase. The high density not only stabilizes the phage via multivalent interactions but also facilitates rebinding of the dissociated phage. Antibodies with slow dissociation kinetics (and good binding affinity) can be promoted by using long washing times and monovalent phage display (as described by Bass et al., Proteins, 8:309-314 (1990) and WO 92 / 09690) and low antigen coating densities (as described by Marks et al., Biotechnol., 10:779-783 (1992)).

[0390] Phage antibodies with different affinities (even slightly different affinities) for SSEA-3 / SSEA-4 / Globo H can be selected. However, random mutagenesis of the selected antibodies (e.g., as performed in some of the affinity maturation techniques described above) may generate many mutants, most of which bind to the antigen, while a few have higher affinity. In the case of limited SSEA-3 / SSEA-4 / Globo H, only a few high-affinity phages can prevail. To retain all mutants with higher affinity, the phage can be incubated with an excess of biotinylated SSEA-3 / SSEA-4 / Globo H, but the molar concentration of the biotinylated SSEA-3 / SSEA-4 / Globo H is lower than the target molar concentration affinity constant of SSEA-3 / SSEA-4 / Globo H. The high-affinity binding phages can then be captured by streptavidin-coated paramagnetic beads. This "equilibrium capture" allows selection of antibodies based on their binding affinity, and its sensitivity allows isolation of pure lines of mutants with only twice the high affinity from a large excess of phages with lower affinity. Discrimination can also be made by manipulating the conditions for washing and solid-phase binding of the phages based on their dissociation kinetics.

[0391] Anti-SSEA-3 / SSEA-4 / Globo H clones can be selected based on activity. In one embodiment, the invention provides anti-SSEA-3 / SSEA-4 / Globo H antibodies that block the binding between the SSEA-3 / SSEA-4 / Globo H ligand and SSEA-3 / SSEA-4 / Globo H, but do not block the binding between the SSEA-3 / SSEA-4 / Globo H ligand and a second protein. The Fv clones corresponding to these anti-SSEA-3 / SSEA-4 / Globo H antibodies can be selected as follows: (1) Isolate anti-SSEA-3 / SSEA-4 / Globo H clones from the phage library described in Section B(I)(2) above, and optionally amplify the isolated phage clone population (by growing the population in a suitable bacterial host); (2) Select SSEA-3 / SSEA-4 / Globo H and the second protein according to the respective requirements for blocking and non-blocking activities; (3) Adsorb the anti-SSEA-3 / SSEA-4 / Globo H phage clones to the immobilized SSEA-3 / SSEA-4 / Globo H; (4) Elute any unwanted clones that recognize SSEA-3 / SSEA-4 / Globo H binding determinants that overlap or share binding determinants with the second protein, using an excess of the second protein; and (5) Elute the clones that remain adsorbed after step (4). Optionally, clones with the desired blocking / non-blocking characteristics can be further enriched by repeating the selection procedure described herein one or more times.

[0392] DNA encoding the Fv clones of the invention is readily isolated and sequenced using known procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). After isolation, the DNA is placed in an expression vector and then transfected into a host cell (such as Escherichia coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) (which do not otherwise produce immunoglobulins) to synthesize the desired monoclonal antibody in the recombinant host cell. Review articles on the recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol. Revs, 130:151 (1992).

[0393] The DNA encoding the Fv clonal line of the present invention can be combined with known DNA sequences encoding the heavy chain and / or light chain constant regions (e.g., appropriate DNA sequences can be obtained from Kabat et al. (supra)) to form a clonal line encoding a full-length or partial-length heavy chain and / or light chain. It should be understood that for this purpose, any isotype of constant region can be used, including IgG, IgM, IgA, IgD, and IgE constant regions, and these constant regions can be obtained from any human or animal species. As used herein, the definitions of "chimeric" and "hybrid" antibodies include an Fv clonal line that is derived from variable domain DNA of one animal (such as human) species and then fused with constant region DNA of another animal species to form a "hybrid" (full-length heavy chain and / or light chain) encoding sequence. In one embodiment, the Fv clonal line derived from human variable DNA is fused with human constant region DNA to form an encoding sequence for all human full-length or partial-length heavy chains and / or light chains.

[0394] Antibodies (natural or synthetic) generated from a naive library can have moderate affinity (about 10 6 to 10 7 M -1 Kd -1 ), but affinity maturation can also be carried out by in vitro mimicking through constructing a second library and reselecting from the second library, as described in Winter et al. (1994) (supra). For example, random mutations can be introduced in vitro by using error-prone polymerases (reported in Leung et al., Technique, 1:11-15 (1989)), using the method of Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be carried out as follows: random mutations are introduced into one or more CDRs in the selected individual Fv clonal lines (e.g., using PCR with primers carrying random sequences spanning the CDRs of interest), and higher affinity clonal lines are screened. WO9607754 (published on March 14, 1996) describes a method for inducing mutations in the complementarity-determining regions of immunoglobulin light chains to establish a light chain gene library. Another effective method is to recombine the VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants obtained from unimmunized donors and screen for high affinity by several rounds of chain shuffling, as described in Marks et al., Biotechnol., 10:779-783 (1992). This technique can generate antibodies and antibody fragments with affinities in the range of 10 -9 M.

[0395] Other methods for generating anti-SSEA-3 / SSEA-4 / Globo H antibodies

[0396] Other methods for generating and evaluating antibody affinity are well known in the art and are described, for example, in Kohler et al., Nature 256:495 (1975); U.S. Patent No. 4,816,567; Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); Munson et al., Anal. Biochem., 107:220 (1980); Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989); Abrahmsen et al., EMBO J., 4:3901 (1985); Methods in Enzymology, Vol. 44 (1976); Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).

[0397] General methods

[0398] Accordingly, one aspect of the invention features an isolated antibody that triple-targets Globo H, SSEA3, and SSEA-4. The triple-targeting antibody specifically binds to Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (Globo H hexasaccharide) and Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-3 pentasaccharide) and Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide). In one example, the triple-targeting antibody is mAb 651.

[0399] Another aspect of the invention features an isolated antibody that dual-targets Globo H and SSEA3. The dual-targeting antibody specifically binds to Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (Globo H hexasaccharide) and Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-3 pentasaccharide). In one example, the dual-targeting antibody is mAb 273.

[0400] In yet another aspect, the invention features an isolated antibody that is specific for SSEA-4. The anti-SSEA-4 antibody binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide). In some examples, the antibody is capable of binding Neu5Gcα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (an analog of the SSEA-4 hexasaccharide). Preferably, the antibody is not murine IgG3 (such as mAb MC-831-70) and the antibody is not murine IgM (such as anti-RM1). Examples of the antibody include (but are not limited to) mAb 45 and 48.

[0401] Another aspect of the invention features an isolated antibody that is specific for SSEA-4 and its fragments. The anti-SSEA-4 antibody binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide) and Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1 (a fragment of the SSEA-4 hexasaccharide). In some examples, the antibody is capable of binding to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galβ1. In some examples, the antibody is capable of binding to Neu5Gcα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (an analog of the SSEA-4 hexasaccharide). In one example, the antibody is mAb 46.

[0402] Antibodies that triple-target Globo H, SSEA-3, and SSEA-4, antibodies that dual-target Globo H and SSEA-3, and anti-SSEA-4 antibodies have been developed and are disclosed herein. The antibodies of the invention can be used for treatment, diagnosis, or as research tools.

[0403] Thus, one aspect of the invention relates to a composition of a homogeneous monoclonal antibody population comprising a single homogeneous N-glycan on the Fc, wherein the structure is an optimized N-glycan structure to enhance the efficacy of effector cell function.

[0404] In a preferred embodiment, the N-glycan is linked to Asn-297 of the Fc region.

[0405] In a preferred embodiment, wherein the N-glycan consists of the structure of Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2.

[0406] The glycoantibodies described herein can be produced in vitro. The glycoantibodies can be produced by Fc glycoengineering. In certain embodiments, the glycoantibodies are engineered from monoclonal antibodies obtained by mammalian cell culture in an enzymatic or chemoenzymatic manner.

[0407] In some embodiments, the Fc region in the glycoantibodies described herein exhibits enhanced binding affinity for FcγRIIA or FcγRIIIA relative to the wild-type Fc region in the corresponding monoclonal antibody.

[0408] In some embodiments, the glycoantibodies described herein exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity relative to wild-type immunoglobulins.

[0409] In some embodiments, the glycoantibodies are selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody can be a humanized antibody, a human antibody, or a chimeric antibody.

[0410] The glycoantibodies described herein can bind to antigens associated with cancer, autoimmune disorders, inflammatory disorders, or infectious diseases. Exemplary cancer-associated antigens can include, for example, Globo-H, SSEA-3, SSEA-4.

[0411] In other aspects, the antibodies disclosed herein can detect glycan variants and derivatives. For example, the reducing end of the glycan is free or linked to a natural tail (such as SSEA4 glycolipid) or an unnatural tail (such as a linker used for preparing glycan arrays or for binding for diagnostic purposes). All of these derivatives can be recognized by the antibody.

[0412] In certain diagnostic and array embodiments, the antibodies of the present invention can thus detect not only the glycans described herein but also their oxidized variants. The antibodies of the present invention can also detect the binding products of these oxidized variants.

[0413] In certain aspects, the present invention provides an isolated humanized monoclonal glycoantibody that specifically binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, and its oxidized variants, and the binding products of these oxidized variants, and their oxidized variants, and the binding products of these oxidized variants; wherein these oxidized variants are the conversion products of the polysaccharide primary alcohol into a carbonyl group, and wherein the binding product is the conversion product of the carbonyl group into an imine having a primary or secondary amine moiety.

[0414] For example, a polysaccharide containing a primary alcohol can be converted into an oxidized variant by methods known to those skilled in the art. As a non-limiting example, the primary alcohol on galactose can be converted into an aldehyde by contacting the polysaccharide with an oxidizing agent (such as sodium periodate (meta-sodium periodate) or another periodate (such as the potassium salt, ammonium salt, manganese salt, lithium salt)). One or more sugar moieties in the polysaccharide can be oxidized. The concentration of the oxidizing agent in water or a suitable buffer can be 1 μM concentration, 5 μM concentration, 10 μM concentration, 25 μM concentration, 50 μM concentration, 100 μM concentration, 200 μM concentration, 500 μM concentration, 750 μM concentration, 1 mM concentration, 5 mM concentration, 10 mM concentration, 25 mM concentration, 50 mM concentration, 100 mM concentration, or 500 mM concentration. The temperature can be 5 to 45 °C, preferably 15 to 40 °C, more preferably 35 to 40 °C. The reaction time can be 10 seconds to 20 minutes, preferably 30 seconds to 10 minutes. Suitable buffers can include or not include saline, phosphate, CHES, MES, borate, acetate, carbonate, formate, citrate, oxalate. A weakly acidic buffer is preferably used. A buffer solution without TRIS or glycine or free sugars is preferably used because these substances are competitive in the reaction. The transformant can be purified by dialysis or diafiltration by methods known to those skilled in the art.

[0415] By methods known to those skilled in the art and as described in the following references, conjugate products can be formed by the reaction of an oxidation product with a suitable amine, hydrazine, acylhydrazine, or hydroxylamine: G. Hermanson, Bioconjugate Techniques, 3rd edition, ISBN: 978-0-12-382239-0, Academic Press, 2013, which is incorporated herein by reference. As a non-limiting example, a primary amine can react with a polysaccharide having a single aldehyde functional group formed by the periodate oxidation of the primary alcohol of galactose within the polysaccharide. The net product is an imine. The imine can optionally be further reduced to an alcohol by methods known to those skilled in the art (e.g., reduction with cyanoborohydride) to form a conjugate product that is more stable to hydrolysis. In some aspects, the amine, hydrazine, acylhydrazine, or hydroxylamine can be further covalently linked to an array, reporter molecule, or biotin for further modification of the conjugate product. In some aspects, the reporter molecule can be a fluorescent molecule. In some aspects, the reporter molecule can be a radioactively labeled molecule. In some aspects, the reporter molecule can be a molecule having unique spectral characteristics (e.g., IR spectrum, Raman spectra, or NMR spectrum). In some aspects, the array can be a solid surface, a chemically modified surface, a polymer-coated surface, beads, gels, particles, or nanoparticles. In some aspects, the nanoparticles can be fluorescent nanoparticles or exhibit photoluminescence. In some aspects, the conjugate product can be a conversion product in which a carbonyl group becomes an imine having a primary or secondary amine moiety.

[0416] Generally, the present invention provides affinity matured SSEA-3 / SSEA-4 / Globo H antibodies. These antibodies have enhanced affinity and specificity for SSEA-3 / SSEA-4 / Globo H. The enhanced affinity and sensitivity allow the molecules of the present invention to be used in applications and methods that benefit from: (a) enhanced sensitivity of the molecules of the present invention and / or (b) tight binding of the molecules of the present invention to SSEA-3 / SSEA-4 / Globo H.

[0417] In one aspect, SSEA4 / SSEA3 / Globo H are three polysaccharides specifically expressed in cancer cells and cancer stem cells. Downregulation of the β-3-GalT5 gene (the main enzyme for synthesizing these three glycolipids) causes apoptosis in cancer cells but not in normal cells. Antibodies, particularly glycoantibodies that preferentially or specifically target SSEA4 and / or simultaneously target SSEA3 / SSEA4 / Globo H, are effective cancer therapeutic agents. In another aspect, the three polysaccharides (SSEA4 / SSEA3 / Globo H, particularly SSEA3) are suitable as cancer stem cell markers.

[0418] In one aspect, SSEA4 and / or the combination of SSEA4 / SSEA3 / Globo H are applicable as therapeutic targets for treating different cancers, including, for example, brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, kidney cancer, bone cancer (osteosarcoma), skin cancer, cervical cancer, ovarian cancer, and prostate cancer.

[0419] In one embodiment, human or humanized therapeutic antibodies against SSEA4 expressed on the cell surface of these exemplary cancer types are provided.

[0420] In another embodiment, human or humanized therapeutic antibodies against SSEA3 / SSEA4 / Globo-H co-expressed on the cell surface of these exemplary cancer types are provided.

[0421] Furthermore, the present invention also relates to immunogenic conjugate compositions targeting SSEA-3 / SSEA-4 / Globo H-related epitopes (natural and modified), which can induce the production of antibodies and / or binding fragments suitable for modulating the synthesis of globo series glycosphingolipids. In addition, the present invention also relates to methods of using the compositions described herein to treat or detect hyperproliferative diseases and / or conditions.

[0422] In one embodiment, SSEA-3 / SSEA-4 / Globo H antibodies are applicable for treating SSEA-3 / SSEA-4 / Globo H-mediated disorders that require partial or complete blockade of one or more SSEA-3 / SSEA-4 / Globo H activities. In one embodiment, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention are used for treating cancer.

[0423] Without the need for mass spectrometry or genetic manipulation, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention allow for sensitive and specific detection of epitopes in immunoassays such as sandwich assays, immunoprecipitation, ELISA, or immunomicroscopy. This in turn provides significant advantages in observing and elucidating the normal functioning of these pathways and detecting when these pathways are functioning abnormally.

[0424] The SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can also be used to determine their role in the development and pathogenesis of diseases. For example, as described above, the SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be used to determine whether the transient expression of TACAs under normal circumstances may be related to one or more disease states.

[0425] The SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be further used to treat one or more diseases in which SSEA-3 / SSEA-4 / Globo H is dysregulated or malfunctioning, without interfering with the normal activity of SSEA-3 / SSEA-4 / Globo H to which the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention are not specific.

[0426] In another aspect, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be used as reagents for detecting the cancer status in various cell types and tissues.

[0427] In yet another aspect, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention are suitable for developing SSEA-3 / SSEA-4 / Globo H antagonists that block in an activity pattern similar to the target antibodies of the present invention. For example, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be used to identify and distinguish other antibodies having the same SSEA-3 / SSEA-4 / Globo H binding characteristics and / or SSEA-3 / SSEA-4 / Globo H pathway blocking ability.

[0428] As another example, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be used to identify other anti-SSEA-3 / SSEA-4 / Globo H antibodies that bind to SSEA-3 / SSEA-4 / Globo H antigenic determinants (including linear and conformational epitopes) that are substantially identical to those exemplified herein.

[0429] The anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be used in the analysis of physiological pathways involving SSEA-3 / SSEA-4 / Globo H in order to screen for small molecule antagonists of SSEA-3 / SSEA-4 / Globo H that exhibit pharmacological effects similar to those of the antibodies in blocking the binding of one or more binding partners to SSEA-3 / SSEA-4 / Globo H.

[0430] Antibodies can be generated using conventional skills in the art, including those techniques described herein, such as hybridoma technology and screening of phage display libraries of binding molecules. These methods are well established in the art.

[0431] Briefly, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be generated by screening a combinatorial library to select synthetic antibody clones with the desired activity. In principle, synthetic antibody clones are selected by screening a phage library containing various fragments of antibody variable regions (Fv) fused to a phage coat protein. These phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any anti-SSEA-3 / SSEA-4 / Globo H antibody of the present invention can be obtained by designing a suitable antigen screening procedure to select the phage clones of interest, and then constructing full-length anti-SSEA-3 / SSEA-4 / Globo H antibody clones using the Fv sequences from the phage clones of interest and suitable constant region (Fc) sequences as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

[0432] In one embodiment, the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention are monoclonal antibodies. Also within the scope of the present invention are antibody fragments of the anti-SSEA-3 / SSEA-4 / Globo H antibodies provided herein, such as Fab, Fab', Fab'-SH, and F(ab')2 fragments, and variants thereof. These antibody fragments can be produced by conventional means such as enzymatic digestion, or can be produced by recombinant techniques. These antibody fragments can be chimeric antibody fragments, human antibody fragments, or humanized antibody fragments. These fragments are suitable for the experimental, diagnostic, and therapeutic purposes described herein.

[0433] A monoclonal antibody can be obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0434] The anti-SSEA-3 / SSEA-4 / Globo H monoclonal antibodies of the present invention can be produced using a variety of methods known in the art, including the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or alternatively can be produced by recombinant DNA methods (e.g., U.S. Patent No. 4,816,567).

[0435] Vectors, host cells, and recombinant methods

[0436] For the recombinant production of the antibodies of the invention, the nucleic acids encoding said antibodies are isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. The DNA encoding the antibodies is readily isolated and sequenced using known procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). A variety of vectors can be utilized. The choice of vector depends in part on the host cell to be used. Host cells include (but are not limited to) cells of prokaryotic or eukaryotic origin (usually mammalian). It should be understood that any isotype of constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and these constant regions can be obtained from any human or animal species.

[0437] Production of antibodies using prokaryotic host cells

[0438] Vector construction

[0439] The polynucleotide sequences encoding the polypeptide components of the antibodies of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated from antibody-producing cells (such as hybridoma cells) and sequenced. Alternatively, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. A variety of vectors available and known in the art can be used for the present invention. The choice of an appropriate vector depends mainly on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed by the vector. Each vector contains different components, depending on its function (amplification or expression of the heterologous polynucleotide or both) and its compatibility with the particular host cell in which it is present. Vector components generally include (but are not limited to): origin of replication, selectable marker gene, promoter, ribosome binding site (RBS), signal sequence, heterologous nucleic acid insertion sequence, and transcription termination sequence.

[0440] In general, plasmid vectors containing replicons and control sequences derived from species compatible with the host cells are used in conjunction with these hosts. The vectors typically carry a replication site and a marker sequence that enables phenotypic selection in the transformed cells. For example, Escherichia coli is typically transformed using pBR322, a plasmid derived from the Escherichia coli species. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and thus provides a means for easy identification of transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives for expression of specific antibodies are described in detail in U.S. Patent No. 5,648,237 to Carter et al.

[0441] In addition, bacteriophage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in conjunction with these hosts. For example, bacteriophages such as λGEM TM -11 can be used to generate recombinant vectors that can be used to transform susceptible host cells such as Escherichia coli LE392.

[0442] The expression vectors of the present invention may comprise two or more promoter-cistron pairs encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are generally divided into two categories: inducible promoters and constitutive promoters. An inducible promoter is a promoter under whose control the transcription level of the cistron begins to increase in response to changes in culture conditions (e.g., the presence or absence of nutrients or temperature changes).

[0443] Many promoters that can be recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain, which is achieved by restriction enzyme digestion to remove the promoter in the source DNA and insertion of the isolated promoter sequence into the vector of the present invention. Native promoter sequences as well as a variety of heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are used because they generally allow for more transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0444] Promoters suitable for prokaryotic hosts include the PhoA promoter, β-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, enabling those skilled in the art to operably join these nucleotide sequences to the cistrons encoding the target light and heavy chains using linkers or adaptors that provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0445] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs the translocation of the expressed polypeptide across the membrane. Generally, the signal sequence can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, the group consisting of alkaline phosphatase, penicillinase, Ipp, or the heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the invention, the signal sequence in both cistrons for the expression system is the STII signal sequence or a variant thereof.

[0446] In another aspect, the production of the immunoglobulins of the present invention can occur in the cytoplasm of the host cell, and thus a secretion signal sequence is not required within each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded, and assembled in the cytoplasm to form a functional immunoglobulin. Certain host strains (such as the Escherichia coli trxB- strain) provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing the correct folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0447] The antibodies of the present invention can also be produced using an expression system in which the quantitative ratio of the expressed polypeptide components can be adjusted to maximize the yield of the secreted and correctly assembled antibodies of the present invention. The adjustment can be accomplished, at least in part, by simultaneously adjusting the translational strength of the polypeptide components.

[0448] A technique for modulating translational strength is disclosed in U.S. Patent No. 5,840,523 to Simmons et al. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants with a range of translational strengths can be generated, thus providing a convenient way to modulate this factor for a desired level of expression of a particular strand. TIR variants can be generated by known mutagenesis techniques, producing codon changes that can alter the amino acid sequence. In some embodiments, the nucleotide sequence changes are silent. TIR changes can include, for example, changes in the numbering or spacing of the Shine-Dalgarno sequence, as well as signal sequence changes. One method of generating a mutant signal sequence is to generate a "codon library" at the start of the coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the change is silent). This can be done by changing the third nucleotide position of each codon; in addition, some amino acids (such as leucine, serine, and arginine) have multiple first and second positions, which can add complexity when preparing the library. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0449] In one embodiment, a set of vectors is generated that have a series of TIR strengths for each cistron. This limited set compares the expression levels of the strands and the yields of the desired antibody product at various combinations of TIR strengths. The TIR strength can be determined by quantifying the expression level of a reporter gene, as detailed in U.S. Patent No. 5,840,523 to Simmons et al. Based on the translational strength comparison, the desired individual TIRs to be combined in the expression vector constructs of the present invention are selected.

[0450] Prokaryotic host cells suitable for expressing the antibodies of the present invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis), Enterobacter, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC deposit number 27,325) and its derivatives, including strain 33D3 having the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41kanR (U.S. Patent No. 5,639,635). Other strains and their derivatives (such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608)) are also suitable. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select an appropriate bacterium considering the replicability of the replicon in the bacterial cell. For example, when using well-known plasmids (such as pBR322, pBR325, pACYC177, or pKN410) to provide the replicon, Escherichia, Serratia, or Salmonella species are suitable as hosts. The host cells should generally secrete a minimal amount of proteolytic enzymes, and other protease inhibitors are preferably incorporated into the cell culture.

[0451] Antibody production

[0452] The host cells are transformed with the above expression vectors and cultured in a known nutrient medium which is adjusted as appropriate to induce the promoter, select transformants or amplify the gene encoding the desired sequence.

[0453] Transformation means introducing DNA into a prokaryotic host such that the DNA can replicate as an extrachromosomal element or as a chromosomal integrant. Depending on the host cells used, transformation is carried out using standard techniques appropriate for these cells. Calcium treatment using calcium chloride is generally used for bacterial cells with a substantial cell wall barrier. Another transformation method is the use of polyethylene glycol / DMSO. Another technique used is electroporation.

[0454] The prokaryotic cells used to produce the polypeptides of the present invention are grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria broth (LB) plus essential nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to allow the growth of cells expressing the ampicillin resistance gene.

[0455] In addition to carbon, nitrogen and inorganic phosphate sources, any necessary supplements may be included at appropriate concentrations, introduced either alone or in combination with other supplements or media such as complex nitrogen sources. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteamine, cystamine, mercaptoacetate, dithiothreitol and dithioerythritol.

[0456] The prokaryotic host cells are cultured at a suitable temperature. For the growth of Escherichia coli, for example, growth occurs at temperatures in the range including (but not limited to) about 20°C to about 39°C, about 25°C to about 37°C and at about 30°C. The pH of the medium can be any pH in the range of about 5 to about 9, depending mainly on the host organism. For Escherichia coli, the pH can be about 6.8 to about 7.4, or about 7.0.

[0457] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the present invention, the PhoA promoter is used to control polypeptide transcription. Thus, the transformed host cells are cultured in a phosphate-restricted medium for induction. In one embodiment, the phosphate-restricted medium is C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133 - 147). As is known in the art, a variety of other inducers can be used depending on the vector construct used.

[0458] In one embodiment, the expressed polypeptide of the invention is secreted into the periplasm of the host cell and recovered from the periplasm of the host cell. Protein recovery generally involves disrupting the microorganism, typically by disruption methods such as osmotic shock, sonication, or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

[0459] In one aspect of the invention, antibodies are produced in large quantities by fermentation methods. A variety of large-scale batch-fed fermentation procedures can be utilized to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, for example, a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to disperse oxygen and nutrients, especially glucose (a common carbon / energy source). Small-scale fermentations generally refer to fermentations carried out in fermenters with a volume capacity of no more than about 100 liters and can range from about 1 liter to about 100 liters.

[0460] During fermentation, protein expression is typically induced after the cells have grown to the desired density under suitable conditions (e.g., OD550 is about 180 - 220, and at this stage the cells are in the early stationary phase). As is known in the art and as described above, a variety of inducers can be used depending on the vector construct employed. The cells can be grown for a shorter period before induction. The cells are typically induced for about 12 - 50 hours, although longer or shorter induction times can be used.

[0461] To enhance the yield and quality of the polypeptides of the present invention, various fermentation conditions can be altered. For example, to enhance the proper assembly and folding of the secreted antibody polypeptides, host prokaryotic cells can be co-transformed with an additional vector that overexpresses chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl-prolyl cis,trans-isomerase with chaperone activity). Chaperone proteins have been shown to promote the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J.Biol.Chem. 275:17100-17105; Ramm and Pluckthun (2000) J.Biol.Chem. 275:17106-17113; Arie et al., (2001) Mol.Microbiol. 39:199-210.

[0462] To minimize proteolysis of the expressed heterologous proteins, especially proteolysis-sensitive proteins, the present invention can use certain host strains lacking proteolytic enzymes. For example, the host cell strain can be modified to create gene mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some Escherichia coli protease-deficient strains are available and are described, for example, in Joly et al., (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0463] In one embodiment, an Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid that overexpresses one or more chaperone proteins is used as the host cell in the expression system of the present invention.

[0464] Antibody Purification

[0465] In one embodiment, the antibody proteins produced herein are further purified to obtain a substantially homogeneous preparation for further analysis and use. Standard protein purification methods known in the art can be used. The following procedures illustrate suitable purification procedures: separation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0466] In one aspect, immunoaffinity purification of the antibody product of the invention is performed using Protein A immobilized on a solid phase. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the Fc region of antibodies with high affinity. Lindmark et al., (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a column containing a glass or silica surface, or a controlled pore glass column or a silica column. In some applications, the column is coated with a reagent such as glycerol to prevent non-specific attachment of contaminants as much as possible.

[0467] As a first step in purification, a preparation derived from cell culture as described above can be applied to the Protein A-immobilized solid phase to allow the antibody of interest to specifically bind to Protein A. The solid phase is then washed to remove contaminants that non-specifically bind to the solid phase. Finally, the antibody of interest is recovered by elution from the solid phase.

[0468] Production of antibodies using eukaryotic host cells

[0469] Vector components generally include (but are not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0470] (i) Signal sequence component

[0471] Vectors for use in eukaryotic host cells may also contain a signal sequence, or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is typically a signal sequence that will be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). When expressed in mammalian cells, mammalian signal sequences as well as viral secretory leader sequences, such as the herpes simplex virus gD signal, can be utilized.

[0472] The DNA of this precursor region is ligated in-frame to the DNA encoding the antibody.

[0473] (ii) Origin of replication

[0474] Generally, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin is often used only because it contains an early promoter.

[0475] (iii) Selectable gene components

[0476] Expression vectors and cloning vectors may contain selectable genes, also known as selectable markers. Typical selectable genes encode proteins that: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b) complement auxotrophies (if necessary); or (c) provide key nutrients not available from complex media.

[0477] One example of a selection scheme uses a drug to block the growth of host cells. Those cells successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection scheme. Examples of this dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0478] Another example of a selectable marker suitable for mammalian cells is a selectable marker that can identify cells capable of taking up antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein-I and metallothionein-II (e.g., the primate metallothionein gene), adenosine deaminase, ornithine decarboxylase, etc.

[0479] For example, cells transformed with a DHFR selectable gene can first be identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Suitable host cells (when using wild-type DHFR) include, for example, Chinese hamster ovary (CHO) cell lines lacking DHFR activity (e.g., ATCC CRL-9096).

[0480] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an antibody, wild-type DHFR protein, and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0481] (iv) Promoter components

[0482] Expression vectors and cloning vectors generally contain a promoter, which is recognized by the host organism and operably linked to a nucleic acid encoding a polypeptide of interest (such as an antibody). Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription start of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal for adding a poly-A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

[0483] In mammalian host cells, transcription of antibody polypeptides from the vector can be controlled, for example, by promoters obtained from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)), heterologous mammalian promoters (such as the actin promoter or immunoglobulin promoter), or heat shock promoters, provided that these promoters are compatible with the host cell system.

[0484] The early and late promoters of the SV40 virus are preferably obtained in the form of an SV40 restriction fragment that also contains the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is preferably obtained in the form of a HindIII E restriction fragment. A system for expressing DNA using bovine papillomavirus as a vector in mammalian hosts is disclosed in U.S. Patent No. 4,419,446. Variations of this system are described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598 - 601 (1982), which is about the expression of human β - interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of Rous Sarcoma Virus can be used as a promoter.

[0485] (v) Enhancer component components

[0486] In higher eukaryotes, transcription of DNA encoding the antibody polypeptides of the invention is increased usually by inserting enhancer sequences into the vector. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, enhancers from eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer (bp 100-270) located behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located behind the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982) for enhancer elements used to activate eukaryotic promoters. The enhancer can be spliced into the expression vector at the 5' or 3' position of the antibody polypeptide coding sequence, but is usually located at the 5' site relative to the promoter.

[0487] (vi) Transcription termination component

[0488] Expression vectors for use in eukaryotic host cells usually also contain sequences necessary for termination of transcription and stabilization of the mRNA. These sequences are commonly obtained from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed in the untranslated portion of the mRNA encoding the antibody as polyadenylation fragments. One suitable transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0489] (vii) Selection and transformation of host cells

[0490] Host cells suitable for cloning or expressing DNA in the vectors of the present invention include the higher eukaryotic cells described herein, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of mammalian host cell lines are the monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver tumor cell line (Hep G2).

[0491] The host cells are transformed with the above-described expression or cloning vectors for antibody production and are cultured in a known nutrient medium that is adjusted as appropriate to induce the promoter, select transformants, or amplify the gene encoding the desired sequence.

[0492] (viii) Culturing the host cells

[0493] Host cells for producing the antibodies of the present invention can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing host cells. In addition, any of the culture media described in the following references can be used as the culture medium for these host cells: Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; or No. 5,122,469; WO 90 / 03430; WO87 / 00195; or U.S. Patent Re. 30,985. Any of these culture media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements at appropriate concentrations known to those skilled in the art can also be included. Culture conditions (such as temperature, pH, and similar conditions) are those previously selected for the host cells used for expression and are obvious to those of ordinary skill in the art.

[0494] (ix) Antibody purification

[0495] When recombinant techniques are used, the antibodies can be produced intracellularly or secreted directly into the culture medium. If the antibodies are produced intracellularly, the first step generally involves removing particulate debris (host cells or lysed fragments), for example, by centrifugation or ultrafiltration. In the case where the antibodies are secreted into the culture medium, the supernatant of these expression systems is usually concentrated first using commercially available protein concentration filters (such as Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0496] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, which are commonly accepted purification techniques. The suitability of an affinity reagent, such as Protein A, as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). All murine isotypes and human γ3 are recommended to be purified using Protein G (Guss et al., EMBO J. 5:1567-1575 (1986)). Although the matrix to which the affinity ligand is attached is most commonly agarose, other matrices can also be utilized. Compared to the flow rate and processing time achievable with agarose, mechanically stable matrices, such as controlled pore glass or poly(styrene-divinylbenzene), can provide a faster flow rate and shorter processing time. In the case where the antibody contains a CH3 domain, then Bakerbond ABX TM resin (J.T. Baker, Phillipsburg, N.J.) is suitable for purification. Other protein purification techniques can also be utilized, such as separation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSE TM chromatography, anion or cation exchange resins (such as polyaspartic acid columns) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

[0497] After any initial purification step, further purification steps can be performed on the mixture containing the antibody of interest and contaminants, if desired, such as low pH hydrophobic interaction chromatography using an elution buffer with a pH between approximately 2.5 - 4.5, generally performed at a low salt concentration (e.g., approximately 0 - 0.25 M salt).

[0498] It should be understood that, in general, the techniques and methods for preparing antibodies for use in research, testing, and clinical applications are well established in the art and are suitable for the specific antibody of interest as described above and / or as would be recognized by those skilled in the art.

[0499] Activity assays

[0500] The physical / chemical properties and biological functions of the antibodies of the present invention can be characterized by various assays known in the art.

[0501] The purified antibodies can be further characterized by a series of assays, including (but not limited to) N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0502] When needed, the biological activity of the antibody is analyzed. In some embodiments, the antigen-binding activity of the antibodies of the invention is tested. Antigen-binding assays known in the art and available herein include, but are not limited to, any direct or competitive binding assay using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, chemiluminescence immunoassay, nanoparticle immunoassay, aptamer immunoassay, and protein A immunoassay.

[0503] Antibody fragment

[0504] The present invention encompasses antibody fragments. In some instances, the use of antibody fragments may offer advantages over whole antibodies. Smaller fragments permit rapid clearance and enhanced access to solid tumors.

[0505] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments could be derived via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from Escherichia coli, thus allowing large amounts of these fragments to be readily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having an extended in vivo half-life are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and sFv are the only antibody fragments that have a complete binding site but lack a constant region; thus, they are useful for reducing non-specific binding during in vivo use. sFv fusion proteins can be constructed to generate fusions of effector proteins at the amino or carboxyl terminus of the sFv. See Antibody Engineering, Borrebaeck ed., supra. Antibody fragments can also be "linear antibodies", such as those described in U.S. Patent No. 5,641,870. These linear antibody fragments can be monospecific or bispecific.

[0506] Humanized antibody

[0507] The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, one or more amino acid residues from non-human sources can be introduced into humanized antibodies. These non-human amino acid residues are commonly referred to as "import" residues, which are typically obtained from "import" variable domains. Humanization can generally follow the methods of Winter and colleagues (Jones et al., (1986) Nature 321:522-525; Riechmann et al., (1988) Nature 332:323-327; Verhoeyen et al., (1988) Science 239:1534-1536), by replacing the corresponding sequences of human antibodies with hypervariable region sequences. Thus, these "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain is replaced with the corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced with residues from the analogous sites of rodent antibodies.

[0508] The choice of human variable domains (light and heavy chains) for generating humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened against a complete library of known human variable domain sequences. The human sequence that is most similar to the rodent sequence is then used as the human framework for the humanized antibody (Sims et al., (1993) J. Immunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196:901). Another approach is to use a specific framework that is derived from the consensus sequence of all human antibodies having a particular subgroup of light or heavy chains. Several different humanized antibodies can use the same framework (Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., (1993) J. Immunol., 151:2623).

[0509] It is often desirable to humanize antibodies while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by using three-dimensional models of the parental and humanized sequences to analyze the parental sequence and various contemplated humanized products. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and present the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these presentations allows analysis of the possible roles of residues when the candidate immunoglobulin sequence functions, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the acceptor and donor sequences and combined to obtain the desired antibody characteristics, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and very substantially involved in affecting antigen binding.

[0510] Human antibody

[0511] The human anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be constructed by combining Fv clonal variable domain sequences selected from a human-derived phage display library with known human constant domain sequences as described above. Alternatively, the human monoclonal anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be produced by the hybridoma method. For example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991) have described human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies.

[0512] Transgenic animals (e.g., mice) that are capable of producing a complete human antibody repertoire upon immunization can now be produced without the production of endogenous immunoglobulins. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete suppression of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into these germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

[0513] Human antibodies can also be obtained by shuffling genes from non-human (e.g., rodent) antibodies, where the human antibodies have an affinity and specificity similar to the original non-human antibody. According to this method (which is also known as "epitope imprinting"), the variable regions of the heavy or light chains of the non-human antibody fragments obtained by phage display technology as described above are replaced with human V domain gene lineages to form a population of non-human chain / human chain scFv or Fab chimeras. Selection with an antigen can isolate the non-human chain / human chain chimeric scFv or Fab, where the human chain restores the antigen-binding site that was damaged after removing the corresponding non-human chain in the primary phage display clone, i.e., the epitope determines (imprints) the selection of the human chain partner. When the method is repeated to replace the remaining non-human chains, a human antibody is obtained (see PCT WO 93 / 06213, published April 1, 1993). Different from the traditional humanization of non-human antibodies by CDR grafting, this technique provides intact human antibodies that do not have FR or CDR residues of non-human origin.

[0514] Bispecific antibody

[0515] A bispecific antibody is a monoclonal antibody that has binding specificity for at least two different antigens. In some embodiments, the bispecific antibody is a human antibody or a humanized antibody. In some embodiments, one of the binding specificities is directed against SSEA-3 / SSEA-4 / Globo H including a specific lysine linkage, and the other is directed against any other antigen. In some embodiments, the bispecific antibody can bind to two different SSEA-3 / SSEA-4 / Globo H with two different lysine linkages. Bispecific antibodies can be prepared in the form of full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0516] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (tetravalent hybridomas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. It is usually quite cumbersome to purify the correct molecule through an affinity chromatography step, and the product yield is low. Similar procedures are disclosed in WO 93 / 08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655 (1991).

[0517] According to different embodiments, an antibody variable domain having the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence. This fusion is, for example, using an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. In certain embodiments, a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of these fusions. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. When optimal yields can be provided using unequal ratios of the three polypeptide chains in the construction, great flexibility is provided in the embodiments to adjust the relative ratios of the three polypeptide fragments. However, if the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important, the coding sequences of two or all three polypeptide chains may be inserted into one expression vector.

[0518] In one embodiment of the method, a bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from the unwanted immunoglobulin chain combinations since the immunoglobulin light chain is only present in half of the bispecific molecules. This method is disclosed in WO 94 / 04690. For other details regarding the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0519] According to another method, the interface between a pair of antibody molecules can be engineered such that the percentage of heterodimers recovered from a recombinant cell culture is maximized. The interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains such as tyrosine or tryptophan. By replacing larger amino acid side chains with smaller amino acid side chains such as alanine or threonine, a compensatory "hole" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.

[0520] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the form of a heteroconjugate can be coupled to avidin and the other to biotin. For example, these antibodies have been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and to treat HIV infection (WO 91 / 00360, WO92 / 200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents and many cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980.

[0521] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical conjugation. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol chelating agent sodium arsenite in order to stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for the selective immobilization of enzymes.

[0522] Recent advances have made it possible to directly recover Fab'-SH fragments from Escherichia coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the preparation of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is secreted separately from Escherichia coli and undergoes directed chemical conjugation in vitro to form a bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the HER2 receptor and normal human T cells, and is also able to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0523] A variety of techniques for the preparation and isolation of bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers, and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. These fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is short enough to prevent the two domains on the same chain from pairing. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0524] Antibodies with more than two valences are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0525] Multivalent antibodies

[0526] Multivalent antibodies can be internalized (and / or excreted) more rapidly by cells expressing the antigen to which the antibody binds than bivalent antibodies. The antibodies of the present invention can be multivalent antibodies having three or more antigen-binding sites (except for antibodies of the IgM class) (e.g., tetravalent antibodies), and multivalent antibodies can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibody can comprise a dimerization domain and three or more antigen-binding sites. The dimerization domain comprises (or includes), for example, an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen-binding sites at the amino terminus of the Fc region. In one embodiment, the multivalent antibody comprises (or includes), for example, three to about eight, or four antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain can comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain can comprise VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodies herein can further comprise at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibodies herein can comprise, for example, about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides encompassed herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0527] Antibody variant

[0528] In some embodiments, the present invention encompasses amino acid sequence modifications of the antibodies described herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can be introduced into the target antibody amino acid sequence during the manufacture of the sequence.

[0529] There is a method called "alanine scanning mutagenesis" as described in Cunningham & Wells (1989) Science, 244:1081-1085, which is applicable to identifying certain residues or regions of an antibody as preferred mutagenesis sites. In this context, a residue or a set of target residues (such as charged residues like Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (such as alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Subsequently, by introducing more or other variants at or for the substitution site, those amino acid positions that demonstrate functional sensitivity to the substitution are improved. Thus, while the sites for introducing amino acid sequence variations are predetermined, the nature of the mutations themselves need not be predetermined. For example, to analyze the efficacy of mutations at a specific site, ala scanning or random mutagenesis is performed at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

[0530] Amino acid sequence insertions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of sequences having single or multiple amino acid residues. Examples of terminal insertions include an antibody having an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of the antibody with an enzyme (such as for ADEPT) or with a polypeptide that extends the serum half-life of the antibody.

[0531] Another class of variants are amino acid substitution variants. In an antibody molecule, these variants have at least one amino acid residue replaced with a different residue. The most interesting substitution mutation sites include the hypervariable regions, but also encompass FR changes. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. If these substitutions result in a change in biological activity, more substantial changes can be introduced (such as those named "Exemplary Substitutions" in Table A, or as further described below with reference to amino acid classes), and the products are screened.

[0532] Table A

[0533]

[0534] Substantial modification of the biological properties of an antibody can be accomplished by selecting substitutions that are significantly different in their effect on maintaining: (a) the structure of the polypeptide backbone in the substitution region, such as the pleated sheet or helical conformation; (b) the molecular charge or hydrophobicity at the target; or (c) the side chain volume. Amino acids can be classified according to the similarity of their side chain properties (in A.L. Lehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)):

[0535] ·(1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

[0536] ·(2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

[0537] ·(3) Acidic: Asp (D), Glu (E)

[0538] ·(4) Basic: Lys (K), Arg (R), His (H).

[0539] Alternatively, naturally occurring residues can be classified based on common side-chain properties:

[0540] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0541] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0542] (3) Acidic: Asp, Glu;

[0543] (4) Basic: His, Lys, Arg;

[0544] (5) Residues affecting chain orientation: Gly, Pro;

[0545] (6) Aromatic: Trp, Tyr, Phe.

[0546] Non-conservative substitutions would require replacement of a member of one of these categories with a member of another category. These substituted residues can also be introduced into conservative substitution sites or into the remaining (non-conservative) sites.

[0547] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Typically, the biological properties of the resulting variant selected for further development are modified (e.g., enhanced) relative to the parental antibody from which the variant is generated. Suitable methods for generating these substitution variants involve affinity maturation using phage display. Briefly, several hypervariable regions (e.g., 6 - 7 regions) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are presented by filamentous phage particles in a form fused to at least a portion of the phage coat protein (e.g., the gene III product of M13) encapsulated within each particle. The phage - displayed variants are then screened for the biological activity (e.g., binding affinity) of the antibody as disclosed herein. To identify candidate hypervariable regions for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues that are significant for antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen - antibody complex to identify the contact points between the antibody and the antigen. According to techniques known in the art, including those detailed herein, these contact residues and adjacent residues are candidates for substitution. After generating these variants, the set of variants is screened using techniques known in the art (including those described herein), and antibodies with favorable properties can be selected for further development in one or more relevant assays.

[0548] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. These methods include (but are not limited to) isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or oligonucleotide - mediated (or site - directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis from earlier prepared antibody variants or non - variant forms.

[0549] It may be desirable to introduce one or more amino acid modifications into the Fc region of the antibodies of the invention to generate Fc region variants. Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) having an amino acid modification (e.g., substitution) at one or more amino acid positions, including the hinge cysteine positions.

[0550] Immunoconjugate

[0551] In another aspect, the invention provides an immunoconjugate or an antibody - drug conjugate (ADC) comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or an antibody conjugated to a radioisotope (i.e., a radio - conjugate).

[0552] Use of antibody-drug conjugates for local delivery of cytotoxic agents or cell growth inhibitors (i.e., drugs that kill or inhibit tumor cells) for the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) allows for the targeted delivery of the drug moiety to tumors and accumulation within cells, where systemic administration of these unbound agents may produce an unacceptable degree of toxicity to normal cells as well as the tumor cells that are intended to be eliminated (Baldwin et al., (1986) Lancet pp. (March 15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Thereby attempting to maximize efficacy while minimizing toxicity. Multiple polyclonal and monoclonal antibodies have been reported to be suitable for these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986), ibid.).The toxins used in antibody-toxin conjugates include bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), small molecule toxins (such as geldanamycin) (Mandler et al., (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al., (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al., (1998) Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342). These toxins can exert their cytotoxic and cytostatic effects through mechanisms including binding to tubulin, binding to DNA, or inhibiting topoisomerase. Some cytotoxic drugs are prone to becoming inactive or less active when bound to large antibodies or protein ligands.

[0553] Antibody derivatives

[0554] The antibodies of the present invention can be further modified to contain other non-protein moieties known and readily available in the art. In one embodiment, the moiety suitable for antibody derivatization is a water-soluble polymer. Non-limiting examples of water-soluble polymers include (but are not limited to) polyethylene glycol (PEG), ethylene glycol / propanediol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylene polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous for manufacture due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers linked to the antibody can be varied, and if more than one polymer is linked, these polymers can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including (but not limited to) the specific properties or functions of the antibody to be modified, whether the antibody derivative can be used for treatment under defined conditions, and the like.

[0555] In another embodiment, conjugates of antibodies with non-protein moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102:11600-11605 (2005)). This radiation can be of any wavelength and includes (but is not limited to) wavelengths that, while not damaging normal cells, can heat the non-protein moiety to a temperature that will kill cells adjacent to the antibody-non-protein moiety conjugate.

[0556] Pharmaceutical formulations

[0557] Therapeutic formulations containing the antibodies of the present invention are prepared by mixing the antibody of the desired purity with a physiologically acceptable carrier, excipient, or stabilizer as selected as needed (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)) and stored in the form of an aqueous solution, a freeze-dried or other dry formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used and include buffers such as phosphates, citrates, histidine, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0558] The formulations herein may also contain more than one active compound required for treating a particular indication, including (but not limited to) those having complementary activities that do not have an adverse effect on each other. These molecules are suitable for being present in combinations in amounts that are effective to achieve the intended purpose.

[0559] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980).

[0560] Formulations intended for in vivo administration must be sterile. This can be readily accomplished by filtration through sterile filtration membranes.

[0561] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the present invention, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide-co-glycolide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM )(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable molecules to be released over a period of more than 100 days, some hydrogels release proteins in a shorter time. When encapsulated immunoglobulins remain in the body for too long, they may denature or aggregate due to exposure to humidity at 37°C, resulting in loss of biological activity and possibly a change in immunogenicity. Depending on the mechanism involved, rational strategies can be designed for stabilization. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds via thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0562] Use

[0563] The antibodies of the present invention can be used, for example, in in vitro, ex vivo and in vivo therapeutic methods. The antibodies of the present invention can be used as antagonists that partially or completely inhibit the activity of a specific antigen in vitro, ex vivo and / or in vivo. In addition, at least some of the antibodies of the present invention can neutralize the antigenic activity of other species. Thus, the antibodies of the present invention can be used, for example, to inhibit the activity of a specific antigen in a cell culture containing the antigen, in a human individual or other mammalian individual (such as chimpanzee, baboon, marmoset, cynomolgus macaque and rhesus macaque, pig or mouse) having an antigen that can cross-react with the antibodies of the present invention. In one embodiment, the antibodies of the present invention can be used to inhibit antigen activity by contacting the antibodies with the antigen such that the antigen activity is inhibited. In one embodiment, the antigen is a human protein molecule.

[0564] In one embodiment, the antibodies of the present invention can be used in a method for inhibiting an antigen in an individual suffering from a disorder in which the antigen activity is harmful, the method comprising administering the antibodies of the present invention to the individual so as to inhibit the antigen activity in the individual. In one embodiment, the antigen is a human protein molecule and the individual is a human individual. Alternatively, the individual can be a mammal that expresses an antigen that binds to the antibodies of the present invention. In addition, the individual can be a mammal that has been introduced with an antigen (such as by administering the antigen or by expressing an antigen transgene). For therapeutic purposes, the antibodies of the present invention can be administered to a human individual. In addition, for veterinary purposes or as an animal model of human diseases, the antibodies of the present invention can be administered to a non-human mammal (such as a primate, pig or mouse) that expresses an antigen that cross-reacts with the antibody. With respect to the latter, these animal models can be suitable for evaluating the therapeutic efficacy of the antibodies of the present invention (such as testing the dosage and duration of administration). The antibodies of the present invention can be used to treat, inhibit, delay the progression of, prevent / delay the recurrence of, improve or prevent diseases, disorders or conditions associated with the abnormal expression and / or activity of SSEA-3 / SSEA-4 / Globo H and SSEA-3 / SSEA-4 / Globo Hylated proteins, and the diseases, disorders or conditions associated with the abnormal expression and / or activity of SSEA-3 / SSEA-4 / Globo H and SSEA-3 / SSEA-4 / Globo Hylated proteins include (but are not limited to) cancer, muscle disorders, ubiquitin pathway-related genetic disorders, immune / inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.

[0565] In one aspect, the blocking antibodies of the present invention are specific for SSEA-3 / SSEA-4 / Globo H.

[0566] In certain embodiments, an immunoconjugate comprising an antibody of the invention conjugated to a cytotoxic agent is administered to a patient. In some embodiments, the immunoconjugate and / or the antigen to which it binds is internalized by cells expressing one or more proteins related to SSEA-3 / SSEA-4 / Globo H on the cell surface, thereby enhancing the therapeutic efficacy of the immunoconjugate in killing target cells to which it binds. In one embodiment, the cytotoxic agent targets or interferes with nucleic acids in the target cell. Examples of such cytotoxic agents include any of the chemotherapeutic agents noted herein (such as maytansinoids or calicheamicin), radioisotopes, or ribonucleases or DNA endonucleases.

[0567] The antibodies of the invention can be used alone or in combination with other compositions in therapy. For example, the antibodies of the invention can be co-administered with another antibody and / or an adjuvant / therapeutic agent (such as a steroid). For example, the antibodies of the invention can be combined with an anti-inflammatory agent and / or a preservative in a treatment regimen, for example for treating any of the diseases described herein, including cancer, muscle disorders, ubiquitin pathway-related genetic disorders, immune / inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders. These combination therapies noted above include co-administration (where two or more agents are incorporated into the same or separate formulations), and separate administration, in which case the antibodies of the invention can be administered before and / or after the administration of an adjuvant therapy.

[0568] The antibodies of the invention (and adjuvant therapeutic agents) can be administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intranasally, and intranasally, and, if local treatment is desired, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Additionally, the antibodies are appropriately administered by pulsed infusion, particularly antibodies at reduced doses. Administration can be by any suitable route (such as by injection, such as intravenous or subcutaneous injection), in part depending on whether the administration is short-term or long-term.

[0569] The location of the binding target of the antibodies of the present invention can be considered in antibody preparation and administration. When the binding target is an intracellular molecule, certain embodiments of the present invention provide an antibody or an antigen-binding fragment thereof to be introduced into the cell where the binding target is located. In one embodiment, the antibodies of the present invention can be expressed intracellularly as intrabodies. As used herein, the term "intrabody" refers to an antibody or an antigen-binding portion thereof that can be expressed intracellularly and is capable of selectively binding to a target molecule as described in the following references: Marasco, Gene Therapy 4:11-15 (1997); Kontermann, Methods 34:163-170 (2004); U.S. Patent Nos. 6,004,940 and 6,329,173; U.S. Patent Application Publication No. 2003 / 0104402, and PCT Publication No. WO2003 / 077945. Intracellular expression of an intrabody is achieved by introducing a nucleic acid encoding the desired antibody or an antigen-binding portion thereof (lacking the wild-type leader sequence and the secretion signal normally associated with the gene encoding the antibody or antigen-binding fragment) into the target cell. Any standard method for introducing nucleic acids into cells can be used, including (but not limited to): microinjection, biolistic injection, electroporation, calcium phosphate precipitation, liposomes, and transfection with retroviruses, adenoviruses, adeno-associated viruses, and vaccinia vectors carrying the nucleic acid of interest. One or more nucleic acids encoding all or a portion of the anti-SSEA-3 / SSEA-4 / Globo H antibodies of the present invention can be delivered to the target cell so as to express one or more intrabodies capable of binding to SSEA-3 / SSEA-4 / Globo H intracellularly and modulating one or more SSEA-3 / SSEA-4 / Globo H-mediated cellular pathways.

[0570] In another embodiment, internalizing antibodies are provided. The antibody may have certain properties that enhance the delivery of the antibody into the cell, or may be modified to have such properties. Techniques for achieving this are known in the art. For example, it is known that cationization of an antibody facilitates its uptake into cells (see, for example, U.S. Patent No. 6,703,019). Lipofection or liposomes can also be used to deliver an antibody into the cell. When using antibody fragments, the minimal inhibitory fragment of the binding domain that specifically binds to the target protein is generally advantageous. For example, based on the variable region sequence of the antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. These peptides can be chemically synthesized and / or produced by recombinant DNA techniques. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993).

[0571] Regulatory polypeptides can be enhanced to enter target cells by methods known in the art. For example, certain sequences (such as those derived from HIV Tat or antennapedia homeodomain proteins) can direct the efficient uptake of heterologous proteins across cell membranes. See, e.g., Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.

[0572] When the target is located in the brain, certain embodiments of the present invention provide antibodies or antigen-binding fragments thereof that can cross the blood-brain barrier. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier, such that antibodies or antigen-binding fragments can be readily introduced into the brain. When the blood-brain barrier remains intact, there are several methods known in the art for delivering molecules across the blood-brain barrier, including (but not limited to) physical methods, lipid-based methods, and receptor- and channel-based methods.

[0573] Physical methods for delivering antibodies or antigen-binding fragments across the blood-brain barrier include (but are not limited to) completely bypassing the blood-brain barrier or creating openings within the blood-brain barrier. Bypass methods include (but are not limited to) direct injection into the brain (see, e.g., Papanastassiou et al., Gene Therapy 9:398-406 (2002)), interstitial infusion / convection-enhanced delivery (see, e.g., Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994)), and implantation of a delivery device into the brain (see, e.g., Gill et al., Nature Med. 9:589-595 (2003); and Gliadel Wafers TM , Guildford Pharmaceutical). Methods for creating openings within the barrier include (but are not limited to) ultrasound (see, e.g., U.S. Patent Publication No. 2002 / 0038086), osmotic pressure (e.g., administration of hypertonic mannitol (Neuwelt, E.A., Implication of the Blood-Brain Barrier and its Manipulation, Volumes 1 and 2, Plenum Press, N.Y. (1989))), permeation through, for example, bradykinin or penetrant A-7 (see, e.g., U.S. Patents...

Claims

1. An isolated monoclonal antibody that binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, wherein the isolated monoclonal antibody comprises an Fc glycoform for enhancing the binding / effector activity of the isolated monoclonal antibody, and wherein the isolated monoclonal antibody comprises a glycoform having the following formula: Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2 wherein the antibody comprises: (i) H-CDR1 having the amino acid sequence of SEQ ID NO:207; (ii) H-CDR2 having the amino acid sequence of SEQ ID NO:208; (iii) H-CDR3 having the amino acid sequence of SEQ ID NO:209; (iv) L-CDR1 having the amino acid sequence of SEQ ID NO:204; (v) L-CDR2 having the amino acid sequence of SEQ ID NO:205; (vi) L-CDR3 having the amino acid sequence of SEQ ID NO:

206.

2. The isolated monoclonal antibody of claim 1, wherein the isolated monoclonal antibody is IgG1 and binds specifically to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1.

3. The isolated monoclonal antibody of claim 2, wherein the isolated monoclonal antibody comprises: (i) VH having the sequence of SEQ ID NO:202 and VL having the sequence of SEQ ID No:203; (ii) VH having the sequence of SEQ ID No.212 and VL having the sequence of SEQ ID No.213; or (iii) VH having the sequence of SEQ ID NO:222 and VL having the sequence of SEQ ID NO:

223.

4. The isolated monoclonal antibody of claim 3, wherein antibodies (ii) and (iii) are humanized antibodies.

5. A pharmaceutical composition comprising the isolated monoclonal antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. Use of an isolated monoclonal antibody of claim 3 for the preparation of a medicament for the treatment of pancreatic cancer.

7. A method for preparing a population of homogeneous antibodies, which are homogeneous antibodies of the isolated monoclonal antibody of claim 1 or 2, the method comprising: (a) contacting the monoclonal antibody with α-trehalosidase and at least one endoglycosidase; (b) generating a detrehalosylated antibody having a single N-acetylglucosamine (GlcNAc); and (c) adding Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc to the GlcNAc of the antibody Fc region to form the homogeneous antibody of the isolated monoclonal antibody having the glycoform as defined in claim 1.

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