US20210198354A1 - Method for treating autoimmune and inflammatory diseases - Google Patents

Method for treating autoimmune and inflammatory diseases Download PDF

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US20210198354A1
US20210198354A1 US17/138,387 US202017138387A US2021198354A1 US 20210198354 A1 US20210198354 A1 US 20210198354A1 US 202017138387 A US202017138387 A US 202017138387A US 2021198354 A1 US2021198354 A1 US 2021198354A1
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Le Sun
Xiaogang Zhang
Maohua Li
Cuijuan ZHANG
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AbMax Biotechnology Co Ltd
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    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

Definitions

  • the present invention relates to de-immunogenicity of anti-Tumor Necrosis Factor-alpha (TNF- ⁇ ) antibodies and applications of using the same for treating inflammatory diseases and other human diseases.
  • TNF- ⁇ Tumor Necrosis Factor-alpha
  • TNF is an immunity-modulating cytokine required for immune processes.
  • the unregulated activities of TNFs can lead to the development of inflammatory diseases.
  • Excess amounts of TNF-expressed in cells are associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease.
  • the function of TNF requires binding to its two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Blocking the interaction between TNF and TNFRs has successfully been developed as a therapy in treating inflammatory or autoimmune diseases.
  • TNF neutralization therapies including the use of a soluble TNFR2-Fc recombinant (Etanercept), a mouse-human chimera mAb (Infliximab), or a human mAb (Adalimumab), have been introduced in the past decades for the management of rheumatoid arthritis and other immune diseases.
  • This invention is about the de-immunogenicity of human anti-Tumor Necrosis Factor-alpha (TNF- ⁇ ) antibody Adalimumab, designed as clones TCX002-L3H4, L1H4. etc, which bind to the same epitope from the one recognized by Adalimumab, but with much lower immunogenicities in vivo.
  • TNF- ⁇ Necrosis Factor-alpha
  • the present invention provides the human anti-Tumor Necrosis Factor-alpha (TNF- ⁇ ) antibodies with reduced immunogenicities and methods of using the same for neutralizing the TNF- ⁇ induced cell death and for treating inflammatory diseases and other human diseases.
  • TNF- ⁇ Necrosis Factor-alpha
  • the present invention features TNF- ⁇ -binding molecules and their DNA and amino acid sequences. Each molecule comprises the CDRs from human anti-TNF- ⁇ monoclonal antibody Adalimumab and the FRs from different human origins.
  • the present invention also provides a method to develop human antibodies with reduced immunogenicities by replacing the FRs of the original human monoclonal antibody with the FRs from different human origins.
  • the present invention also provides one example of using the method to develop human anti-TNF- ⁇ antibodies with reduced immunogenicities by replacing the FRs of human anti-TNF- ⁇ monoclonal antibody Adalimumab with the FRs from different human origins.
  • the present invention features de-immunized human anti-TNF- ⁇ antibodies with one of the amino acid sequences of light chains shown in SEQ ID NO.11-15 or 23, and one of the amino acid sequences of heavy chains shown in SEQ ID NO.16-20.
  • the present invention features de-immunized human anti-TNF- ⁇ antibodies with one of the DNA sequences of light chains L1-L5 shown in SEQ ID NO.1-5, and one of the DNA sequences of heavy chains h1-h5 shown in SEQ ID NO.6-10.
  • the present invention features de-immunized human anti-TNF- ⁇ antibody with the the amino acid sequence of light chains shown in SEQ ID NO.13, and the amino acid sequences of heavy chain shown in SEQ ID19.
  • the present invention features de-immunized human anti-TNF- ⁇ antibody with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID.9
  • the present invention provides the sequences for 10 de-immunized human anti-TNF- ⁇ antibodies, named as L3h2, L3h4, L5h2, L4h1, L4h2, L4h4, L1h3, L2h1, L0h4 and L2h5, which have similar affinities as the original and can block the binding of TNF- ⁇ to its receptors TNFRs p55 and p75.
  • the de-immunized human anti-TNF- ⁇ antibody L3h2 with the amino acid sequence of light chains shown in SEQ ID NO.13 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • the de-immunized human anti-TNF- ⁇ antibody L3h4 with the amino acid sequence of light chains shown in SEQ ID NO.13 and the amino acid sequences of heavy chain shown in SEQ ID No.19 and with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • the de-immunized human anti-TNF- ⁇ antibody L5h2 with the amino acid sequence of light chains shown in SEQ ID NO.15 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.5, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • the de-immunized human anti-TNF- ⁇ antibody L4h1 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.16, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.6.
  • the de-immunized human anti-TNF- ⁇ antibody L4h2 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • the de-immunized human anti-TNF- ⁇ antibody L4h4 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.19, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • the de-immunized human anti-TNF- ⁇ antibody L1h3 with the amino acid sequence of light chains shown in SEQ ID NO.11 and the amino acid sequences of heavy chain shown in SEQ ID No.18, and with the DNA sequence of light chain shown in SEQ ID NO.1, and the DNA sequence of heavy chain shown in SEQ ID No.8.
  • the de-immunized human anti-TNF- ⁇ antibody L2h1 with the amino acid sequence of light chains shown in SEQ ID NO.12 and the amino acid sequences of heavy chain shown in SEQ ID No.16, and with the DNA sequence of light chain shown in SEQ ID NO.2, and the DNA sequence of heavy chain shown in SEQ ID No.6.
  • the de-immunized human anti-TNF- ⁇ antibody L2h5 with the amino acid sequence of light chains shown in SEQ ID NO.12 and the amino acid sequences of heavy chain shown in SEQ ID No.20, and with the DNA sequence of light chain shown in SEQ ID NO.2, and the DNA sequence of heavy chain shown in SEQ ID No.10.
  • the de-immunized human anti-TNF- ⁇ antibody L0h4 with the amino acid sequence of light chains shown in SEQ ID NO.23 and the amino acid sequences of heavy chain shown in SEQ ID No.19, and with the DNA sequence of light chain shown in SEQ ID NO.21, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • the present invention features the expression plasmid containing the de-immunized anti-TNF- ⁇ antibody sequences.
  • the present invention also covers the plasmid, the host cells containing the de-immunized anti-TNF- ⁇ antibody sequences.
  • the invention also provides de-immunized anti-TNF- ⁇ antibodies for treatment of human diseases targeting TNF- ⁇ .
  • TNF- ⁇ -binding molecules or antibodies of the present invention can be used to inhibit the death of cells.
  • TNF-A-binding molecules or antibodies of the present invention can be used to treat human diseases including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease.
  • These methods comprise administrating an effective amount of a TNF- ⁇ -binding molecule or antibody of the present invention to a subject in need thereof.
  • the present invention also features pharmaceutical and diagnostic compositions comprising a TNF- ⁇ -binding molecule or antibody of the present invention.
  • the present invention provides the method of de-immunogenicity of anti-TNF- ⁇ monoclonal antibody, including:
  • FIG. 1 Digestions of Plasmids.
  • FIG. 1 a shows double digestions of pJH16 plasmid with Kpn I and Age I.
  • FIG. 1 b shows double digestions of pJH16 with Kpn I and BamH I.
  • FIG. 1 c shows double digestions of the heavy chain of Adailimumab in lane 1, and shows double digestions of the light chain of Adalimumab.
  • M the DNA markers in lane 2.
  • FIG. 2 Sequence blasts the light chains of the modified vs the one of Adalimumab.
  • FIG. 3 Sequence blasts the heavy chains of the modified vs the one of Adalimumab.
  • FIG. 4 The EC50s of modified human anti-TNF- ⁇ monoclonal antibodies.
  • FIGS. 5 a and 5 b show inhibitions of TNF- ⁇ -induced cell toxicity in L929 cells by modified human anti-TNF- ⁇ monoclonal antibodies.
  • FIG. 6 PK study of modified human anti-TNF- ⁇ monoclonal antibodies.
  • Fully human anti-TNF- ⁇ monoclonal antibodies can be any combination of one light chain from any one of L0-L5 (SEQ ID No.1-5, 21) and one heavy chain from any one of h1-h5 (SEQ ID No.6-10).
  • the plasmids and the expression vectors were subjected to enzyme digestions at 37 C overnight. Results of digestions of light chain, heavy chain, and the expression vectors are shown in FIG. 1 .
  • the bands of target genes and expression vectors were cut-out and extracted using Qiagen Gel Extraction Kit, then performed the ligations overnight using T4 DNA ligation system and transformed into E. coli DH5 ⁇ . Colonies were picked for DNA sequencing and the alignments of sequencing data matched the designed gene 100%.
  • the expression levels of human IgGs in the culture supernants were examined on Day 3 and the expression levels ranged between 423.5-2624 ng/ml.
  • L0h1 refers to the combination of light chain L0 from the adalimumab light chain variable region and the heavy chain h1 from a modified anti-TNF- ⁇ antibody.
  • CHO cells was electro-transfected and selected under MTX pressure (purchased from Sigma) in the selective Opti-CHO medium (purchased from Invitrogen). Five selecting gradients were set as 50 nM, 100 nM, 200 nM, 400 nM and 800 nM. After each round, the expression levels of IgG in the culture supernatants on Day 7 were examined using Sandwich ELISA method. The results showed that stable expressions of IgGs were observed with all of the combinations but the levels were different (Table 3).
  • Affinities The EC50s of the newly invented human anti-TNF- ⁇ antibodies were compared with the one of Adalimumab using Indirect ELISA.
  • the wells of 96-well plates were coated with 300 ng/ml of TNF- ⁇ in PBS overnight at 4 C. After wash, the wells were blocked with 5% skim milk in PBS for 1 hour at room temperature.
  • Various concentrations of antibodies diluted in 5% skim milk-PBS were added to the wells and incubated for 1 hour at room temperature. After another wash, HRP-conjugated goat-anti-human IgG secondary antibodies were added and incubated for another 1 hour. After through wash, the substrates were added and the absorbances at 450 nm were measured.
  • some of the newly invented human anti-TNF- ⁇ antibodies have very similar EC50 as Adalimumab.
  • the specificities of the newly invented human anti-TNF- ⁇ antibodies were examined by Indirect ELISA against TNF- ⁇ and other cytokines.
  • the wells of 96-well plates were coated with 1000 ng/ml of rhTNF ⁇ , rhTNF ⁇ , rIFN ⁇ , IL-1 ⁇ , IL-1 ⁇ , IL-2, IL-4 and IL-8 in PBS overnight at 4 C. After wash, the wells were blocked with 5% skim milk in PBS for 1 hour at room temperature.
  • Different human anti-TNF- ⁇ antibodies diluted in 5% skim milk-PBS were added to the wells and incubated for 1 hour at room temperature.
  • L929 cells were seeded at 50,000 cells/well of 96-well plate in RPMI-1640-10% FBS and incubated at 37° C. 5% CO2. 4 hours later, discard the medium and added 100 ⁇ l/well of different concentrations of ADALIMUMAB or the invented human anti-TNF- ⁇ antibodies in RPMI-1640-10% FBS plus Actinomysin D 1 ug/ml at 37° C. 5% CO2. One day's later, the cell numbers in each well were determined by CKK assay.
  • both ADALIMUMAB and the newly invented human anti-TNF- ⁇ antibodies could inhibit TNF- ⁇ induced apoptosis of L929 cells.
  • mice were tail vent-injected with 125 I-labeled all 10 new human anti-TNF- ⁇ antibodies and Adalimumab (370 kBq, 2 ⁇ g), 5 mice per group. At various time points (5, 12, 30 min, 1, 2, 4, 8, 11, 22, 34, 48, 72 h), the blood samples were collected and the radioactivities were measured. As shown in FIG. 6 , the PK of newly invented human anti-TNF- ⁇ antibodies were similar or better than the one of Adalimumab.
  • the invention features human anti-TNF- ⁇ antibodies which share the CDRs of the amino acid sequences from Adalimumab but with different FRs from other human IgGs.
  • the newly invented human anti-TNF- ⁇ antibodies have the same specificities, similar affinities and inhibitory activities against TNF- ⁇ but much lower immunogenicities than Adalimumab.
  • the invention also features method of de-immunogenicity of human antibodies by replacing the high immunogenic FR sequences with lower ones from other human IgGs without alter the activities of the antibody significantly. Reduced immunogenicity will significantly reduce the level of anti-drug antibody in the patients treated with anti-TNF- ⁇ drug, extend drug's half-life and increase the efficacy of the biological drugs.

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Abstract

A method for treating autoimmune and inflammatory diseases in a human includes administering an effective amount of a low immunogenic human anti-TNF-α antibody to the human, thereby targeting TNF-α. The low immunogenic human anti-TNF-α antibody includes CDR regions of heavy and light chains from human Adalimumab, and also includes an additional human light chain amino acid sequence, and an additional human heavy chain amino acid sequence. The antibody has reduced immunogenicity compared to Adalimumab.

Description

    INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
  • Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57.
    • SEQUENCE LISTING IN ELECTRONIC FORMAT
  • The present application is being filed along with an Electronic Sequence Listing as an ASCII text file via EFS-Web. The Electronic Sequence Listing is provided as a file entitled 2020-12-29_Sequence listing—CNKH016.001D1.txt created and last saved on Dec. 29, 2020, which is approximately 32.3 KB in size. The information in the Electronic Sequence Listing is incorporated herein by reference in its entirety in accordance with 35 U.S.C. § 1.52(e).
    • BACKGROUND OF THE INVENTION Field of the Invention
  • The present invention relates to de-immunogenicity of anti-Tumor Necrosis Factor-alpha (TNF-α) antibodies and applications of using the same for treating inflammatory diseases and other human diseases.
    • Description of the Related Art
  • TNF is an immunity-modulating cytokine required for immune processes. The unregulated activities of TNFs can lead to the development of inflammatory diseases. Excess amounts of TNF-expressed in cells are associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease. The function of TNF requires binding to its two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Blocking the interaction between TNF and TNFRs has successfully been developed as a therapy in treating inflammatory or autoimmune diseases. TNF neutralization therapies, including the use of a soluble TNFR2-Fc recombinant (Etanercept), a mouse-human chimera mAb (Infliximab), or a human mAb (Adalimumab), have been introduced in the past decades for the management of rheumatoid arthritis and other immune diseases.
  • However, although it is fully human antibody, high immunogenicity has been observed in human patients treated with Adalimumab. Anti-drug antibody (ADA) to Adalimumab was detected in up to 75% of the patients. It was also reported that the annual loss of response to Adalimumab was calculated to be 24%. ADA was considered as the causes of treatment failures, and it is believed that ADAs might reduce drug efficacy by competing with the endogenous ligand (neutralizing antibodies, Nab) and/or by forming immune complex, which accelerate the clearance of the drug from the circulation. Therefore there is need to develop a better anti-TNF antibody with lower immunogenicity and longer efficacy.
  • This invention is about the de-immunogenicity of human anti-Tumor Necrosis Factor-alpha (TNF-α) antibody Adalimumab, designed as clones TCX002-L3H4, L1H4. etc, which bind to the same epitope from the one recognized by Adalimumab, but with much lower immunogenicities in vivo.
    • SUMMARY OF THE INVENTION
  • The present invention provides the human anti-Tumor Necrosis Factor-alpha (TNF-α) antibodies with reduced immunogenicities and methods of using the same for neutralizing the TNF-α induced cell death and for treating inflammatory diseases and other human diseases. In one aspect, the present invention features TNF-α-binding molecules and their DNA and amino acid sequences. Each molecule comprises the CDRs from human anti-TNF-α monoclonal antibody Adalimumab and the FRs from different human origins.
  • The present invention also provides a method to develop human antibodies with reduced immunogenicities by replacing the FRs of the original human monoclonal antibody with the FRs from different human origins.
  • In addition, the present invention also provides one example of using the method to develop human anti-TNF-α antibodies with reduced immunogenicities by replacing the FRs of human anti-TNF-α monoclonal antibody Adalimumab with the FRs from different human origins.
  • The present invention features de-immunized human anti-TNF-α antibodies with one of the amino acid sequences of light chains shown in SEQ ID NO.11-15 or 23, and one of the amino acid sequences of heavy chains shown in SEQ ID NO.16-20.
  • Furthermore, the present invention features de-immunized human anti-TNF-α antibodies with one of the DNA sequences of light chains L1-L5 shown in SEQ ID NO.1-5, and one of the DNA sequences of heavy chains h1-h5 shown in SEQ ID NO.6-10.
  • Furthermore, the present invention features de-immunized human anti-TNF-α antibody with the the amino acid sequence of light chains shown in SEQ ID NO.13, and the amino acid sequences of heavy chain shown in SEQ ID19.
  • Furthermore, the present invention features de-immunized human anti-TNF-α antibody with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID.9
  • The present invention provides the sequences for 10 de-immunized human anti-TNF-α antibodies, named as L3h2, L3h4, L5h2, L4h1, L4h2, L4h4, L1h3, L2h1, L0h4 and L2h5, which have similar affinities as the original and can block the binding of TNF-α to its receptors TNFRs p55 and p75.
  • Whereas, the de-immunized human anti-TNF-α antibody L3h2 with the amino acid sequence of light chains shown in SEQ ID NO.13 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • Whereas, the de-immunized human anti-TNF-α antibody L3h4 with the amino acid sequence of light chains shown in SEQ ID NO.13 and the amino acid sequences of heavy chain shown in SEQ ID No.19 and with the DNA sequence of light chain shown in SEQ ID NO.3, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • Whereas, the de-immunized human anti-TNF-α antibody L5h2 with the amino acid sequence of light chains shown in SEQ ID NO.15 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.5, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • Whereas, the de-immunized human anti-TNF-α antibody L4h1 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.16, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.6.
  • Whereas, the de-immunized human anti-TNF-α antibody L4h2 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.17, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.7.
  • Whereas, the de-immunized human anti-TNF-α antibody L4h4 with the amino acid sequence of light chains shown in SEQ ID NO.14 and the amino acid sequences of heavy chain shown in SEQ ID No.19, and with the DNA sequence of light chain shown in SEQ ID NO.4, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • Whereas, the de-immunized human anti-TNF-α antibody L1h3 with the amino acid sequence of light chains shown in SEQ ID NO.11 and the amino acid sequences of heavy chain shown in SEQ ID No.18, and with the DNA sequence of light chain shown in SEQ ID NO.1, and the DNA sequence of heavy chain shown in SEQ ID No.8.
  • Whereas, the de-immunized human anti-TNF-α antibody L2h1 with the amino acid sequence of light chains shown in SEQ ID NO.12 and the amino acid sequences of heavy chain shown in SEQ ID No.16, and with the DNA sequence of light chain shown in SEQ ID NO.2, and the DNA sequence of heavy chain shown in SEQ ID No.6.
  • Whereas, the de-immunized human anti-TNF-α antibody L2h5 with the amino acid sequence of light chains shown in SEQ ID NO.12 and the amino acid sequences of heavy chain shown in SEQ ID No.20, and with the DNA sequence of light chain shown in SEQ ID NO.2, and the DNA sequence of heavy chain shown in SEQ ID No.10.
  • Whereas, the de-immunized human anti-TNF-α antibody L0h4 with the amino acid sequence of light chains shown in SEQ ID NO.23 and the amino acid sequences of heavy chain shown in SEQ ID No.19, and with the DNA sequence of light chain shown in SEQ ID NO.21, and the DNA sequence of heavy chain shown in SEQ ID No.9.
  • The present invention features the expression plasmid containing the de-immunized anti-TNF-α antibody sequences.
  • The present invention also covers the plasmid, the host cells containing the de-immunized anti-TNF-α antibody sequences.
  • The invention also provides de-immunized anti-TNF-α antibodies for treatment of human diseases targeting TNF-α.
  • The TNF-α-binding molecules or antibodies of the present invention can be used to inhibit the death of cells.
  • In addition, the TNF-A-binding molecules or antibodies of the present invention can be used to treat human diseases including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease. These methods comprise administrating an effective amount of a TNF-α-binding molecule or antibody of the present invention to a subject in need thereof.
  • Furthermore, the present invention also features pharmaceutical and diagnostic compositions comprising a TNF-α-binding molecule or antibody of the present invention.
  • The present invention provides the method of de-immunogenicity of anti-TNF-α monoclonal antibody, including:
      • 1. Analysis the FR sequences of anti-TNF-α monoclonal antibody Adalimumab and identify the sequences with high immunogenicities.
      • 2. Align the FR sequences of anti-TNF-α monoclonal antibody Adalimumab against the ones of human IgGs in NCBI database, and find the ones with high homologies but lower immunogenicities.
      • 3. Replace the high immunogenic FR sequence(s) of anti-TNF-α monoclonal antibody Adalimumab with low immunogenic FR sequences from other human antibodies.
      • 4. Perform 3D structure modeling of the newly designed antibody sequences against the anti-TNF-α monoclonal antibody Adalimumab using Pymol program to identify the ones with closest resembling of the original antibody.
      • 5. Once the variable region sequences confirmed, chemically synthesize both the rariable sequences with artificially added restriction enzyme sites (Kpn I and BamH I for light chain variable region, Kpnl and Agel for heavy chain variable region), ligate to vector pJH16 to obtain the expression plasmids for heavy chain and light chain of human antibody (Results see FIG. 1). Screen for positive clones after transformation by sequenceing and restriction enzyme digestions.
      • 6. Extract the plasmids using the kit from Qiagen following the instruction from the manufacturer.
      • 7. Transient co-transfect the different combinations of the human light and heavy chain expression plasmids produced different human anti-TNF-α monoclonal antibodies with different expression levels and affinities for TNF-α(see FIG. 4).
      • 8. Based on above data, a few combinations were selected to develop stable cell lines for over-expression of human anti-TNF-α.
      • 9. The human anti-TNF-α monoclonal antibodies featured in this invention bind to the same antigenic eptiope as Adalimumab but with reduce immunogenicities and different 3D structures, longer half-lives, could be a better biotherapeutics.
      • 10. The present invention features method to modify the immunogenicity of Adalimumab in human patients by replacing some of the amino acid sequences in Adalimumab with other human sequences.
      • 11. The present invention provides examples to show the modified human anti-TNF-α monoclonal antibodies have similar affinities as Adalimumab but extend the half-lives with prolonger efficacies.
    BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 Digestions of Plasmids. FIG. 1a shows double digestions of pJH16 plasmid with Kpn I and Age I. FIG. 1b shows double digestions of pJH16 with Kpn I and BamH I. FIG. 1c shows double digestions of the heavy chain of Adailimumab in lane 1, and shows double digestions of the light chain of Adalimumab. M, the DNA markers in lane 2.
  • FIG. 2 Sequence blasts the light chains of the modified vs the one of Adalimumab.
  • FIG. 3 Sequence blasts the heavy chains of the modified vs the one of Adalimumab.
  • FIG. 4 The EC50s of modified human anti-TNF-α monoclonal antibodies.
  • FIGS. 5a and 5b show inhibitions of TNF-α-induced cell toxicity in L929 cells by modified human anti-TNF-α monoclonal antibodies.
  • FIG. 6 PK study of modified human anti-TNF-α monoclonal antibodies.
    •  DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • It should be understood that the above-described embodiments and the following examples are given by way of illustration, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the present description.
  • Unless specified, all the techniques used are common practices and can be performed by skilled personel. All of the materials and reagents can be purchased commercially.
    • Example 1 Analysis and Modification of the Immunogenicity of the Sequences Adalimumab
  • Used a program to examine the sequences of Adalimumab and found that the immunogenicity score is 16.
  • Used the same software to study the immunogenicities of the FRs of Adalimumab, identified the sequences with high immunogenicities, and searched human antibody sequence database for potential human sequences with lower immunogenicity.
  • Replaced the high immunogenic sequences in Adalimumab with the low immunogenic ones, and designed 5 human light chains L1-L5 (SEQ ID No.1-5) and 5 human heavy chains h1-h5 (SEQ ID No.6-10) for fully human anti-TNF-α monoclonal antibodies.
  • Perform 3D structure modeling of the newly designed antibody sequences against the ones of Adalimumab using Pymol program to identify the ones with closest resembling of the original antibody.
  • Fully human anti-TNF-α monoclonal antibodies can be any combination of one light chain from any one of L0-L5 (SEQ ID No.1-5, 21) and one heavy chain from any one of h1-h5 (SEQ ID No.6-10).
    • Example 2 Construction of the Expression Plasmids of Fully Human Anti-TNF-α Monoclonal Antibodies
  • Added the restriction sites of Kpn I and BamH I to the light chain variable region sequences and the restriction sites of Kpn I and Age I to the heavy chain variable region sequences obtained in Example 1. All the variable region of the light and heavy chain sequences were inserted into the plasmids. Cut the heavy chain variable region sequences from the plasmids and inserted into the corresponding sites of the expression vector pJH16 using the restriction sites of Kpn I and Age I. Cut the light chain variable region sequences from the vector and inserted into the corresponding sites of the expression vector pJH16 using the restriction sites of Kpn I and BamH I, to obtain the fully human monoclonal antibody heavy and light chain expression plasmids. The plasmids and the expression vectors were subjected to enzyme digestions at 37 C overnight. Results of digestions of light chain, heavy chain, and the expression vectors are shown in FIG. 1. The bands of target genes and expression vectors were cut-out and extracted using Qiagen Gel Extraction Kit, then performed the ligations overnight using T4 DNA ligation system and transformed into E. coli DH5α. Colonies were picked for DNA sequencing and the alignments of sequencing data matched the designed gene 100%.
    • Example 3 Transient Expression and Purification of Fully Human Anti-TNF-α Monoclonal Antibodies
  • Extracted the plasmids from the transformed E. coli DH5α, as shown in Example 2, using the Ultrapure Plasmid Prep kit from Qiagen.
  • Co-transfected the 293F cells with different combinations of the human light and heavy chain expression plasmids using lipofecting reagents from Invitogen. Total 31 combinations tried.
  • The expression levels of human IgGs in the culture supernants were examined on Day 3 and the expression levels ranged between 423.5-2624 ng/ml.
  • TABLE 1
    Expression Levels of Human Antibodies (ng/ml)
    Comb. Conc. Comb. Conc. Comb. Conc. Comb. Conc. Comb. Conc. Comb. Conc.
    L0h1 1530 L1h1 1371 L2h1 1988 L3h1 2624 L4h1 810.7 L5h1 439.1
    L0h2 11172 L1h2 487.6 L2h2 755.8 L3h2 1208 L4h2 1130 L5h2 423.5
    L0h3 2021 L1h3 873.3 L2h3 662.2 L3h3 602.9 L4h3 2206 L5h3 797
    L0h4 1109 L1h4 1257 L2h4 476 L3h4 1638 L4h4 1381 L5h4 475.9
    L0h5 1408 L1h5 868 L2h5 677.7 L3h5 1282 L4h5 1423 L5h5 952.2
    Adh0l0 1892
    Note:
    The table is a combination of different combinations of light and heavy chains. For example, L0h1 refers to the combination of light chain L0 from the adalimumab light chain variable region and the heavy chain h1 from a modified anti-TNF-α antibody.) Performed indirect ELISA against TNF-α coated on 96-well plate, and found some of them (L0h4, L3h4, L3h2, L4h4, etc) have strong signals as Adalimumab, and some of them lost the binding affinity (L0h2) (Data see Table 2)
  • TABLE 2
    ELISA Screening of Different Combinations against TNF-α
    Comb. L0h1 L0h2 L0h3 L0h4 L0h5 NC
    OD 2.158 2.182 0.057 0.054 0.68 0.768 3.339 3.133 1.03 0.873 0.09 0.059
    Comb. L1h1 L1h2 L1h3 L1h4 L1h5 NC
    OD 1.926 2.401 2.268 2.459 1.413 1.431 2.621 2.552 0.824 1.051 0.045 0.057
    Comb. L2h1 L2h2 L2h3 L2h4 L2h5 NC
    OD 1.891 2.384 2.802 2.704 0.709 0.973 2.235 2.848 0.894 1.255 0.047 0.051
    Comb. L3h1 L3h2 L3h3 L3h4 L3h5 L0h0
    Comb. 2.178 2.329 2.434 2.498 0.888 0.815 2.616 2.664 0.959 1.104 3.008 3.244
    OD L4h1 L4h2 L4h3 L4h4 L4h5 L0h0
    1.978 2.182 2.968 2.546 1.617 1.607 2.904 2.757 0.714 0.972 2.877 3.041
    Comb. L5h1 L5h2 L5h3 L5h4 L5h5 L0h0
    OD 0.864 1.583 1.857 1.821 1.366 1.404 1.528 1.643 0.885 0.903 2.926 3.172
    • Example 4 Stable Expression and Purification of Fully Human Anti-TNF-α Monoclonal Antibodies
  • Based on above data, 10 combinations were selected to develop stable cell lines for over-expression of human anti-TNF-α.
  • CHO cells was electro-transfected and selected under MTX pressure (purchased from Sigma) in the selective Opti-CHO medium (purchased from Invitrogen). Five selecting gradients were set as 50 nM, 100 nM, 200 nM, 400 nM and 800 nM. After each round, the expression levels of IgG in the culture supernatants on Day 7 were examined using Sandwich ELISA method. The results showed that stable expressions of IgGs were observed with all of the combinations but the levels were different (Table 3).
  • TABLE 3
    IgG Levels of different combinations at different stages
    opti-cho IgG 50 nM IgG 100 nM IgG
    Comb. (ng/ml) (ng/ml) (ng/ml)
    adh010 30 134 346
    L3h2 92.7 122 237
    L3h4 87.4 251 367
    L5h2 30.8 129 452
    L4h1 104 176 258
    L4h2 127 318 523
    L4h4 72.5 939 734
    L1h3 97 160 270
    L2h1 64 208.6 471
    L0h4 30 389 598
    L2h5 29.2 226 476
  • When the process was complete, limiting dilution was performed for monoclonal cloning. Cells were seeded at 96-well plate and cultured at 37° C. 5% CO2. 14 days later, 50 μl of supernatant was collected for antibody production testing using sandwich ELISA method. Clones with higher expressing levels were selected for further expansion.
  • Used a Protein-A affinity chromatography column to purify the human anti-TNF-α antibodies from the culture supernatants of the 11 stable cell lines. The concentrations of antibodies were determined by OD280/1.4. The purities of the antibodies were examined by SDS-PAGE analysis.
    • Example 5 Biological Activities of Human Anti-TNF-α Antibodies
  • 1. Affinities: The EC50s of the newly invented human anti-TNF-α antibodies were compared with the one of Adalimumab using Indirect ELISA. The wells of 96-well plates were coated with 300 ng/ml of TNF-α in PBS overnight at 4 C. After wash, the wells were blocked with 5% skim milk in PBS for 1 hour at room temperature. Various concentrations of antibodies diluted in 5% skim milk-PBS were added to the wells and incubated for 1 hour at room temperature. After another wash, HRP-conjugated goat-anti-human IgG secondary antibodies were added and incubated for another 1 hour. After through wash, the substrates were added and the absorbances at 450 nm were measured. As shown in Table 4, some of the newly invented human anti-TNF-α antibodies have very similar EC50 as Adalimumab.
  • TABLE 4
    EC50s of human anti-TNF-a antibodies
    Comb. 10h4 12h1 13h2 13h4 14h1 14h2 14h4 adh0l0
    EC50(nM) 0.37 0.43 0.40 0.34 0.47 0.60 0.69 0.49
  • 2. Specificities: The specificities of the newly invented human anti-TNF-α antibodies were examined by Indirect ELISA against TNF-α and other cytokines. The wells of 96-well plates were coated with 1000 ng/ml of rhTNFα, rhTNFβ, rIFN γ, IL-1α, IL-1β, IL-2, IL-4 and IL-8 in PBS overnight at 4 C. After wash, the wells were blocked with 5% skim milk in PBS for 1 hour at room temperature. Different human anti-TNF-α antibodies diluted in 5% skim milk-PBS were added to the wells and incubated for 1 hour at room temperature. After another wash, HRP-conjugated goat-anti-human IgG secondary antibodies were added and incubated for another 1 hour. After through wash, the substrates were added and the absorbances at 450 nm were measured. As shown in Table 5, all of the newly invented human anti-TNF-α antibodies are very specific for TNF-α.
  • TABLE 5
    Specificities of human anti-TNF-α antibodies
    Ab
    Cytokine Adalimumab L0h4 L2h1 L3h2 L3h4
    rTNFα 2.877 3.041 3.339 3.238 2.251 2.325 2.434 2.498 2.804 2.789
    rTNFβ 0.097 0.089 0.058 0.064 0.081 0.082 0.071 0.065 0.064 0.071
    rTNFγ 0.082 0.078 0.062 0.068 0.065 0.071 0.057 0.068 0.068 0.065
    IL-1α 0.059 0.065 0.080 0.072 0.057 0.062 0.064 0.072 0.072 0.077
    IL-1β 0.067 0.058 0.059 0.068 0.063 0.071 0.059 0.073 0.068 0.063
    IL-2 0.053 0.059 0.074 0.069 0.073 0.075 0.067 0.061 0.069 0.073
    IL-4 0.049 0.05 0.055 0.049 0.072 0.069 0.078 0.069 0.059 0.062
    IL-8 0.063 0.057 0.067 0.060 0.058 0.061 0.069 0.074 0.060 0.058
    Ab
    Cytokine L4h1 L4h2 L4h4 NC NC
    rTNFα 2.018 2.121 2.754 2.802 2.826 2.855 0.054 0.051 0.044 0.051
    rTNFβ 0.065 0.064 0.082 0.071 0.064 0.071 0.068 0.065 0.058 0.055
    rTNFγ 0.068 0.068 0.071 0.067 0.068 0.065 0.052 0.057 0.054 0.061
    IL-1α 0.072 0.072 0.062 0.064 0.072 0.077 0.058 0.063 0.052 0.053
    IL-1β 0.073 0.068 0.071 0.069 0.068 0.063 0.069 0.063 0.059 0.061
    IL-2 0.061 0.069 0.075 0.067 0.069 0.073 0.049 0.052 0.059 0.062
    IL-4 0.069 0.059 0.069 0.078 0.069 0.072 0.060 0.058 0.062 0.058
    IL-8 0.074 0.060 0.061 0.069 0.060 0.058 0.064 0.051 0.054 0.057
    • 3. Inhibition of TNF-α Induced Apotosis.
  • L929 cells were seeded at 50,000 cells/well of 96-well plate in RPMI-1640-10% FBS and incubated at 37° C. 5% CO2. 4 hours later, discard the medium and added 100 μl/well of different concentrations of ADALIMUMAB or the invented human anti-TNF-α antibodies in RPMI-1640-10% FBS plus Actinomysin D 1 ug/ml at 37° C. 5% CO2. One day's later, the cell numbers in each well were determined by CKK assay.
  • As shown in FIG. 5, both ADALIMUMAB and the newly invented human anti-TNF-α antibodies could inhibit TNF-α induced apoptosis of L929 cells.
    • Example 6 Immunogenicity and PK in Mice
  • 1. Immunogenicity: Mice were injected with all 10 new human anti-TNF-α antibodies and Adalimumab with the adjuvent. 14 days' later, the tail bleeds were examined by ELISA against their antigens respectively. As shown in Table 6, the anti-drug antibody titers of some newly invented human anti-TNF-α antibodies were at least 5-time lower than the one of Adalimumab.
  • TABLE 6
    ADA Titers of human anti-TNF-α antibodies in mice
    Titers 1:500 1:1000 1:5000 1:10000 1:50000 NC
    Comb. L3h2 0.974 0.459 0.056 0.064 0.051 0.042
    L3h4 0.676 0.385 0.044 0.043 0.046 0.046
    L5h2 0.854 0.435 0.042 0.047 0.047 0.047
    L4h1 0.699 0.311 0.054 0.058 0.047 0.045
    L4h2 1.207 0.607 0.062 0.049 0.042 0.042
    L4h4 0.713 0.379 0.059 0.048 0.048 0.047
    L0h4 1.016 0.591 0.048 0.067 0.054 0.056
    L1h3 1.156 0.548 0.043 0.080 0.053 0.055
    L2h1 0.781 0.389 0.041 0.056 0.059 0.057
    L2h5 0.802 0.410 0.032 0.066 0.053 0.047
    L0h0 2.614 1.311 0.2614 0.144 0.131 0.144
  • 2. Pharmakintics: Mice were tail vent-injected with 125 I-labeled all 10 new human anti-TNF-α antibodies and Adalimumab (370 kBq, 2 μg), 5 mice per group. At various time points (5, 12, 30 min, 1, 2, 4, 8, 11, 22, 34, 48, 72 h), the blood samples were collected and the radioactivities were measured. As shown in FIG. 6, the PK of newly invented human anti-TNF-α antibodies were similar or better than the one of Adalimumab.
    • INDUSTRIAL APPLICATIONS
  • The invention features human anti-TNF-α antibodies which share the CDRs of the amino acid sequences from Adalimumab but with different FRs from other human IgGs. The newly invented human anti-TNF-α antibodies have the same specificities, similar affinities and inhibitory activities against TNF-α but much lower immunogenicities than Adalimumab. The invention also features method of de-immunogenicity of human antibodies by replacing the high immunogenic FR sequences with lower ones from other human IgGs without alter the activities of the antibody significantly. Reduced immunogenicity will significantly reduce the level of anti-drug antibody in the patients treated with anti-TNF-α drug, extend drug's half-life and increase the efficacy of the biological drugs.

Claims (11)

What is claimed is:
1. A method for treating a disease selected from the group consisting of autoimmune and inflammatory diseases in a human, the method comprising administering an effective amount of a low immunogenic human anti-TNF-α antibody to the human, thereby targeting TNF-α,
wherein the low immunogenic human anti-TNF-α antibody comprises CDR regions of heavy (SEQ ID NO.23) and light (SEQ ID NO.24) chains from human Adalimumab, and further comprises (a) a human light chain amino acid sequence selected from SEQ ID NOs. 11-15 and 23, and (b) a human heavy chain amino acid sequence selected from SEQ ID NOs. 16-20; and
wherein the antibody has reduced immunogenicity compared to Adalimumab.
2. The method of claim 1, wherein the human light chain amino acid sequence is SEQ ID NO. 13, and wherein the human heavy chain amino acid sequence is SEQ ID NO. 19.
3. The method of claim 1, further comprising:
administering to the human an additional agent selected from the group consisting of a chemotherapeutic agent, an anti-cell proliferation agent, an immunotherapeutic agent and a combination thereof.
4. The method of claim 3, wherein the at least one therapeutic agent and the additional agent are co-administered to the human.
5. The method of claim 3, wherein the at least one therapeutic agent and the additional agent are co-formulated and are co-administered to the human.
6. The method of claim 1, wherein the administration route is selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, and a combination thereof.
7. The method of claim 1, wherein the disease is selected from the group consisting of rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease.
8. The method of claim 1, wherein the disease is rheumatoid arthritis.
9. The method of claim 1, wherein the disease is inflammatory bowel disease.
10. The method of claim 1, wherein the disease is psoriatic arthritis.
11. The method of claim 1, wherein the low immunogenic human anti-TNF-α antibody comprises at least one of the amino acid sequences of L3h4 (SEQ ID NOs. 13 and 19).
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