This invention is directed to deimmunized antibodies that are useful as immunotherapeutic drugs against Human Immunodeficiency Virus (HIV) and CD4-mediated autoimmune disorders. More specifically, antibodies expressed by clones, Clone 7 containing the recombinant genes B4DIVHv1/VK1CHO#7, Clone 16 containing the recombinant genes B4DIVHv1/VK1#16, and clone 21 containing the recombinant genes B4DIVHv1/VK1#21, are derived from mouse monoclonal B4 antibody (mAb B4). The antibodies were produced by removing particular murine determinants recognized as foreign by the human immune system. These recombinant antibodies were generated by the chimerization and deimmunization of the Fv region of mouse monoclonal antibody (mAb) B4. For improved safety, the coding sequence may further be mutated to express an aglycosylated IgG.sub.1 antibody that is unable to bind complement. The deimmunized antibodies retain the specificity of the murine mAb B4 for a receptor complex involving CD4 on the surface of the host T cells, and retain the characteristic ability of mAb B4 to neutralize primary isolates of HIV.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is directed to deimmunized antibodies derived from mouse monoclonal antibody B4 that are useful for immunotherapy against AIDS and other CD4-mediated disorders. The present invention is also directed to methods of treatment using the deimmunized antibodies. More particularly, antibodies expressed by clones #7, #16 and #21 containing the recombinant genes B4DIVHv1k /VK1#7, B4DIVHv1/VK1#16, and B4DIVHv1/VK1#21 are derived from mouse monoclonal B4 antibody (U.S. Pat. No. 5,912,176, issued Jun. 15, 1999). Mouse monoclonal B4 antibody is characterized by its specificity for the HIV receptor complex on CD4.sup.+ T cells and by its ability to neutralize in vitro and in vivo primary isolates of HIV and related immunodeficiency viruses [17,18].

The deimmunized recombinant antibodies were generated by a process that included expression from the fusion of the cDNA or polynucleotide encoding the Fv fragment of mAb B4 to the cDNA or polynucleotide for the human IgG.sub.1 Fc fragment. The nucleotide sequence of the resulting chimeric genes with the murine Fv region was then deimmunized by directed mutagenesis to remove "foreign to human" B cell and T cell epitopes from the variable domains of the murine heavy (V.sub.H) and .kappa. (V.sub..kappa.) chains so that it is suitable for treatment in human subjects. Four alternative cDNA or polynucleotide sequences are provided for the deimmunized V.sub.H chain and three for the deimmunized V.sub..kappa. chain. A deimmunized antibody was also engineered into an N-aglycosylated IgG.sub.1 form by using site-directed mutagenesis to alter a glycosylation site so that it is unable to bind complement. Thus, the aglycosylated deimmunized antibody does not evoke complement-mediated lysis of bound CD4.sup.+ cells.

These novel deimmunized antibodies retain the specificity and neutralization activities of the original murine monoclonal antibody.

These antibodies, expressed by NSO mouse myeloma cells and Chinese Hamster Ovary (CHO) cell clones, were adapted to serum-free medium for large-scale production for use in humans. The deimmunized antibodies may also be produced on a large scale in transgenic plants to reduce the cost. The antibodies of the invention may be used for prophylaxis of HIV exposure, for immunotherapy of HIV infection, and for immunotherapy of CD4-mediated autoimmune disorders such as rheumatoid arthritis.

DETAILED DESCRIPTION OF THE INVENTION

The deimmunization of the Fv fragment of murine mAb B4 was achieved by the identification and elimination of potentially immunogenic murine T and B-cell epitopes. Removal of the T cell epitopes was achieved following the identification of such epitopes from the variable regions of the therapeutic antibodies. The amino acid sequences of the variable region were analyzed for the presence of MHC class II-binding motifs by a 3-dimensional "peptide threading" method [22,25]. Removal of at least one or all of the B cell epitopes from the variable region was achieved by the `veneering` of surface residues where this will not interfere with antibody recognition [22,24]. The constant regions of the murine antibody (C.sub.H and C.sub..kappa.) were entirely removed by replacement of the murine constant regions with human IgG.sub.1 constant regions through the chimerization of the DNA sequence for the mAb B4 variable region with that for human IgG.sub.1 constant regions.

Example 1 below (see Original Patent) describes in detail the recovery, cloning and sequencing of the DNA encoding the variable regions of the heavy and light chains of murine mAb B4. The DNA and amino acid sequences of the murine B4 V.sub.H are shown in FIG. 1 (see Original Patent). The DNA and amino acid sequences of the murine B4 V.sub..kappa. are shown in FIG. 2 (see Original Patent). The locations of the CDRs on both chains were determined by reference to other antibody sequences [32]. B4 V.sub.H and V.sub..kappa. can be respectively assigned to the Mouse Heavy Chains Subgroup V(B) and the Mouse Kappa Chains Subgroup III [32]. If desired, intermediate chimeric antibodies containing fully human constant regions and fully murine variable regions can be obtained as described in Example 2 herein. Confirmation that the correct variable regions were obtained was shown by the binding of the resulting chimeric antibody to recombinant soluble CD4 (rsCD4).

The cDNA or polynucleotide sequences encoding murine B4 V.sub.H and V.sub..kappa. were compared to the sequences of the directory of human germline V.sub.H [33] and V.sub..kappa. [34] sequences and also to human germline J region sequences [35]. The reference human framework selected for B4 V.sub.H was DP14 with human J.sub.H6. The reference human framework selected for B4 V.sub..kappa. was B1. The J region sequence was human J.sub..kappa.2.

Following identification of the reference human framework sequences, certain nucleotide sequences for non-identical amino acid residues within the B4 V.sub.H and V.sub..kappa. frameworks were changed to the corresponding nucleotide sequence in the human reference sequence. Residues which were considered to be critical for antibody structure and binding were excluded from this process and not altered. These include identifying the B4 V.sub.H and V.sub..kappa. complementarity determining regions (CDRs) listed as SEQ ID NOs:20, 22, 24, 26, 28 and 30 in FIG. 18 (see Original Patent). The CDR residues and those in the immediate framework neighborhood of the CDRs, such as those at the N-terminus for instance, were retained [24]. Other murine residues that were retained at this stage were largely non-surface, buried residues.

This process produced a sequence that is broadly similar to a `veneered` antibody as the surface residues are mainly human and the buried residues are those in the original murine sequence. These sequences were then subjected to peptide threading to identify potential T cell epitopes, through analysis of binding to 18 different human MHC class II allotypes. Primary deimmunized V.sub.H and V.sub..kappa. sequences were defined (B4DIVHv.1, B4DIVKv.1). As generation of the primary deimmunized sequences requires a small number of nucleotide sequence substitutions that might affect the binding of the final deimmunized molecule, three other variant V.sub.Hs and two other V.sub..kappa.s were designed. The comparative amino acid sequences of murine and deimmunized V regions are shown in FIG. 5 (see Original Patent) for for V.sub.H and FIG. 6 (see Original Patent) for V.sub..kappa.. The comparative nucleotide sequences of the murine and deimmunized V regions are shown in FIG. 7 (see Original Patent) for V.sub.H and FIG. 8 (see Original Patent) for V.sub..kappa..

The method of constructing the nucleotide sequence fragment for the deimmunized variable regions including 5' flanking sequence, the leader signal peptide, leader intron and the murine immunoglobulin promoter, and 3' flanking sequence, the splice site and intron sequences is detailed in Example 3 (see Original Patent).

Once the deimmunized heavy and light chain V-region genes were constructed, they were respectively transferred to the expression vectors containing human IgG.sub.1 C.sub.H or C.sub..kappa. regions. Markers for selection in mammalian cells (FIGS. 3 and 4 (see Original Patent)) were also intoduced into the vectors. The recombinant vectors were then transfected into NSO cells. The expressed deimmunized antibodies were then purified from the culture media and were tested for binding to rsCD4 and for neutralizing activity against HIV-1.

The twelve variants of mAb B4 showed different binding affinities to rsCD4 by ELISA assay. The twelve variant B4 deimmunized antibodies also showed different neutralizing activities against SI primary isolates from HIV-1 subgroups A, B, C, D and E and T cell line isolate HIV-1.sub.MN by the MT-2 microplaque assay [36]. The comparisons of the four heavy chain variants (VHv.1-4), all combined with deimmunized .kappa chain version 1 (VKv.1), showed that the antibody having the version 1 heavy chain had high neutralizing activity against all of the HIV-1 primary isolates, with activities approaching that of murine mAb B4 for HIV-1.sub.VL135, HIV-1.sub.UG029 and HIV-1.sub.TH036 (Table 1 (see Original Patent)).

The deimmunized B4 antibodies were ineffective against the T cell line-adapted HIV-1.sub.MN virus as was the murine parent antibody. Deimmunized antibodies comprising .kappa. chain versions 2 and 3 either were comparable or had lower neutralizing activities (Table 2 (see Original Patent)). The comparisons of the 12 variants showed that the antibody having the DIVHv.1 heavy chain and DIVKv.1 had high neutralizing activity against all of the HIV-1 primary isolates, with activities against HIV-1 approaching that of murine mAb B4.

For the production of the present deimmunized B4 monoclonal antibodies, any appropriate expression system, including eukaryotic cells, for example, animal cells, such as established mammalian cell lines, or plant cells, may be used. Preferably, the present deimmunized antibodies are expressed in mammalian cells such as NSO cells or CHO cells. NSO , a non-immunoglobulin producing mouse myeloma can be obtained from the European Collection of Animal Cell Culture, Porton UK (ECACC no. 85110505). CHO cells lacking the enzyme dihydrofolate reductase (CHOdfhr.sup.-) can be obtained from the American Type Culture Collection (ATCC no. CRL-9096).

For high-level expression of recombinant antibodies, dihydrofolate reductase (dhfr)-deficient CHO cells are one of the most widely used mamalian expression systems for gene amplification. Recombinant CHO cells are commonly used because the productivity of these cells has been found to increase with gene copy number and has been calculated to have specific antibody productivity as high as 100 .mu.g/10.sup.6 cells per day [42].

In such cases, a conventional promoter useful for expression in mammalian cells can be used. For example, a viral expression system including the human cytomegalovirus immediate early (CMV) promoter is preferred [37]. Alternatively, other promoters suitable for gene expression of the present invention in mammalian cells can be used. These promoters include viral promoters such as retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40) or mammalian cell-derived promoters such as human elongation factor 1.alpha. promoter [38,39]. Immunoglobulin promoter could be used in lymphatic cells such as NSO cells. The vectors containing the Ig promoter, leader signal peptide, leader intron, the splice site, intron sequences and suitable restriction sites for the insertion of any variable region cassette were designed and constructed as described in Example 2. These vectors were expressed in NSO cells for convenient screening for expression of deimmunized antibodies by ELISA and bioactivity.

High level recombinant protein expression in mammalian cells can be obtained by selection of stable cell lines followed by amplification of transfected DNA. A recombinant gene can be co-amplified with a selection marker gene during DNA amplification. CHOdhfr.sup.- cells and the amplificable selection marker gene for dihydrofolate reductase (DHFR) can be used to establish cell lines that produce clinically useful amounts of product [40]. Alternatively, the glutamine synthetase (GS) gene also can be used as a dominant selection marker. Due to insufficient endogenous GS activity of myeloma and hybridoma cell lines, these cell lines have an absolute requirement for glutamine. Transfected GS gene is able to confer the ability to grow in an appropriate, glutamine free medium [41]. Preferably, the CHOdfhr.sup.- cells are used to express B4 deimmunized antibody, and the dhfr gene is used as the selection and amplification marker.

The plasmids for expression of antibody and DHFR in CHOdhfr.sup.- cells were constructed as described in Example 5. The expression levels of DHFR and antibody genes are amplified for greater production of deimmunized B4 antibodies by stepwise increases in methotrexate (MTX) concentrations [42].

Recombinant CHO (rCHO) cells with specific antibody productivity of 10-30 pg/cell/day were obtained through several rounds of selection in medium containing stepwise increases in MTX level.

Although expression of the deimmunized antibodies is preferably in mammalian cells, such as NSO or CHO cells, to reduce the cost of production on a large scale, expression in transgenic plants may also be employed. See Example 10.

All antibodies are glycoproteins. IgG.sub.1 contains a biantennary complex N-linked carbohydrate within CH2 [43]. Presence of this carbohydrate is important for effector functions such as complement fixation and antigen-dependent-cellular-cytotoxicity (ADCC) which result in elimination of the target cell [44]. B4 antibody is targeted to the CD4 receptor complex and it may cause the destruction of CD4.sup.+ cells and immunosuppression of CD4.sup.+ cell function through the effector functions of IgG.sub.1 that are responsible for binding complement. Therefore, an N-aglycosylated deimmunized B4 monoclonal antibody has the potential to be less depleting of CD4.sup.+ cells and less immunotoxic than an N-glycosylated IgG.sub.1 antibody. Removing the N-glycosylation site in the Fc region of IgG.sub.1 by mutation has been shown to abolish the ability of IgG.sub.1 to bind the human FcR1, to activate complement, and to bind C1 q [45].

Two general approaches have been used to remove carbohydrate: tunicamycin treatment of the antibody-producing cells to inhibit the attachment of carbohydrate precursor to asparagines [46], or removal of Asn.sub.297 by mutation to another amino acid. The present deimmunized antibodies were modified by removal of the Asn.sub.297 site for N-linked glycosylation. The AAC codon for Asn.sub.297 of the heavy chain expression vector was mutated to the CAC codon for His by site-directed mutagenesis PCR. The N-aglycosylated deimmunized antibodies were expressed in CHO cells, and N-aglycosyated antibodies were secreted by these cells (FIG. 16, see Original Patent).

The functional properties and safety feature of the deimmunized monoclonal antibodies are important considerations associated with their usage as human therapeutic drugs, and they need to be characterized. Within this context, they have been evaluated for their in vitro capabilities to: (1) neutralize HIV-1 viruses, (2) fix complement, and (3) stimulate human lymphocytes to elicit immune responses.

The HIV-1 neutralization activity of the deimmunized monoclonals derived from CHO clones as compared to the murine mAb B4 counterpart was assessed using the MT-2 microplaque assay [36]. The results of this study shown in Table 3 (see Original Patent) reveal that the deimmunized antibody DH1DK1CHO#21 expressed by Clone 21 exhibited virus neutralization capability comparable to the murine antibody produced by the B4 hybridoma. This was evident from the amount of these monoclonal antibodies required to achieve 50% neutralization of the representative viruses selected from the five clades (A to E) of HIV-1 tested. The aglycosylated deimmunized antibody DH1DK1CHO#7 had exhibited significantly enhanced neutralizing capability against the viruses 23135 from lade B, UG046 from clade D and TH036 from clade E as compared to the murine B4 antibody (Table 3, see Original Patent).

A complement consumption assay was employed to evaluate the complement fixation property of the deimmunized antibodies as compared to their native counterpart, murine mAb B4 (Example 7, see Original Patent). The results obtained from this assay showed that deimmunization of B4 had led to a reduction of the complement fixation property (FIG. 17 (see Original Patent)). This was evident from the higher amount of complement remaining in the supernatant collected from the reaction mixture performed with the deimmunized antibodies in comparison to the reaction performed with the murine monoclonal. That result suggests that the deimmunized antibodies bound to the CD4 complex on human CD4.sup.+ cells were less able to initiate complement fixation than the original murine antibody which more effectively fixed complement when bound to the CD4 cell surface complex. Thus, the supernatant assayed from the reaction mixture performed with murine B4 had contained less complement able to lyse the sensitized SRBC. The results obtained from this study means that the deimmunized antibodies DH1DK1CHO#7 and DH1DK1CHO#16 have greater safety than murine mAb B4 in view of their reduced capacities for fixing complement, and that the N-aglycosylated deimmunized antibody, as expected, has the least capacity for binding complement.

The immunogenicity of murine monoclonal antibodies in their application as human therapeutic drugs could lead to the generation of both human T and B cell responses directed against the therapeutic antibodies. The T cell responses elicited could result in the production of cytokines capable of modulating the immune responses the human subjects may be initiating against particular infectious agents or cancer at the time. A disturbance caused by the presence of murine antibody-induced cytokines may therefore result in the induction of imbalanced or even impaired immune responses required to fight off the invading pathogens or cancer.

The B cell responses elicited would generate human anti-mouse antibodies in the host. This would lead to the formation of antigen-antibody immune complexes and the deposit of these complexes in undesirable tissue sites to cause inflammation and/or pathological conditions in the human subject. In addition, the human anti-mouse antibodies would bind to the administered drug and reduce the effective concentration, thus preventing the therapeutic drug from reaching its desired targets, i.e., CD4.sup.+ cells.

In view of the above considerations, we tested the immunogenicity of the deimmunized monoclonal antibodies and mAb B4 in an in vitro human peripheral blood mononuclear cell (PBMC) culture system (see Example 8). The results obtained from this study are shown in Table 4 (see Original Patent). It was found that the purified murine B4 antibody stimulated human PBMC to produce IL-10 and TNF-.alpha.; while IFN-.gamma. and IL-2 generally accepted to be secreted by antigen-activated Th1 CD4 and another cytokine, IL-4, produced by Th2 CD4 cells, were not detected.

In contrast, the deimmunized antibodies DH1DK1CHO#16 and aglycosylated DH1DK1CHO#7 were both found to be non-immunogenic as judged by their failure to induce the human PBMC to secrete any of the 5 cytokines tested. These findings add to the safety value of these deimmunized monoclonal antibodies in their usage as therapeutic drugs.

The regulatory and the cost advantages of serum-free mammalian cell growth medium has been well established. The use of animal sera in cell culture processes brings along with it the potential for introduction of adventitious agents such as viruses and other transmissible agents (e.g., bovine spongiform encephalopathy) [47]. Additionally, the use of animal sera as a raw material impacts negatively on the cost of large-scale cell-culture processes. Finally, because protein biotherapeutics produced by mammalian cells are secreted into the medium, the use of serum-free medium greatly simplifies the development and robust execution of downstream protein purification processes.

The serum-dependent growth properties of rCHO cells require that cell line adaptation be carried out to obtain phenotypes appropriate for large-scale, serum-free, suspension culture-based manufacturing processes. The process described in Example 6 led to the successful adaptation of an rCHO clone for suspension growth and antibody expression in serum-free medium, with the production of deimmunized antibodies at a level equal to that of cells grown in monolayer cells in medium containing 10 fetal calf serum. The antibody produced in serum-free suspension culture had HIV-1 neutralization activity equal to those of antibodies secreted by monolayer cells cultured in medium containing 10% serum.

Collectively, these results strongly support the efficacy and safety of deimmunized antibodies as therapeutic drugs capable of reducing virus load in HIV-infected patients, and preventing establishment of infection following exposure.

The route(s) of administration useful in a particular application are apparent to one or ordinary skill in the art. Routes of administration of the antibodies include, but are not limited to, parenteral, and direct injection into an affected site. Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous.

The present invention includes compositions of the deimmunized antibodies described above, suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for intravenous, intramuscular, intraperitoneal, or subcutaneous injection, or direct injection into a joint or other area.

In providing the deimmunized antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like. The determination of the optimum dosage and of the optimum route and frequency of administration is well within the knowledge of those skilled in the art. Similarly, dosages for other deimmunized antibodies within the scope of the present invention can be determined without excessive experimentation.

The deimmunized antibodies disclosed herein may be administered to a human patient, in a pharmaceutically acceptable dosage form, suitable for intravenous, subcutaneous or intramuscular administration. Such dosage forms encompass pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, mannitol, sorbic acid, hydrochloric acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sodium phosphate monobasic, sodium phosphate dibasic, dibasic sodium phosphate dihydrate, sodium hydroxide, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol. The deimmunized antibodies will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

Pharmaceutical compositions may be prepared and formulated in dosage forms by methods known in the art; for example, see Remington's Pharmaceutical Sciences [51].

In general, it is desirable to provide the recipient with an initial candidate dosage of deimmunized antibodies which is in the range from about 1 to 100 mg/kg, preferably about 1 to 25 mg/kg, more preferably about 1 to 10 mg/kg, most preferably about 2 to 5 mg/kg of deimmunized antibody, whether, for example, by one or more separate administrations, or by continuous infusion. In general, the antibodies will be administered intravenously (IV) or intramuscularly (IM). For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved. The dose may be readministered at intervals ranging from once a week to once every six months. The determination of the optimum dosage and of optimum route and frequency of administration is well within the knowledge of those skilled in the art. Similarly, dosages for other deimmunized antibodies within the scope of the present invention can be determined without excessive experimentation.

The deimmunized antibodies of the present invention may also be used in combination with antibodies with specificity for HIV such as antibodies to gp41 and gp120 of HIV. These include, but are not limited to, antibodies designated as 2F5, which is specific for gp41, and 2G12, which is specific for gp120, that are described by Hofmann-Lehmann, et al. [27].
 

Claim 1 of 7 Claims

1. A deimmunized recombinant monoclonal antibody expressed from the fusion of nucleic acids encoding a deimmunized Fv fragment of a murine antibody B4 comprising V.sub.H and V.kappa. variable chains and complementarity determining regions of the variable chains of said murine antibody B4 SEQ ID NOS: 20, 22, 24, 26, 28 and 30 and nucleic acids encoding human immunoglobulin constant regions, wherein said deimmunized Fv fragment of murine antibody B4 is deimmunized by replacing the murine heavy V.sub.H chain from the Fv domains of said murine antibody B4 with a nucleotide sequence encoding a deimmunized V.sub.H chain of murine antibody B4 selected from the group consisting of DIVHv.1 of SEQ ID NO: 5, DIVHv.2 of SEQ ID NO: 6, DIVHv.3 of SEQ ID NO: 7, and DIVHv.4 of SEQ ID NO: 8 and replacing the V.kappa. chain from the Fv domains of said murine antibody B4 with a nucleotide sequence encoding a deimmunized V.kappa. chain of murine antibody B4 selected from the group consisting of DIVKv.1 of SEQ ID NO: 9, DIVKv.2 of SEQ ID NO: 10 and DIVKv.3 of SEQ ID NO: 11.

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