Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to the field of fully human monoclonal antibodies, method of making same, and their use in preventive and therapeutic applications in anthrax. More particularly, antibodies have binding specificity for anthrax protective antigen (PA) toxin are provided.
• PA protective antigen
• Anthrax is a zoonotic soil organism endemic to many parts of the world. The B. anthracis organism was one of the first biological warfare agents to be developed and continues to be a major threat in this regard. Although vaccine strains have been developed, currently there are concerns regarding their efficacy and availability.
• a passive immunization strategy may be useful in conferring medium-term protection, and can also have benefits for non-immunized patients who seek treatment after the point at which antibiotic therapy alone is effective.
• B. anthracis spores germinate in the alveolar macrophages, then migrate to lymph nodes where they multiply and enter the bloodstream.
• the vegetative bacteria excrete the tripartite exotoxin that is responsible for the etiology of the disease.
• PA protective antigen
• EF edema factor
• LF lethal factor
• PA can bind either LF or EF, forming lethal toxin (LeTx) or edema toxin (EdTx).
• LeTx lethal toxin
• EdTx edema toxin
• Lethal factor is a zinc-metalloprotease that is essential for the lethal effect of the anthrax toxin on macrophages.
• Protective antigen contains the binding domain of anthrax toxin, which binds to a recently identified receptor on the cell surface and allows translocation of LF or EF into the cell by endocytosis.
• Evidence that the hu-PBL-SCID system can be used to obtain recall antibody responses dates from the original publication of the method by Mosier and co-workers. Mosier et al., Nature 335:256 1988.
• the portion of an anthrax exotoxin is selected from the group consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF).
• PA protective antigen
• LF lethal factor
• EF edema factor
• a fully human immunoglobulin or fragment thereof, that recognizes at least a portion of an anthrax exotoxin is disclosed, wherein the immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain comprising the amino acid sequence shown in FIG. 5.
• the immunoglobulin or fragment thereof is encoded by the nucleotide sequence shown in FIG. 5.
• the immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain comprising CDR1, CDR2, and CDR3; wherein the CDR1 is comprised of the amino acid sequence, as shown in FIG. 5; the CDR2 is comprised of the amino acid sequence, as shown in FIG. 5; and the CDR3 is comprised of the amino acid sequence, as shown in FIG. 5.
• the CDR1, CDR2, and CDR3 regions of the immunoglobulin are encoded by the nucleotide sequences, as shown respectively in FIG. 5.
• a fully human immunoglobulin or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein the immunoglobulin or fragment thereof comprises an immunoglobulin light chain comprising the amino acid sequence shown in FIG. 6.
• the immunoglobulin or fragment thereof is encoded by the nucleotide sequence shown in FIG. 6.
• the immunoglobulin or fragment thereof comprises an immunoglobulin light chain comprising CDR1, CDR2, and CDR3; wherein the CDR1 is comprised of the amino acid sequence, as shown in FIG. 6; the CDR2 is comprised of the amino acid sequence, as shown in FIG. 6; and the CDR3 is comprised of the amino acid sequence, as shown in FIG. 6.
• the CDR1, CDR2, and CDR3 regions of the immunoglobulin are encoded by the nucleotide sequences, as shown respectively in FIG. 6.
• the fully human immunoglobulin or fragment thereof is a single chain that recognizes at least a portion of an anthrax exotoxin.
• a fully human immunoglobulin or fragment thereof is disclosed, that recognizes at least a portion of an anthrax exotoxin, wherein the immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain comprising at least one complementary determining region selected from the group consisting of CDR1, CDR2 and CDR3; wherein the CDR1 is comprised of the amino acid sequence, as . shown in FIG.
• a fully human immunoglobulin or fragment thereof is disclosed, . that recognizes at least a portion of an anthrax exotoxin, wherein the immunoglobulin or fragment thereof comprises an immunoglobulin light chain comprising at least one complementary determining region selected from the group consisting of CDRl, CDR2 and CDR3; wherein the CDRl is comprised of the amino acid sequence, as shown in FIG. 6; the CDR2 is comprised of the amino acid sequence, as shown in FIG. 6; and the CDR3 is comprised of the amino acid sequence, as shown in FIG. 6.
• a fully human immunoglobulin or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein the immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain or light chain comprising the amino acid sequence shown in FIG. 8.
• a fully human immunoglobulin or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein said immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain or light chain comprising the amino acid sequence shown in FIG. 9.
• a fully human immunoglobulin or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein said immunoglobulin or fragment thereof comprises an immunoglobulin heavy chain or light chain comprising the amino acid sequence shown in FIG. 10.
• the nucleotide sequences shown respectively in FIG. 5 and FIG. 6 are disclosed. These nucleotide sequences encode a heavy chain variable region and a light chain variable region, respectively, of a fully human immunoglobulin or fragment thereof, that recognizes at least a portion of an anthrax exotoxin.
• the nucleotide sequences shown in FIG. 8 are disclosed.
• nucleotide sequences encode a heavy chain variable region and a light chain variable region, respectively, of a fully human immunoglobulin or fragment thereof, that recognizes at least a portion of an anthrax exotoxin.
• nucleotide sequences shown in FIG. 9 are disclosed.
• nucleotide sequences encode a heavy chain variable region and a light chain variable region, respectively, of a fully human immunoglobulin or fragment thereof, that recognizes at least a portion of an anthrax exotoxin.
• nucleotide sequences shown in FIG. 10 are disclosed.
• nucleotide sequences encode a heavy chain variable region and a light chain variable region, respectively, of a fully human immunoglobulin or fragment thereof, that recognizes at least a portion of an anthrax exotoxin.
• a method for generating a fully human monoclonal antibody which specifically recognizes at least a portion of an anthrax exotoxin. The method comprises administering peripheral blood mononuclear cells from one or more human donors exposed to anthrax to an immuno- compromised animal; isolating at least one lymphocytic cell from the animal; and fusing the at least one lymphocytic cell with a hybridoma fusion partner, thereby generating a fully human monoclonal antibody.
• the method may further comprise screening the generated antibodies; transforming at least a portion of said lymphocytic cells with EBV; characterizing the animal's immune response using a test bleed; administering one or more booster injections of anthrax antigen to the animal; administering one or more injections of anti-CD8 to the animal; and using a double selection method to select against undesirable cells, wherein the double selection method comprises using HAT selection or using ouabain.
• the human donor is an anthrax-vaccinated donor and/or the human donor has been inadvertently exposed to anthrax.
• the animal is a SCID mouse.
• the hybridoma fusion partner is derived from a mouse myeloma MOPC21.
• the hybridoma fusion partner is a myeloma.
• the hybridoma fusion partner is P3x63Ag8.653.
• the portion of an anthrax exotoxin is selected from the group consisting of PA, LF and EF.
• a method is disclosed for inhibiting the assembly of the protective antigen (PA) of an anthrax exotoxin on receptors in a human.
• the method comprises administering to such human the antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS.
• a pharmaceutical composition for vaccinating a mammal against anthrax is disclosed in accordance with another embodiment of the invention.
• the pharmaceutical composition comprises the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10.
• a pharmaceutical composition for treating a mammal exposed to an anthrax exotoxin is disclosed in accordance with another embodiment of the invention.
• the pharmaceutical composition comprises the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10.
• a method of vaccinating a mammal against anthrax is disclosed.
• the method comprises administering to the mammal an immunizing dose of the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10.
• a method of treating a mammal exposed to anthrax is also disclosed.
• the method comprises administering to the mammal a therapeutic dose of the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10.
• a method of identifying the presence of anthrax exotoxin in a sample is disclosed in accordance with another embodiment of the present invention.
• the method comprises contacting at least a portion of the sample with the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10; and determining binding of anthrax exotoxin with the antibody, wherein the binding is an indicator of the presence anthrax in the sample.
• a kit to identify the presence of anthrax exotoxin in a sample is also disclosed.
• the kit comprises the fully human monoclonal antibody of any of the immunoglobulins or fragments thereof described above, including those described in FIGS. 5, 6, 8, 9, and 10; and an assay system to determine the binding of anthrax exotoxin with the antibody, wherein the binding is an indicator of the presence anthrax in the sample.
• the anthrax exotoxin is tripartite.
• the exotoxin is naturally-occurring or synthetic.
• Figure 1 shows a timeline of the engraftment of SCID mice with human PBMC from anthrax- vaccinated donors.
• Figures 2A-H show anti-anthrax toxin levels in donor plasma compared to the engrafted mice.
• Figure 3 shows testing of the presence of neutralizing PA bioactivity in donor and HuPBL-SCID engrafted mice sera.
• Figure 4 shows a dose-response curve of the inhibition of anthrax PA toxin bioactivity with 21 D9 MAb.
• Figure 5 shows the full nucleotide sequence and amino acid sequence of the 21D9 MAb heavy chain variable region.
• Figure 6 shows the full nucleotide sequence and amino acid sequence of the 21D9 MAb light chain variable region.
• Figure 7 shows survival data in a rat protection model.
• Figure 8 shows the full nucleotide sequence and amino acid sequence of the 1C6 Mab VH and VK chain variable regions.
• Figure 9 shows the full nucleotide sequence and amino acid sequence of the 4H7 Mab VH and VL chain variable regions.
• Figure 10 shows the full nucleotide sequence and amino acid sequence of the 22G12 Mab VH and VL chain variable regions.
• Preferred Embodiment Antibodies which bind to one or more components of the tripartite anthrax exotoxin, the methods of making said antibodies, and the methods of using said antibodies are provided.
• the antibodies provide protection either as single agents or combined in a cocktail.
• Anthrax as defined herein, shall be given its ordinary meaning and shall also include the tripartite anthrax toxin, synthetic or naturally- occurring, and shall also be defined broadly to include one or more of the following components, synthetic or naturally-occurring: protective antigen (PA), lethal factor (LF) and edema factor (EF).
• PA protective antigen
• LF lethal factor
• EF edema factor
• antibodies to “anthrax” shall include antibodies to any portion of one or more components of the anthrax toxin.
• the singular forms “a”, “an”, and “the” include plural reference, unless the context clearly dictates otherwise.
• a reference to “a host cell” includes a plurality of such host cells
• a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art.
• FIG. 1 a method of preparing a fully human monoclonal antibody which specifically recognizes at least a portion of the protective antigen (PA) of an anthrax exotoxin is provided.
• PA protective antigen
• this method includes obtaining peripheral blood mononuclear cells from human donors. After obtaining the peripheral blood mononuclear cells from donors, the blood cells are administered to an immuno-compromised animal. The lymphocytic cells are isolated and fused with a hybridoma fusion partner.
• blood cells from donors who have been exposed to anthrax are obtained. Such exposure may have occurred naturally through exposure, or may have occurred by vaccination. Moreover, in one embodiment, exposure may have occurred decades, years or days prior to obtaining the donor's blood cells.
• the "memory" of said exposure is captured or recalled and is selectably expanded by immunizing the engrafted SCID mice.
• said recall technology is used to generate human monoclonal antibodies.
• the human donor has been vaccinated against anthrax.
• unexposed or na ⁇ ve blood cells are used.
• the unexposed blood cells are exposed to anthrax ex vivo or in vitro, prior to engraftment in the immuno-deficient mouse.
• said initially unexposed cells are transformed into exposed cells and can be used in accordance with the recall technology described above.
• peripheral blood mononuclear cells are obtained from a donor.
• other cell types are obtained, including but not limited to lymphocytes, splenocytes, bone marrow, lymph node cells, and immune cells.
• the blood cells are administered to an immuno-compromised or immuno-deficient animal.
• the animal is a SCID mouse.
• the animal's immune response is characterized using a test bleed.
• the generated antibodies are screened and isolated.
• the lymphocytic cells are transformed with EBV.
• one or more booster injections of anthrax antigen are administered to the immuno-compromised animal.
• one or more injections of anti-human CD8 is administered to the animal.
• a double selection method to select against undesirable cells is used, including, but not limited to using HAT and ouabain.
• the hybridoma fusion partner is the mouse myeloma P3x63Ag8.653.
• the hybridoma fusion partner is derived from the mouse myeloma P3x63Ag8.653.
• a series of human anti-anthrax PA toxin antibodies is provided.
• a monoclonal antibody (IJ8:21D9, or "21D9" is provided. As illustrated in FIG.
• antibody 21D9 was effective in RAW cell assays in toxic inhibition.
• Antibody 21D9 was also shown to protect in vitro a mouse macrophage cell line from toxin challenge.
• the IC 50 of 21D9 was found to be in the picomolar range and in approximately equimolar stoichiometry with the input PA toxin.
• the equilibrium dissociation constant (K ) as determined - by BiaCore analysis revealed this embodiment to bind antigen with high affinity in the picomolar range.
• Deduced amino acid sequence from the 2ID9 hybridoma heavy and light chain cDNA allowed assignment to known VH and VL gene families, although significant mutation away from these germline sequences was also observed thereby indicating the occurrence of somatic hypermutation.
• the mechanism by which 21D9 provides protection is also provided.
• Antibody 21D9, and other antibodies described herein, can be used for human use in vivo for prophylaxis and treatment of Anthrax Class A biowarfare toxins.
• a method for preventing anthrax infection is provided.
• a method for vaccinating mammals to prevent anthrax infection is provided.
• a method to treat mammals who have been exposed to anthrax is provided.
• antibody 22G12, and methods of making and using same, are provided.
• antibody 1C6, and methods of making and using same are provided.
• antibody 4H7, and methods of making and using same are provided.
• Preferred embodiments provide a fully human monoclonal antibody that specifically binds to a component of an anthrax exotoxin or combinations of components thereof
• the anthrax exotoxin can be in tripartite form; and the anthrax toxin can be naturally-occurring or synthetic.
• the anthrax exotoxin comprises protective antigen (PA), edema factor (EF), and lethal factor (LF).
• PA protective antigen
• EF edema factor
• LF lethal factor
• a monoclonal antibody is produced by rescuing the genes encoding antibody variable region from the antibody-producing cells and establishing stable recombinant cell lines producing whole IgG/kappa or IgG/lambda.
• antibody-producing cells recovered from the immunized animal are subjected to cell fusion with an appropriate fusion partner.
• the resulting hybridomas are then screened in terms of the activity of the produced antibodies.
• the hybridomas subjected to selection are screened first in terms of the binding activity to a component of the tripartite anthrax exotoxin.
• the hybridomas are selected based on ability to bind to PA, LF and/or EF proteins in an immunoassay, such as an ELISA test and also a bipassay.
• the hybridoma shows protection in the bioassay.
• the cells from positive wells are used to isolate mRNA. From the mRNA, cDNA is reverse transcribed.
• variable domains are PCR amplified.
• the amino acid sequences constituting the variable regions of the antibodies having a desired binding activity to PA, LF and/or EF and the nucleotide sequences encoding the same is provided.
• FIGS. 5, 6, 8, 9, and 10. show the nucleotide sequence and amino acid sequence of the 21D9 MAb heavy chain variable region.
• FIG. 6 shows the 1 nucleotide sequence and amino acid sequence of the 21D9 MAb light chain variable region.
• cDNA encoding the immunoglobulin variable regions containing the nucleotide sequences shown in FIG. 5 and FIG. 6 is provided.
• these amino acid sequences or cDNA nucleotide sequences are not necessarily identical but may vary so long as the specific binding activity to PA, LF and/or EF is maintained.
• variation in nucleotide sequence is accommodated.
• the site corresponding to CDR is highly variable. In the CDR region, even entire amino acids may vary on some occasions.
• each immunoglobulin molecule consists of heavy chains having a larger molecular weight and light chains having a smaller molecular weight.
• the heavy and light chains each carries a region called "a variable region" in about 110 amino acid residues at the N-terminus, which are different between the molecules.
• Variable regions of a heavy chain and a light chain are designated VH and VL, respectively.
• the antigen-binding site is formed by forming a dimer through electrostatic interaction between the heavy chain variable region VH and the light chain variable region VL.
• the variable region consists of three complementarity determining regions (CDRs) and four frameworks.
• the CDR forms a complementary steric structure with the antigen molecule and determines the specificity of the antibody.
• the three CDRs inserted between the four framework regions (FRs) are present like a mosaic in the variable region (E. A.
• the cDNAs bearing the nucleotide sequences coding the variable regions in immunoglobulin molecules can be cloned from hybridomas that produce the monoclonal antibody to PA, LF and/or EF of the tripartite anthrax exotoxin.
• PCR can be performed.
• ELISA can be used to determine binding to PA, LF and/or EF of the tripartite anthrax exotoxin.
• affinities of an antibody that can bind to PA, LF and/or EF of the tripartite anthrax exotoxin can be determined with kinetic and thermodynamic studies using apparatus, such as BiaCore (Biacore, Piscataway, NJ) surface plasmon resonance apparatus for measuring binding affinity and binding kinetics.
• BiaCore BiaCore
• a monoclonal antibody that can block oligomerization of the PA component of anthrax exotoxin is provided. Accordingly, a monoclonal antibody of preferred embodiments can have preventive or therapeutic uses.
• a preferred monoclonal antibody can be used in a pharmaceutical composition as a vaccination for a mammal against anthrax or as a treatment for a mammal exposed to anthrax exotoxin. Accordingly, preferred embodiments provide methods of vaccinating a mammal against anthrax and/or treating a mammal exposed to anthrax.
• a monoclonal antibody of several embodiments can be administered as a pharmaceutical composition. Thus, in one embodiment, the antibody can be administered by several different routes, including but not limited to: orally, parenterally and topically.
• parenterally shall be given its ordinary meaning and shall also include subcutaneous, intravenous, intraarterial, injection or infusion techniques, without limitation.
• topically shall be given its ordinary meaning and shall also encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and the mucous membranes of the mouth and nose.
• dosage levels of preferred antibody in a pharmaceutical composition may be varied so as to administer an amount of a preferred antibody that is effective to achieve the desired therapeutic response for a particular patient.
• the selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
• the effective daily dose may be divided into multiple doses for purposes of administration, e.g., two to four separate doses per day. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, diet, time and route of administration, combination with other drugs and the severity of the particular disease being treated.
• the pharmaceutical formulation can be in a variety of forms, including, but not limited to, injectable fluids, suppositories, powder, tablets, capsules, syrups, suspensions, liquids and elixirs.
• Preferred embodiments of the present invention provide a kit for identifying the presence of anthrax exotoxin in a sample.
• a monoclonal antibody which specifically recognizes at least a portion of a component of an anthrax exotoxin.
• a sample is contacted with a monoclonal antibody which specifically recognizes at least a portion of a component of an anthrax exotoxin. If an anthrax exotoxin is present, then the binding of the anthrax exotoxin with the monoclonal antibody can be determined.
• the disclosure below is of specific examples setting forth preferred methods for making compounds according to several embodiments of the present invention. These examples are not intended to limit the scope, but rather to exemplify preferred embodiments.
• EXAMPLE 1 INDIRECT ELISA Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated with 50 ⁇ l of Bacillus anthracis Protective Antigen (PA) and Lethal Factor (LF)(List Biological Laboratories (City, State)) at a concentration of l ⁇ g/mL in PBS overnight at 4°C. Plates were washed four times with PBS with Tween 20 at 0.1% and 50 ⁇ l of diluted sera was added to the wells for one hour at room temperature.
• PA Bacillus anthracis Protective Antigen
• LF Lethal Factor
• PA 100 ng/ml
• LF 50 ng/ml
• PA 100 ng/ml
• LF 50 ng/ml
• This lOO ⁇ l volume was subsequently transferred into a 96 well flat bottom tissue culture plate containing lxlO 4 RAW 264.7 cells/well in 100 ⁇ l of the same medium. The culture was incubated for 3 hours at 37°C.
• LDH Lactate dehydrogenase
• mice were immunized with a combination of PA and LF (i.p.) 2 ⁇ g each adsorbed to Alum (Imject®, Pierce, Rockford, IL) and subsequently boosted (i.p.) on day 7, 19 and day 26.
• Mice were inoculated with 0.5 ml of EBV obtained from spent conditioned culture medium of the B95-8 marmoset cell line on day 7. Test bleeds were obtained from the orbital sinus on days 14 and 29.
• Double selection to select against the EBV-LCL and the unfused P3x63ag8.653 fusion partner was carried out using a combination of HAT selection and ouabain. A concentration of 8 ⁇ M ouabain (Sigma, St. Louis, MO) was used.
• ouabain Sigma, St. Louis, MO
• One skilled in the art will appreciate that other poisons or toxins that interfere with the Na+/K+ ATPase can also be used in accordance with several embodiments of the present invention.
• other selection methods can also be used.
• VARIABLE REGION 21D9 IGG AND IGK cDNA CLONING AND EXPRESSION Total RNA was prepared from specific ELISA positive hybridomas using RNeasy Mini Kit (Qiagen, Valencia, CA). Mixture of VH and VL cDNAs were synthesized and amplified in a same tube using One-Step RT-PCR Kit (Qiagen, Valencia, CA). Cycling parameters were 50 °C for 35min, 95 °C for 15min, 35 cycles of 94°C for 30 sec, 52 °C for 20 sec and 72 °C for 1 min 15sec, and 72 °C for 5min. Primers were used for RT-PCR.
• VK1F GACATCCRGDTGACCCAGTCTCC b.
• VK36F GAAATTGTRWTGACRCAGTCTCC
• VK2346F GATRTTGTGMTGACBCAGWCTCC
• VK5F GAAACGACACTCACGCAGTCTC
• VL ⁇ Forward a.
• VLl CAGTCTGTGYTGACGCAGCCGCC b.
• VL2 CAGTCTGYYCTGAYTCAGCCT c.
• VL3 TCCTATGAGCTGAYRCAGCYACC d.
• VL1459 CAGCCTGTGCTGACTCARYC
• VL78 CAGDCTGTGGTGACYCAGGAGCC f
• VL6 AATTTTATGCTGACTCAGCCCC
• RT-PCR was followed by nested PCR with High Fidelity Platinum PCR Mix (Invitrogen, Carlsbad, CA). A micro liter of RT-PCR products was used for VH ⁇ , VLK or VL ⁇ specific cDNA amplification in the separate tube. At substantially the same time, restriction enzyme sites were introduced at both ends. Cycling parameters were 1 cycle of 94°C for 2 minutes, 6 °C for 30 seconds and 68°C for 45 seconds, 35 cycles of 94°C for 40 sseconds, 54 °C for 25 seconds and 68°C for 45 seconds, and 68°C for 5 minutes.
• C ⁇ ER GACSGATGGGCCCTTGGTGGA VH ⁇ PCR products are digested with BsrG I and Apa I and ligated into pEEGl.l vector that is linearlized by Spl I and Apa, I double digestion.
• AgelVKlF TTTTACCGGTGTGACATCCRGDTGACCCAGTCTCC
• AgeIVK36F TTTTACCGGTGTGAAATTGTRWTGACRCAGTCTCC
• AgeIVK2346F TTTTACCGGTGTGATRTTGTGMTGACBCAGWCTCC
• AgeIVK5F TTTTACCGGTGTGAAACGACACTCACGCAGTCTC
• SplKFR4R12 TTTCGTACGTTTGAYYTCCASCTTGGTCCCYTG
• SplKFR4R3 TTTCGTACGTTTSAKATCCACTTTGGTCCCAGG
• SplKFR4R4 TTTCGTACGTTTGATCTCCACCTTGGTCCCTCC
• SplKFR4R5 TTTCGTACGTTTAATCTCCAGTCGTGTCCCTTG
• VLK PCR products are digested with Age I and Spl I and ligated into pEEKl.l vector linearlized by Xma I and Spl I double digestion.
• ApalVLl ATATGGGCCCAGTCTGTGYTGACGCAGCCGCC
• ApaIVL2 ATATGGGCCCAGTCTGYYCTGAYTCAGCCT
• ApaIVL3 ATATGGGCCCAGTATGAGCTGAYRCAGCYACC
• ApaIVL1459 ATATGGGCCCAGCCTGTGCTGACTCARYC
• ApaIVL78 ATATGGGCCCAGDCTGTGGTGACYCAGGAGCC f.
• AvrllVLlIR TTTCCTAGGACGGTGACCTTGGTCCCAGT b.
• AvrIIVL6IR TTTCCTAGGACGGTCACCTTGGTGCCACT d.
• AvrirVLmixIR TTTCCTAGGACGGTCARCTKGGTBCCTCC
• VL ⁇ PCR products are digested with Apa I and Avr II and ligated into pEELg vector linearlized by Apa I and Avr II double digestion.
• the positive clones were identified after transient co-transfection by determining expression in the supernatants by indirect ELISA on PA coated plates.
• CHO Kl cells were transfected with different combinations of IgG and IgK cDNAs using Lipofectamine-2000 (Invitrogen, Carlsbad, CA). The supernatants were harvested about 48 hours to about 72 hours after transfection. Multiple positive clones were sequenced with the ABI 3700 automatic sequencer (Applied Biosystems, Foster City, CA) and analyzed with Sequencher v4.1.4 software (Gene Codes, Ann Arbor, MI).
• EXAMPLE 8 STABLE CELL LINE ESTABLISHMENT Ig heavy chain or light chain expression vector were double digested with Not I and Sal I, and then both fragments were ligated to form a double gene expression vector.
• CHO-K1 cells in 6 well-plate were transfected with the double gene expression vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 24 firs transfection cells were transferred to 10 cm dish with selection medium (D.MEM supplemented with 10% dialyzed FBS, 50 ⁇ M L-methionine sulphoximine (MSX), penicillin/streptomycin, GS supplement). Two weeks later MSX resistant transfectants were isolated and expanded.
• selection medium D.MEM supplemented with 10% dialyzed FBS, 50 ⁇ M L-methionine sulphoximine (MSX), penicillin/streptomycin, GS supplement.
• Anti-PA antibody high producing clones were selected by measuring the supernatant with PA specific ELISA assay. MSX concentration was increased from 50 ⁇ M to 100 ⁇ M to enhance the antibody productivity. EXAMPLE 9 SERUM FREE ADAPTATION PROCEDURE Cells were thawed out from liquid nitrogen storage, the cells were in 10% FBS in
• Purification was carried out by filtering the spent culture media through a 0.2 ⁇ filter and then loaded directly to a HiTrap Protein A column (Pharmacia), followed by washing with 20 mM Sodium phosphate pH 7.4, and the antibody eluted with 0.1M glycine HCI pH3.4 and immediately neutralized with 1/10 volume of IM Tris-HCl pH 8.0.
• the fraction protein content was determined by absorbance at 280 nm, the fractions containing antibody were pooled and dialyzed against phosphate buffered saline pH 7.4 (2x 500 volumes) and filter sterilized through 0.2 ⁇ filter.
• the antibody was further characterized by SDS-PAGE and the purity exceeded 95%.
• EXAMPLE 10 AFFINITY DETERMINATIONS Affinity constants were determined using the principal of surface plasmon resonance (SPR) with a Biacore 3000 (Biacore Inc.).
• a Biacore CM5 chip was used with affinity purified goat anti-human IgG+A+M (Jackson Immuno Research) conjugated to two flowcells of the CM5 chip according to manufacturer's instructions.
• An optimal concentration of an antibody preparation is first introduced into one of the two flowcells, and is captured by the anti-human Ig.
• a defined concentration of antigen is introduced into both flowcells for a defined period of time, using the flowcell without antibody as a reference signal.
• EXAMPLE 1 1 HUMAN IGG QUANTIFICATION BY IMMUNOENZYMETRIC ASSAY
• Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated overnight at 4°C with 50 ⁇ l of Goat anti-Human IgG, Fc ⁇ specific, (cat# 109-005-098, Jackson Immuno Research, West Grove, Pennsylvania) at l ⁇ g/mL in PBS. Plates were washed four times with PBS-0.1% Tween 20. Meanwhile, in a separate preparation plate, dilutions of standards (in duplo) and unknowns were prepared in 100 ⁇ l volume of PBS with 1 mg/ml BSA.
• a purified monoclonal human IgGlK myeloma protein (cat# 1-5154 Sigma, St. Louis, MO) was used as the standard and a different IgG IK myeloma protein (Athens Research, Athens, Georgia) served as an internal calibrator for comparison. Diluted test samples (50 ⁇ l) were transferred to the wells of the assay plate and incubated for one hour at room temperature. Plates were washed as before and 50 ⁇ l of the detecting antibody (1 :4000 in PBS with lmg/ml BSA.) Goat anti-Human Kappa-HRP (Cat.
• FIG. 2A-H shows comparison results of the anti-anthrax toxin levels in the donor plasma as compared to the sera of engrafted mice.
• FIG. 2A-H shows that the mouse sera level of functional immunoreactive (Indirect ELISA) antibody is considerably greater higher than that observed in the donor. A range of levels of immunoreactive antibody was observed in the engrafted mice.
• Test bleeds from engrafted mice were also evaluated for the presence of anti-PA/LF protective antibody in the mouse macrophage RAW cell bioassay ( Figure 3).
• the presence of appropriate seropositivity is one preferred criterion for selecting appropriate animals for fusion to generate human hybridomas.
• a series of 14 individual fusions was carried out with cells obtained from various compartments (either peritoneal wash (PW), spleen (SP), or LCL tumors (TU) within the peritoneal cavity) of the engrafted mice.
• the cells were pooled from several engrafted mice determined to be producing specific anti-anthrax toxin antisera by Indirect ELISA and RAW Cell bioassay prior to fusion. A summary of the fusion results is shown in Table 2.
• Hybridomas were initially selected based on ability to bind to PA (83kD) protein adsorbed to polystyrene microtiter plate wells in an indirect ELISA. A wide range of values for the relative amount of specific anti-PA antibody in the supernatants was observed. In parallel, each of the supernatants was tested individually at a in the Anthrax toxin protection RAW cell bioassay. A dose-response curve of hybridoma-derived 21D9 in the RAW cell bioassay was used to evaluate the effective in vitro IC 50 protective concentration using a cocktail of the PA (83kD) and LF toxins.
• the 21D9 antibody was found to bind to the intact (83kD) form as well as the cleaved (63kD) form of the PA toxin, but to a lesser degree the heptamer using BiaCore (Pharmacia, Peapack, NJ). Additionally, there was no evidence that the antibody was able to inhibit LF binding to PA (63 kD) heptamer as determined by sequential incubations in the BiaCore. This finding potentially implicates the domain 2 on the PA toxin as the epitope blocked by this antibody.
• the nucleotide sequences of the 21D9 MAb heavy and light chains variable regions were determined (FIG. 5 and FIG. 6).
• Anthrax exotoxins the dominant virulence factors produced by Bacillus anthracis are a tripartite combination of protective antigen (PA), lethal factor (LF) and edema factor (EF). These toxins are thought to have a critical role in anthrax pathogenesis; initially to impair the immune system, permitting the anthrax bacterium to evade immune surveillance to disseminate and reach high concentrations; and later in the infection the toxins may contribute directly to death in the host animals including humans. Antibodies that neutralize the PA component of the exotoxin could provide an effective protection from anthrax toxin exposure, early and potentially late in the infection.
• PA protective antigen
• LF lethal factor
• EF edema factor
• the generation of a panel of very potent fully human anti-PA neutralizing antibodies derived from PBMCs obtained from vaccinated donors is provided.
• the antibodies were generated through the combined use of in vivo immunization of SCID mice reconstituted with human PBMC (U.S. Patent Nos. 5,476,996 5,698,767 5,811,524, 5,958,765, 6,413,771, 6,537,809), subsequent recovery of human B cells expressing anti-PA antibodies and immortalization via cell fusion with the mouse myeloma cells.
• Human immunoglobulin cDNAs were isolated and subcloned into the mammalian expression vector.
• Recombinant antibodies were first screened by in vitro neutralization assay using RAW264.7 mouse macrophage cell line. Furthermore, selected antibodies were evaluated for neutralization of lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model. Analysis of the variable regions indicated that antibodies recovered from SCID mice were diverse and hyper-mutated. Among these antibodies, a single IV administration of AVP-21D9 or AVP-22G12 was found to confer full protection with only 0.5x (AVP-21D9) or lx (AVP-22G12) molar excess relative to PA in the rat toxin challenge prophylaxis model. Aglycosylated PA neutralizing antibodies also protected rats from lethal toxin challenge.
• these potent fully human anti-PA toxin-neutralizing antibodies generated may be used for in vivo human use for prophylaxis and/or treatment against Anthrax Class A bioterrorism toxins.
• EXAMPLE 12 CHARACTERIZATION OF A PANEL OF POTENT ANTHRAX TOXIN NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES FROM IMMUNIZED DONORS
• antibodies that bind to the PA component of the tripartite anthrax-toxin and which provide protection as single agents are provided.
• antibody 21D9 is provided.
• antibody 22G12 is provided.
• antibody 1C6 is provided.
• these antibodies are used as single agent in preventing and/or treating anthrax infection.
• combination of two or more of these antibodies are used to treat mammals who have been exposed to aerosolized Bacillus anthracis spores, or exposed to other forms of anthrax.
• antibodies that bind to PA with a range of high affinities from about 82 pM to about 700 pM, as determined by surface plasmon resonance (BiaCore 3000), is provided.

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