Patent Grant
10689441
This invention relates generally to anti-Toll-like Receptor 4 (TLR4 antagonists) including antibodies that specifically bind Toll-like Receptor 4 (TLR-4), to methods of using the anti-TLR4 antagonists as therapeutics and to methods of using the anti-TLR4 antagonists in methods of treating, inhibiting, delaying the progression of, or otherwise ameliorating a symptom of Graft-versus-host disease (GvHD) in a subject and/or improving survival of stem cell-transplanted or bone marrow-transplanted subjects.
Stem cell transplantation such as allogeneic hematopoietic stem cell transplantation (allo-HCT) and allo bone marrow transplantation (BMT) is a curative therapy for human blood-borne cancer. Unfortunately, donor immune cells may consider normal host tissues as non-self and initiate immune attack against those tissues, resulting in the phenomenon of GvHD. Although progress has been made in reducing GvHD after allo-HCT and allo-BMT, it remains a major complication and limits more widespread application of the therapies in hematologic malignancy. GvHD occurs when donor immune cells attacks the normal tissues of the recipient. Severe tissue damage and death are the consequences of the disease.
Thus, there remains a need for methods to prevent GvHD and improve survival of the allo-HCT or allo-BMT patients.
The invention provides methods of inhibiting, delaying the progression of, or otherwise ameliorating a symptom of Graft-versus-Host disease (GvHD) and/or improving survival of subjects who are receiving, have received and/or will be receiving transplanted biological materials, including for example, subjects who have received and/or are receiving a stem cell transplant, a bone marrow transplant or other stem cell-related transplant, using anti-Toll-like Receptor 4 (TLR4 antagonists) including antibodies that specifically bind Toll-like receptor 4 (TLR4). In some embodiments, the GvHD is acute. In some embodiments, the GvHD is chronic. In some embodiments, the GvHD is a subtype of GvHD and/or a disease that is associated with or otherwise considered a side effect of GvHD, such as by way of non-limiting example, idiopathic pneumonia syndrome (IPS), bronchiolitis obliterans (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), acute pancreatitis, and/or acute hepatitis.
The invention provides methods of inhibiting, delaying the progression of, or otherwise ameliorating a symptom of GvHD and/or improving survival of stem cell-transplanted subjects, including bone marrow transplant subjects, by administering to the subject the anti-TLR4 antagonist. In some embodiments, the GvHD is acute. In some embodiments, the GvHD is chronic. In some embodiments, the GvHD is a subtype of GvHD and/or a disease that is associated with or otherwise considered a side effect of GvHD, such as by way of non-limiting example, IPS, BOS, BOOP, acute pancreatitis, and/or acute hepatitis. In some embodiments, the anti-TLR4 antagonist is or is derived from an antibody or immunologically active fragment thereof that binds TLR4. In some embodiments, the anti-TLR4 antagonist is a peptide-based antagonist. In some embodiments, the anti-TLR4 antagonist a nucleic acid-based antagonist. In some embodiments, the anti-TLR4 antagonist is a small molecule inhibitor of TLR4. For example, suitable small molecule inhibitors include, by way of non-limiting example, TAK-242 (resatorvid) and/or E5564 (Eritoran).
In some embodiments, the methods also include the step of administering to the subject who has been implanted with a stem cell-related biological material, e.g., one or more stem cell transplantations and/or one or more bone marrow transplantations, one or more additional doses of an anti-TLR4 antagonist, wherein the antagonist is administered in an amount sufficient to inhibit, delay the progression of, or otherwise ameliorate a symptom of GvHD and/or improve survival of the transplanted biological material the subject. The additional dose(s) of anti-TLR4 antagonist can be administered before, during, or after the transplant. The additional dose(s) of anti-TLR4 antagonist can be the same anti-TLR4 antagonist or a different anti-TLR4 antagonist.
The invention provides methods of inhibiting, delaying the progression of, or otherwise ameliorating a symptom of GvHD and/or improving survival of stem cell-transplanted subjects, including bone marrow transplant subjects, by administering to the subject the antibody or immunologically active fragment thereof that specifically binds a Toll-like receptor 4 (TLR4) polypeptide.
In some embodiments, the methods also include the step of administering to the subject who has been implanted with a stem cell-related biological material, e.g., one or more stem cell transplantations and/or one or more bone marrow transplantations, one or more additional doses of an antibody or immunologically active fragment thereof that specifically binds TLR4, wherein the antibody is administered in an amount sufficient to inhibit, delay the progression of, or otherwise ameliorate a symptom of GvHD and/or improve survival of the transplanted biological material the subject. The additional dose(s) of anti-TLR4 antibody can be administered before, during, or after the transplant. The additional dose(s) of anti-TLR4 antibody can be the same anti-TLR4 antibody or a different anti-TLR4 antibody.
The invention provides methods of inhibiting GvHD and/or improving survival of GvHD subjects by contacting the stem cell-related biological material to be transplanted, e.g., one or more stem cell transplantations and/or one or more bone marrow transplantations, with an anti-TLR4 antagonist to produce a transplantable composition, implanting the transplantable composition at a desired location in the subject, and administering to the subject one or more additional doses of an anti-TLR4 antagonist, wherein the antagonist is administered in an amount sufficient to inhibit GvHD and/or improve survival of the GvHD subjects. The additional dose(s) of anti-TLR4 antagonist can be administered during the transplant, after the transplant or both. The additional dose(s) of anti-TLR4 antagonist can be the same anti-TLR4 antagonist or a different anti-TLR4 antagonist. In some embodiments, the anti-TLR4 antagonist is or is derived from an antibody or immunologically active fragment thereof that binds TLR4. In some embodiments, the anti-TLR4 antagonist is a peptide-based antagonist. In some embodiments, the anti-TLR4 antagonist a nucleic acid-based antagonist. In some embodiments, the anti-TLR4 antagonist is a small molecule inhibitor of TLR4.
The invention provides methods of inhibiting GvHD and/or improving survival of GvHD subjects by contacting the stem cell-related biological material to be transplanted, e.g., one or more stem cell transplantations and/or one or more bone marrow transplantations, with an antibody or immunologically active fragment thereof that specifically binds a Toll-like receptor 4 (TLR4) polypeptide to produce a transplantable composition, implanting the transplantable composition at a desired location in the subject, and administering to the subject one or more additional doses of an antibody or immunologically active fragment thereof that specifically binds TLR4, wherein the antibody is administered in an amount sufficient to inhibit GvHD and/or improve survival of the GvHD subjects. The additional dose(s) of anti-TLR4 antibody can be administered during the transplant, after the transplant or both. The additional dose(s) of anti-TLR4 antibody can be the same anti-TLR4 antibody or a different anti-TLR4 antibody.
The invention also provides methods of treating a subject who has received or will receive a transplant of stem cell-related biological material by administering to the subject one or more doses of an anti-TLR4 antagonist, wherein the antagonist is administered in an amount sufficient to inhibit GvHD and/or improve survival of the GvHD subject. In some embodiments, the anti-TLR4 antagonist is or is derived from an antibody or immunologically active fragment thereof that binds TLR4. In some embodiments, the anti-TLR4 antagonist is a peptide-based antagonist. In some embodiments, the anti-TLR4 antagonist a nucleic acid-based antagonist. In some embodiments, the anti-TLR4 antagonist is a small molecule inhibitor of TLR4.
The invention also provides methods of treating a subject who has received or will receive a transplant of stem cell-related biological material by administering to the subject one or more doses of an antibody or immunologically active fragment thereof that specifically binds a Toll-like receptor 4 (TLR4) polypeptide, wherein the antibody is administered in an amount sufficient to inhibit GvHD and/or improve survival of the GvHD subject.
In some embodiments, the GvHD is acute. In some embodiments, the GvHD is chronic. In some embodiments, the GvHD is a subtype of GvHD and/or a disease that is associated with or otherwise considered a side effect of GvHD, such as by way of non-limiting example, IPS, BOS, BOOP, acute pancreatitis, and/or acute hepatitis.
In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human.
In some embodiments, the stem cell-related biological material to be transplanted is one or more cells or cell types, one or more tissues or tissue types, or an organ or portion thereof. For example, the biological material to be transplanted is allogeneic biological material.
In some embodiments, the biological material to be transplanted is bone marrow cells. In some embodiments, the bone marrow cells are allogeneic bone marrow cells.
In some embodiments, the biological material to be transplanted is hematopoietic stem cells. In some embodiments, the hematopoietic stem cells are allogeneic hematopoietic stem cells.
In some embodiments, the anti-TLR4 antagonist is administered prophylactically to a subject before the biological material has been transplanted, for example, in subjects known or suspected of being “high risk” for GvHD, such as, by way of non-limiting example, older recipients and/or recipients receiving transplanted materials from non-family related donors.
In some embodiments, the anti-TLR4 antagonist that is used to contact the biological material prior to transplantation, i.e., the first anti-TLR4 antagonist, is the same anti-TLR4 antagonist that is administered to the subject before, during and/or after the biological material has been transplanted, i.e., the second anti-TLR4 antagonist. In some embodiments, the first and second anti-TLR4 antagonists are administered at the same dosage. In some embodiments, the first and second anti-TLR4 antagonists are administered at a different dosage.
In some embodiments, the anti-TLR4 antibody that is used to contact the biological material prior to transplantation, i.e., the first anti-TLR4 antibody, is the same anti-TLR4 antibody that is administered to the subject before, during and/or after the biological material has been transplanted, i.e., the second anti-TLR4 antibody. In some embodiments, the first and second anti-TLR4 antibodies are administered at the same dosage. In some embodiments, the first and second anti-TLR4 antibodies are administered at a different dosage.
In some embodiments, the anti-TLR4 antagonist that is used to contact the biological material prior to transplantation, i.e., the first anti-TLR4 antagonist, is a different antibody than the anti-TLR4 antagonist that is administered to the subject before, during and/or after the biological material has been transplanted, i.e., the second anti-TLR4 antagonist. In some embodiments, the first and second anti-TLR4 antagonists are administered at the same dosage. In some embodiments, the first and second anti-TLR4 antagonists are administered at a different dosage.
In some embodiments, the anti-TLR4 antibody that is used to contact the biological material prior to transplantation, i.e., the first anti-TLR4 antibody, is a different antibody than the anti-TLR4 antibody that is administered to the subject before, during and/or after the biological material has been transplanted, i.e., the second anti-TLR4 antibody. In some embodiments, the first and second anti-TLR4 antibodies are administered at the same dosage. In some embodiments, the first and second anti-TLR4 antibodies are administered at a different dosage.
Administration of an anti-TLR4 antagonist, including an anti-TLR4 antibody or fragment thereof, to a patient who is receiving, has received and/or will be receiving stem cell-related transplanted material is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of an anti-TLR4 antagonist, including an anti-TLR4 antibody or fragment thereof, to a patient who is receiving, has received and/or will be receiving stem cell-related transplanted material is considered successful if one or more of the symptoms associated with GvHD is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of an anti-TLR4 antagonist, including an anti-TLR4 antibody or fragment thereof, to a patient who is receiving, has received and/or will be receiving stem cell-related transplanted material is considered successful if one or more of the symptoms associated with GvHD is considered successful if GvHD enters remission or does not progress to a further, i.e., worse, state.
Symptoms of acute and chronic GvHD range from mild to severe. Symptoms of acute GvHD, which usually happens within the first three months after a transplant, include by way of non-limiting example, abdominal pain or cramps, nausea, vomiting, diarrhea, dry or irritated eyes, jaundice, skin rash, itching, and/or redness on areas of the skin. Symptoms of chronic GvHD, which usually starts more than 3 months after a transplant, and, in some instances, can last a lifetime, include by way of non-limiting example, dry eyes or vision changes, dry mouth, white patches inside the mouth, sensitivity to spicy foods, fatigue, muscle weakness, chronic pain, skin rash with raised, discolored areas, as well as skin tightening or thickening, shortness of breath, vaginal dryness, and/or weight loss.
In some embodiments, the anti-TLR4 antagonist, e.g., an antibody or immunologically active fragment thereof that specifically binds TLR4, is administered before, during and/or after transplantation in combination with one or more additional agents. In some embodiments, the anti-TLR4 antagonist, e.g., an anti-TLR4 antibody, and the additional agent(s) are administered simultaneously. For example, the anti-TLR4 antagonist, e.g., an anti-TLR4 antibody, and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the anti-TLR4 antagonist, e.g., an anti-TLR4 antibody, and the additional agent(s) are administered sequentially.
In some embodiments, the additional agent(s) is an immunosuppressive agent. For example, the additional agent(s) is selected from methotrexate, cyclosporin A, tacrolimus, sirolimus, everolimus, a corticosteroid, anti-thymocyte globulin, Infliximab, Etanercept and Adalimumab. The additional agent(s) can also include any compound or other molecule that exhibits an immunosuppressive effect.
The anti-TLR4 antibodies or immunologically active fragments thereof provided herein are capable of blocking, e.g., neutralizing, receptor activation and subsequent intracellular signaling induced TLR4 ligands, e.g., LPS or any other TLR4 ligand described herein. In some embodiments, the antibody is an antibody or an immunologically active fragment thereof. In some embodiments, the antibody or immunologically active fragment thereof that binds TLR4 is a monoclonal antibody. In some embodiments, the antibody or immunologically active fragment thereof that binds TLR4 is a mouse, chimeric, humanized, fully human monoclonal antibody, domain antibody, single chain, Fab, Fab′ and F(ab′)2 fragments, scFvs, or an Fab expression library. In some embodiments, the anti-TLR4 antibodies also bind the human TLR4/MD-2 receptor complex.
In some embodiments, the antibody or an immunologically active fragment thereof binds a human TLR4 polypeptide. In some embodiments, the human TLR4 polypeptide comprises the amino acid sequence:
In some embodiments, the antibody or immunologically active fragment thereof that binds TLR4 comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of GGYSWH (SEQ ID NO: 1); a VH CDR2 region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of YIHYSGYTDFNPSLKT (SEQ ID NO: 2); and a VH CDR3 region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of KDPSDAFPY (SEQ ID NO: 3); a variable light chain complementarity determining region 1 (VL CDR1) region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of RASQSISDHLH (SEQ ID NO: 4); a VL CDR2 region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of YASHAIS (SEQ ID NO: 5); and a VL CDR3 region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of QQGHSFPLT (SEQ ID NO: 6). In some embodiments, the antibody or immunologically active fragment thereof that binds TLR4 further comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the heavy chain variable amino acid sequence QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYT DFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVS S (SEQ ID NO: 7) and an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the light chain variable amino acid sequence EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPS RFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIK (SEQ ID NO: 8). In some embodiments, the antibody or immunologically active fragment thereof that binds TLR4 further comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the heavy chain amino acid sequence MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIR QPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCAR KDPSDAFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 9) and an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the light chain amino acid sequence MEWSWVFLFFLSVTTGVHSEIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKP DQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).
In some embodiments, anti-TLR4 antibody or immunologically active fragment thereof is or is derived from an antibody as described in PCT/IB2005/004206, filed Jun. 14, 2005 and published as WO 2007/110678, the contents of which are hereby incorporated by reference in their entirety.
In some embodiments, anti-TLR4 antibody or immunologically active fragment thereof is or is derived from an antibody as described in PCT application PCT/IB2008/003978, filed May 14, 2008 and published as WO 2009/101479, the contents of which are hereby incorporated by reference in their entirety.
In some embodiments, anti-TLR4 antibody or immunologically active fragment thereof is or is derived from the anti-TLR4 antibody known as HTA125, which is described, for example, in Shimazu, et al., J. Exp. Med., vol. 189:1777-1782 (1999); Nijhuis et al., Clin. Diag. Lab. Immunol., vol. 10(4): 558-63 (2003); and Pivarcsi et al., Intl. Immunopharm., vol. 15(6):721-730 (2003), the contents of each of which are hereby incorporated by reference in their entirety.
In some embodiments, the anti-TLR4 antibody or immunologically active fragment thereof is or is derived from a domain antibody such as, for example, the domain antibodies that bind TLR4 described in PCT application PCT/EP2009/055926, filed May 15, 2009 and published as WO 2009/13848, the contents of which are hereby incorporated by reference in their entirety.
In some embodiments, the anti-TLR4 antibody or immunologically active fragment thereof binds to an epitope comprising one or more amino acid residues on human TLR4 between residues 289 and 375 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 328 and 329 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 349 through 351 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 369 through 371 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 328, 329, 349 through 351 and 369 through 371 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 293 through 295 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 296 and 297 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 319 through 321 of SEQ ID NO: 11. For example, in some embodiments, the antibody or immunologically active fragment thereof binds to an epitope that comprises at least residues 293 through 295, 296, 297 and 319 through 321 of SEQ ID NO: 11.
In some embodiments, anti-TLR4 antibody or immunologically active fragment thereof is or is derived from an antibody as described in PCT/US2013/034543, filed Mar. 29, 2013 and published as WO 2013/14911, the contents of which are hereby incorporated by reference in their entirety.
The anti-TLR4 antibodies of the invention also include antibodies that include a heavy chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical an amino acid sequence shown herein, and/or a light chain variable amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical an amino acid sequence shown herein.
In some embodiments, the anti-TLR4 antibodies described herein also include at least one specific amino acid substitution within, for example, an Fc region or an FcR binding fragment thereof (e.g., a polypeptide having amino acid substitutions within an IgG constant domain) such that the modified antibody elicits alterations in antigen-dependent effector function while retaining binding to antigen as compared to an unaltered antibody. For example, the altered antibodies elicit the prevention of proinflammatory mediator release. In a preferred embodiment, the altered antibodies are human and of the IgG1 isotype.
The anti-TLR4 antibodies of the invention include an altered antibody in which at least one amino acid residue in the constant region of the Fc portion of the antibody has been modified. For example, at least one amino acid in the CH2 domain of the Fc portion has been replaced by a different residue, i.e., an amino acid substitution. In the altered antibodies described herein, one or more of the amino acid residues that correspond to residues 325, 326 and 328 is substituted with a different residue as compared to an unaltered antibody. The numbering of the residues in the gamma heavy chain is that of the EU index (see Edelman, G. M. et al., 1969; Kabat, E, A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller., 1991. Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, Bethesda, Md., NIH Publication n. 91-3242). In a preferred embodiment, EU amino acid position 325 of the gamma heavy chain constant region is substituted with serine, and EU amino acid position 328 of the gamma heavy chain constant region is substituted with phenylalanine, such that the EU positions 325 to 328 of the gamma heavy chain constant region of the altered human IgG1 antibody comprise the amino acid sequence SKAF (SEQ ID NO: 76).
Pharmaceutical compositions according to the invention can include an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.
The present invention provides antagonists to Toll like Receptor 4 (TLR4), such as, for example, monoclonal antibodies (mAbs) that specifically bind Toll like Receptor 4, and more specifically, human TLR4. These anti-TLR4 antagonists, including anti-TLR4 antibodies are used in methods of inhibiting GvHD and/or improving survival of GvHD subjects.
In some embodiments, the anti-TLR4 antagonists are antibodies and immunologically active fragments thereof that bind TLR4. Anti-TLR4 antibodies include antibodies that bind the human TLR4/MD-2 receptor complex and also bind TLR4 independently of the presence of MD-2.
TLR4 antibodies of the invention include, for example, antibodies having the combination of heavy chain and light chain sequences shown below.
Exemplary antibodies of the invention include, for example, the anti-TLR4 antibodies described in PCT/IB2005/004206, filed Jun. 14, 2005 and published as WO 2007/110678, the anti-TLR4 antibodies described in PCT application PCT/IB2008/003978, filed May 14, 2008 and published as WO 2009/101479, the contents of each of which are hereby incorporated by reference in their entirety, and commercially available antibodies such as HTA125.
Exemplary antibodies of the invention include, for example, the antibody referred to herein as NI-0101, which binds the human TLR4/MD2 complex and also binds TLR4 independently of the presence of MD-2. The sequences of the NI-0101 (hu15c1) antibody are shown below, with the CDR sequences underlined in the VH and VL amino acid sequences:
GYSWHW
IRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSL
HLH
WYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAE
The NI-0101 (hu15c1) antibody includes VH CDRs having the sequences GGYSWH (SEQ ID NO: 1), YIHYSGYTDFNPSLKT (SEQ ID NO: 2), and KDPSDAFPY (SEQ ID NO: 3), and VL CDRs having the sequences RASQSISDHLH (SEQ ID NO: 4), YASHAIS (SEQ ID NO: 5) and QQGHSFPLT (SEQ ID NO: 6).
The amino acid and nucleic acid sequences of the heavy chain variable (VH) and light chain variable (VL) regions of the anti-TLR4/MD-2 antibodies are shown below. The amino acids encompassing the complementarity determining regions (CDR) as defined by Chothia et al. 1989, E. A. Kabat et al., 1991 are highlighted in underlined and italicized text below. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).
Anti-TLR4 antibodies include the antibodies described in co-pending U.S. application Ser. No. 11/009,939, filed Dec. 10, 2004 and Ser. No. 11/151,916, filed Jun. 15, 2004 and in WO 05/065015, filed Dec. 10, 2004 and PCT/US2005/020930, filed Jun. 15, 2004, each of which is hereby incorporated by reference in its entirety. Several exemplary antibodies include the antibodies referred to therein as 18H10, 16G7, 15C1 and 7E3.
Anti-TLR4 antibodies include the antibodies described in co-pending U.S. application Ser. No. 11/151,916, filed Jun. 15, 2004 (U.S. Patent Publication No. US 2008-0050366 A1) and in PCT/IB2005/004206, filed Jun. 15, 2004 (PCT Publication No. WO 07/110678), each of which is hereby incorporated by reference in its entirety. The sequences of several exemplary antibodies are shown below.
Anti-TLR4 antibodies include the antibodies described in PCT/IB2008/003978, filed May 14, 2008 (PCT Publication No. WO 2009/101479), the contents of which are hereby incorporated by reference in their entirety. These anti-TLR4 antibodies are modified to include one or more mutations in the CDR3 portion. The sequences of several exemplary antibodies are shown below.
Antibodies of the invention interfere with or otherwise antagonize signaling via human TLR4 and/or human TLR4/MD-2 complexes. In some embodiments, antibodies of the invention also bind cynomolgus monkey TLR4 and/or cynomolgus monkey TLR4/MD-2 complexes. In some embodiments, the antibody binds to an epitope that includes one or more amino acid residues on human and/or cynomolgus monkey TLR4 having the following sequences:
Antibodies of the invention interfere with or otherwise antagonize signaling via human and/or cynomolgus monkey TLR4 and/or human and/or cynomolgus monkey TLR4/MD-2 complexes. In some embodiments, the antibody binds to an epitope that includes one or more amino acid residues on human and/or cynomolgus monkey TLR4 between residues 289 and 375 of SEQ ID NO: 11-(human TLR4) and/or SEQ ID NO: 77 (cynomolgus TLR4). For example, TLR4 antibodies specifically bind to an epitope that includes residue 349 of SEQ ID NO: 11 (human) and/or SEQ ID NO: 77 (cynomolgus). In some embodiments, the epitope also includes additional residues, for example, residues selected from the group consisting of at least residues 328 and 329 of SEQ ID NO: 11 (human) and/or SEQ ID NO: 77 (cynomolgus); at least residue 351 of SEQ ID NO: 11 (human) and/or SEQ ID NO: 77 (cynomolgus); and at least residues 369 through 371 of SEQ ID NO: 11 (human) and/or SEQ ID NO: 77 (cynomolgus), and any combination thereof.
In some embodiments, the anti-TLR4 antibody or immunologically active fragment thereof is or is derived from monoclonal antibodies recognizing human and/or cynomolgus monkey TLR4/MD-2 receptor expressed on the cell surface. The antibodies are capable of blocking, e.g., neutralizing, receptor activation and subsequent intracellular signaling induced TLR4 ligands, e.g., LPS or any other TLR4 ligand described herein. Antibodies of the invention include antibodies that bind human and cynomolgus monkey TLR4/MD-2 receptor complex and also bind TLR4 independently of the presence of MD-2.
In some embodiments, the anti-TLR4 antibody or immunologically active fragment thereof interferes with or otherwise antagonizes signaling via human and/or cynomolgus monkey TLR4/MD-2 receptor expressed on the cell surface, e.g., by blocking receptor activation and subsequent intracellular signaling induced by LPS. Exemplary monoclonal antibodies of these embodiments include: 1A1, 1A6, 1B12, 1C7, 1C10, 1C12, 1D10, 1E11, 1E11 N103D, 1G12, 1E11.C1, 1E11.C2, 1E11.C3, 1E11.C4, 1E11.C5, 1E11.C6, 1E11.E1, 1E11.E2, 1E11.E3, 1E11.E4, 1E11.E5, 1E11.C2E1, 1E11.C2E3, 1E11.C2E4 and 1E11.C2E5. The sequences of these antibodies are shown below.
These antibodies have distinct specificities. Some antibodies show specificity for both the human and cynomolgus monkey TLR4 and/or both the human and cynomolgus monkey TLR4/MD-2 receptor complex, and they have been shown to inhibit receptor activation and subsequent intracellular signaling via LPS. For example, 1C12, 1E11, 1E11 N103D, 1E11.C1, 1E11.C2, 1E11.C3, 1E11.C4, 1E11.C5, 1E11.C6, 1E11.C2E1, 1E11.C2E2, 1E11.C2E3, 1E11.C2E4 and 1E11.C2E5 bind both human and cynomolgus monkey TLR4 independently of the presence of human or cynomolgus monkey MD-2. 1A1, 1A6, 1B12, 1C7, 1C10, 1D10 and 1G12 only bind to cynomolgus monkey TLR4 independently of the presence of cynomolgus monkey MD-2. 1E11.E1, 1E11.E2, 1E11.E3, 1E11.E4 and 1E11.E5 bind only to human TLR4 independently of the presence of human MD-2.
In some embodiments, the invention provides an isolated antibody that specifically binds Toll-like receptor 4 (TLR4), wherein the antibody binds to an epitope that includes at least residue 349 of SEQ ID NO: 11 and an epitope that includes at least residue 349 of SEQ ID NO: 77. In some embodiments, the antibody includes a heavy chain with three complementarity determining regions (CDRs) including a variable heavy chain complementarity determining region 1 (CDRH1) amino acid sequence of GYSITGGYS (SEQ ID NO: 49); a variable heavy chain complementarity determining region 2 (CDRH2) amino acid sequence of IHYSGYT (SEQ ID NO: 56); and a variable heavy chain complementarity determining region 3 (CDRH3) amino acid sequence of ARKDSG(X1)(X2)(X3)PY (SEQ ID NO: 57), where X1 is N, Q, D or E, X2 is any hydrophobic amino acid, and X3 is any hydrophobic amino acid; and a light chain with three CDRs including a variable light chain complementarity determining region 1 (CDRL1) amino acid sequence of QSISDH (SEQ ID NO: 68); a variable light chain complementarity determining region 2 (CDRL2) amino acid sequence of YAS (SEQ ID NO: 69); and a variable light chain complementarity determining region 3 (CDRL3) amino acid sequence of QQGHSFPLT (SEQ ID NO: 6). In some embodiments, the epitope further includes at least residues 328 and 329 of SEQ ID NO: 11 and SEQ ID NO: 77. In some embodiments, the epitope further includes at least residue 351 of SEQ ID NO: 11 and SEQ ID NO: 77. In some embodiments, the epitope further includes one or more residues between residues 369 through 371 of SEQ ID NO: 11 and SEQ ID NO: 77. In some embodiments, the epitope further includes at least residues 369 through 371 of SEQ ID NO: 11 and SEQ ID NO: 77. In some embodiments, the antibody specifically binds to an epitope that includes at least residues 328, 329, 349, 351 and 369 through 371 of SEQ ID NO: 11 and SEQ ID NO: 77. In some embodiments, the antibody further includes an amino acid substitution in the gamma heavy chain constant region at EU amino acid position 325 and an amino acid substitution at EU amino acid position 328. In some embodiments, the amino acid substituted at EU amino acid position 325 is serine, and wherein the amino acid substituted at EU amino acid position 328 is phenylalanine.
In some embodiments, the three heavy chain CDRs include an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) amino acid sequence selected from the group consisting of G(F/Y)PI(R/G/W)(Y/F/G)GYS (SEQ ID NO: 48), GYSITGGYS (SEQ ID NO: 49); GFPIRYGYS (SEQ ID NO: 50); GYPIRFGYS (SEQ ID NO: 51); GYPIRHGYS (SEQ ID NO: 52); GFPIGQGYS (SEQ ID NO: 53); GYPIWGGYS (SEQ ID NO: 54) and GYPIGGGYS (SEQ ID NO: 55), a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) amino acid sequence of IHYSGYT (SEQ ID NO: 56); and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) amino acid sequence selected from the group consisting of ARKDSG(N/Q/D/E)X1X2PY (SEQ ID NO: 57) where X1 and X2 are each independently any hydrophobic amino acid, ARKDSGNYFPY (SEQ ID NO: 58); ARKDSGRLLPY (SEQ ID NO: 59); ARKDSGKWLPY (SEQ ID NO: 60); ARKDSGHLMPY (SEQ ID NO: 61); ARKDSGHNYPY (SEQ ID NO: 62); ARKDSGKNFPY (SEQ ID NO: 63); ARKDSGQLFPY (SEQ ID NO: 64); ARKDSGHNLPY (SEQ ID NO: 65); ARKDSGDYFPY (SEQ ID NO: 66) and ARKDSGRYWPY (SEQ ID NO: 67). The three light chain CDRs include an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) amino acid sequence of QSISDH (SEQ ID NO: 68); a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) amino acid sequence of YAS (SEQ ID NO: 69); and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) amino acid sequence selected from the group consisting of QQG(Y/N)(D/E)(F/Y)PXT (SEQ ID NO: 70) where X is any hydrophobic amino acid, QQGHSFPLT (SEQ ID NO: 6); QQGNDFPVT (SEQ ID NO: 71); QQGYDEPFT (SEQ ID NO: 72); QQGYDFPLT (SEQ ID NO: 73); QQGYDYPLT (SEQ ID NO: 74) and QQGYEFPLT (SEQ ID NO: 75). The antibodies bind to human and cynomolgus monkey TLR4/MD-2 complex, to human and cynomolgus TLR4 when not complexed with human and cynomolgus MD-2, to human TLR4/MD-2 complex, to human TLR4 when not complexed with human MD-2, to cynomolgus monkey TLR4/MD-2 complex or cynomolgus TLR4 when not complexed with cynomolgus MD-2.
An exemplary TLR4 monoclonal antibody is the 1E11 antibody described herein. As shown below, the 1E11 antibody includes a heavy chain variable region (SEQ ID NO: 79) encoded by the nucleic acid sequence shown in SEQ ID NO: 78, and a light chain variable region (SEQ ID NO: 81) encoded by the nucleic acid sequence shown in SEQ ID NO: 80.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1A1 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1A1 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGRLLPY (SEQ ID NO: 59). The light chain CDRs of the 1A1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1A6 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1A6 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGKWLPY (SEQ ID NO: 60). The light chain CDRs of the 1A6 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1B12 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1A6 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGHLMPY (SEQ ID NO: 61). The light chain CDRs of the 1B12 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1C7 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1C7 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGHNYPY (SEQ ID NO: 62). The light chain CDRs of the 1C7 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1C10 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1C10 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGKNFPY (SEQ ID NO: 63). The light chain CDRs of the 1C10 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1C12 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1C12 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGQLFPY (SEQ ID NO: 64). The light chain CDRs of the 1C12 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1D10 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1D10 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGHNLPY (SEQ ID NO: 65). The light chain CDRs of the 1D10 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11 N103D antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11 N103D antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGDYFPY (SEQ ID NO: 66). The light chain CDRs of the 1E11 N103D antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1G12 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1G12 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGRYWPY (SEQ ID NO: 67). The light chain CDRs of the 1E11 N103D antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C1 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C1 antibody have the following sequences: GFPIRYGYS (SEQ ID NO: 50); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C2 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C2 antibody have the following sequences: GYPIRFGYS (SEQ ID NO: 51); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C3 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C3 antibody have the following sequences: GYPIRHGYS (SEQ ID NO: 52); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C4 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C4 antibody have the following sequences: GFPIGQGYS (SEQ ID NO: 53); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C5 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C5 antibody have the following sequences: GYPIWGGYS (SEQ ID NO: 54); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.C6 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C6 antibody have the following sequences: GYPIGGGYS (SEQ ID NO: 55); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11.C1 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGHSFPLT (SEQ ID NO: 6).
An exemplary TLR4 monoclonal antibody is the 1E11.E1 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.E1 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGNDFPVT (SEQ ID NO: 71).
An exemplary TLR4 monoclonal antibody is the 1E11.E2 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.E2 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYDEPFT (SEQ ID NO: 72).
An exemplary TLR4 monoclonal antibody is the 1E11.E3 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.E3 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYDFPLT (SEQ ID NO: 73).
An exemplary TLR4 monoclonal antibody is the 1E11.E4 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.E4 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYDYPLT (SEQ ID NO: 74).
An exemplary TLR4 monoclonal antibody is the 1E11.E5 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.E5 antibody have the following sequences: GYSITGGYS (SEQ ID NO: 49); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYEFPLT (SEQ ID NO: 75).
An exemplary TLR4 monoclonal antibody is the 1E11.C2E1 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C2E1 antibody have the following sequences: GYPIRFGYS (SEQ ID NO: 51); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGNDFPVT (SEQ ID NO: 71).
An exemplary TLR4 monoclonal antibody is the 1E11.C2E3 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C2E3 antibody have the following sequences: GYPIRFGYS (SEQ ID NO: 51); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYDFPLT (SEQ ID NO: 73).
An exemplary TLR4 monoclonal antibody is the 1E11.C2E4 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C2E4 antibody have the following sequences: GYPIRFGYS (SEQ ID NO: 51); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYDYPLT (SEQ ID NO: 74).
An exemplary TLR4 monoclonal antibody is the 1E11.C2E5 antibody described herein.
The amino acids encompassing the complementarity determining regions (CDR) are as defined by M. P. Lefranc (See Lefranc, M.-P., Current Protocols in Immunology, J. Wiley and Sons, New York supplement 40, A1.P.1-A.1P.37 (2000) LIGM:230). The heavy chain CDRs of the 1E11.C2E5 antibody have the following sequences: GYPIRFGYS (SEQ ID NO: 51); IHYSGYT (SEQ ID NO: 56); and ARKDSGNYFPY (SEQ ID NO: 58). The light chain CDRs of the 1E11 antibody have the following sequences: QSISDH (SEQ ID NO: 68); YAS (SEQ ID NO: 69); and QQGYEFPLT (SEQ ID NO: 75).
In some embodiments, the TLR4 antibodies are formatted in an IgG isotype. In some embodiments, the TLR4 antibodies are formatted in an IgG1 isotype.
An exemplary IgG1-formatted antibody is the IgG1-formatted 1E11 antibody comprising the heavy chain sequence of SEQ ID NO: 178 and the light chain sequence of SEQ ID NO: 179, as shown below:
An exemplary IgG1-formatted antibody is the IgG1-formatted 1E11.C11 antibody comprising the heavy chain sequence of SEQ ID NO: 182 and the light chain sequence of SEQ ID NO: 183, as shown below:
In some embodiments, TLR4 antibodies of the invention specifically bind human and/or cynomolgus TLR4/MD-2 complex, wherein the antibody binds to an epitope that includes one or more amino acid residues on human and/or cynomolgus TLR4 between residues 325 and 374 of SEQ ID NO: 11 (human) and SEQ ID NO: 77 (cynomolgus). Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as 1A1, 1A6, 1B12, 1C7, 1C10, 1C12, 1D10, 1E11, 1E11 N103D, 1G12, 1E11.C1, 1E11.C2, 1E11.C3, 1E11.C4, 1E11.C5, 1E11.C6, 1E11.E1, 1E11.E2, 1E11.E3, 1E11.E4, 1E11.E5, 1E11.C2E1, 1E11.C2E3, 1E11.C2E4 and 1E11.C2E5.
The anti-TLR4 antibodies of the invention include an altered antibody in which at least the amino acid residue at EU position 325 and at least the amino acid residue at EU position 328 in the CH2 domain of the Fc portion of the antibody has been modified. For example, at least the amino acid residue at EU position 325 has been substituted with serine, and at least the amino acid residue at EU position 328 has been substituted with phenylalanine
These anti-TLR4 antibodies with a modified Fc portion elicit modified effector functions e.g., a modified Fc receptor activity, as compared to an unaltered antibody. For example, the human Fc receptor is CD32A. In some embodiments, these anti-TLR4 antibodies elicit a prevention of proinflammatory mediators release following ligation to CD32A as compared to an unaltered antibody. Thus, these anti-TLR4 antibodies elicit a modified Fc receptor activity, such as the prevention of proinflammatory mediators release while retaining the ability to bind a target antigen. In some embodiments, these anti-TLR4 antibodies are neutralizing antibodies, wherein the anti-TLR4 antibody elicits a modified Fc receptor activity, while retaining the ability to neutralize one or more biological activities of a target antigen.
For example, anti-TLR4 antibodies of the invention include monoclonal antibodies that bind the human TLR4/MD-2 receptor complex. This receptor complex is activated by lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria. The anti-TLR4 antibodies of the invention inhibit receptor activation and subsequent intracellular signaling via LPS. Thus, the anti-TLR4 antibodies neutralize the activation of the TLR4/MD-2 receptor complex. In particular, the invention provides anti-TLR4 antibodies that recognize the TLR4/MD-2 receptor complex expressed on the cell surface. These anti-TLR4 antibodies block LPS-induced and other TLR4 ligand-induced pro-inflammatory cytokine (e.g., IL-6, IL-8, TNFα) production. In addition, some anti-TLR4 antibodies of the invention also recognize TLR4 when not complexed with MD-2. The altered antibody is, e.g., a humanized antibody.
Monoclonal antibodies of the invention (e.g., murine monoclonal, humanized antibodies or fully human monoclonal antibodies) specifically bind TLR4. Also included in the invention are antibodies that bind to the same epitope as the antibodies described herein. For example, antibodies of the invention that specifically bind TLR4 and/or the TLR4/MD-2 complex bind to an epitope that includes one or more amino acid residues on human TLR4 (SEQ ID NO: 11)
Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a monoclonal antibody (e.g., a murine monoclonal or humanized antibody) has the same specificity as a monoclonal antibody described herein by ascertaining whether the former prevents the latter from binding to the TLR4/MD-2 complex or to TLR4 when not complexed to MD-2. If the monoclonal antibody being tested competes with the monoclonal antibody of the invention, as shown by a decrease in binding by the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or a closely related, epitope. An alternative method for determining whether a monoclonal antibody has the specificity of monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with the TLR4/MD-2 complex or a soluble TLR4 protein (with which it is normally reactive), and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind the TLR4/MD-2 complex or to bind TLR4 and TLR4 complexed with MD-2. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd>10−6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab, Fab′ and F(ab′)2 fragments, scFvs, and an Fab expression library.
The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
The term “monoclonal antibody” (mAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
The term “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).
As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide. An antibody is said to specifically bind an antigen when the dissociation constant is ≤1 μM; preferably ≤100 nM and most preferably ≤10 nM.
As used herein, the terms “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff/Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is said to specifically bind to the Toll-like Receptor 4 (TLR4)/MD-2 complex or to TLR4 when not complexed to MD-2, when the equilibrium binding constant (Kd) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the invention include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown herein, and nucleic acid molecules encoding the light chain immunoglobulin molecules shown herein.
The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the invention comprise the heavy chain immunoglobulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.
The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The term “control sequence” as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
The term oligonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the invention are either sense or antisense oligonucleotides.
The term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes Oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotide can include a label for detection, if desired.
The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, and “substantial identity”. A “reference sequence” is a defined sequence used as a basis for a sequence comparison a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window”, as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.
As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”, sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences”.
As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.
Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic, and asparagine-glutamine.
As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long′ more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term “analog” as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has specific binding to TLR4/MD2 complex or TLR4 alone, under suitable binding conditions. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986), Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH-(cis and trans), —COCH2—, CH(OH)CH2—, and —CH2SO—, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).
The term “antineoplastic agent” is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.
As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
The term patient includes human and veterinary subjects.
Use of Anti-TLR4 Antibodies
It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.
Therapeutic formulations of the invention, which include an anti-TLR4 antibody, are used to inhibit GvHD and/or improve survival of a GvHD subject.
Efficaciousness of treatment is determined in association with any known method for diagnosing or treating GvHD or other transplant related disorders. Inhibiting GvHD or improving survival of a GvHD subject indicates that the antibody confers a clinical benefit.
Anti-TLR4 antibodies are administered in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macro emulsions.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
Pharmaceutical Compositions
The antibodies or soluble chimeric polypeptides of the invention (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or soluble chimeric polypeptide and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The Examples and data provided herein assess the role of TLR4 blockade in inhibiting GvHD and improving survival of GvHD subjects. Briefly, B6D2F1 and C57BL/6 mice (female, 8 weeks of age) were lethally irradiated and administered 5×106 bone marrow derived cells and 6×106 splenocytes from either syngeneic (B6D2F1) or allogeneic (C57Bl/6) donors. Mice transplanted with allogeneic cells were treated intravenously with 100 mg/kg of anti-TLR4 monoclonal antibody, 5E3, or isotype control either prophylactically (day −1, 3, 7, 14, 21, 28 and 35) or therapeutically (day 7, 10, 14, 21, 28 and 35). Body weight (
While the studies described herein use allogeneic bone marrow cells, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Materials and Methods for the Generation of 5E3 Monoclonal Antibody:
The 5E3 monoclonal antibody is a monoclonal antibody that binds mouse TLR4. (See Daubeuf et al., “TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock,” J Immunol vol. 179:6107-6114 (1997).
Animals:
B6D2F1 and C57BL/6 mice (female, 8 weeks of age) were purchased from Charles River laboratories and housed in a conventional facility with free access to food and water. All experiments were conducted under protocols reviewed and approved by institutional animal care and use committee.
Isolation of Mouse Bone Marrow Cells:
Place the femurs from the donor B6D2F1 and C57BL/6 mice in a small dish (35×10 mm) on ice containing RPMI 1640+2% FBS. Flush the femurs with 10 ml of RPMI+2% FBS using a 26G needle. Pipette cells up and down several times to disperse aggregates. Pass the cells through a sterile 40-μm nylon Cell Strainer (Falcon 352340). Bring the volume to 50 ml with medium and centrifuge at 2000 rpm (900×g), 10 min, 4° C. Wash the cell pellet twice with 50 ml of serum-free RPMI. Centrifuge at 2000 rpm, 5 min, 4° C. Resuspend cells in 20 ml medium and count cells. Centrifuge again and resuspend cells to 5×107 cells/ml.
Isolation of Mouse Splenocytes:
Place the femurs from the donor B6D2F1 and C57BL/6 mice in a small dish (35×10 mm) on ice containing RPMI 1640+2% FBS. Move the spleen to a sterile wire mesh screen (200 um bar width and 340 um open space). Gently push the spleen through the screen with the plunger of a 10 ml syringe into the petri dish. Rinse the screen with 3 ml of RPMI 1640+2% FBS. Transfer the cell suspension to a centrifuge tube and centrifuge to pellet the cells. Lyse the red blood cells using ACK (Ammonium-Chloride-Potassium) Lysing Buffer (1 ml of Tris-NH4Cl per 0.1 ml of packed cells for 2 min at room temperature. Wash cells three times with medium, count cells and resuspend cells to 6×107 cells/ml.
Allo-Bone Marrow Transplantation:
Recipient B6D2F1 female mice were immobilized in individual boxes and exposed to 900 rads administered over 9 minutes utilizing a gamma irradiator. Isolated bone marrow cells and splenocytes from the same donor mouse (from B6D2F1 for syngeneic and from C57BL/6 for allogeneic) were mixed 1:1 in volume and 200 μl of the cell mix (containing 5×106 bone marrow derived cells and 6×106 splenocytes) was administered into the irradiated recipients.
Administration of Monoclonal Antibodies:
B6D2F1 mice transplanted with allogeneic cells (from C57BL/6) were treated intravenously (i.v.) with 100 mg/kg of anti-TLR4 monoclonal antibody, 5E3, or isotype control either prophylactically (day −1, 3, 7, 14, 21, 28 and 35) or therapeutically (day 7, 10, 14, 21, 28 and 35). On the days specified above and immediately prior to the administration of the antibodies, the antibodies were thawed at room temperature and diluted with PBS to reach required antibody concentrations for injection. Mice were injected with 200 μl of antibody/PBS solution (i.v.). Body weight and survival were followed weekly starting from day 0 for 6 weeks.
Statistical Analysis:
Statistical analysis was performed using GraphPad Prism version 5.0d. Survival curves were analyzed by Log-rank (Mantel-Cox) test in comparison to the isotype control treated allo-transplantation group. The p-values were shown in the figures.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application No. 61/912,617, filed Dec. 6, 2013. The contents of this application are hereby incorporated by reference in their entirety. The contents of the text file named “NOV1034001US_SeqList.txt”, which was created on Feb. 4, 2015 and is 159 KB in size, are hereby incorporated by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3773919 | Boswell et al. | Nov 1973 | A |
| 4522811 | Eppstein et al. | Jun 1985 | A |
| 5151510 | Stec | Sep 1992 | A |
| 7312320 | Elson | Dec 2007 | B2 |
| 7348316 | Rossignol | Mar 2008 | B2 |
| 20080050366 | Elson et al. | Feb 2008 | A1 |
| 20120142098 | Elson | Jun 2012 | A1 |
| 20120177648 | Kosco-Vilbois | Jul 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 03106692 | Dec 2003 | WO |
| WO 2005065015 | Jul 2005 | WO |
| WO 2005117975 | Dec 2005 | WO |
| WO 2007110678 | Oct 2007 | WO |
| WO 2009101479 | Aug 2009 | WO |
| WO 2009138494 | Nov 2009 | WO |
| WO 2013149111 | Oct 2013 | WO |
| Entry |
|---|
| Goldberg et al, FASEB J. 2007, vol. 21, pp. 2840-2848. |
| Zhao et al., Cell Mol Immunol. Mar. 2013; 10(2): 165-175. |
| Dunn-Siegrist et al., (Journal of Biological Chemistry, 2007, vol. 282, No. 48, pp. 34817-34827. |
| Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):2 W-8 (2000). |
| Bowie et al. “A Method to Identify Protein Sequences That Fold into a Known Three-Dimensional Stucture”, 1991, Science, vol. 253, p. 164-171. |
| Charman WN “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000). |
| Daubeuf et al. “TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock,” J. Immunol., vol. 179, p. 6107-6114 (1997). |
| Davies et al. “Antibod-Antigen Complexes”, Annual Rev Biochem vol. 59, p. 439-473 (1990). |
| Devetten M P et al. “Graft-versus-host disease: How to translate new insights into new therapeutic strategies”, Biology of Blood and Marrow Transplantation, vol. 10, p. 815-825 (2004). |
| Di W., et al. “Relationship between TLR4 and acute graft-versus-host disease in murine haploidentical hematopoietic stem cell transplantation model”, Acta Universitatis Medicinalis Nanjing Natural Science, vol. 33, No. 4, p. 462-466 (2013). |
| Fauchere, “Elements for the Rational Design of Peptide Drugs”, Advances in Drug Research, vol. 15, p. 29-69 (1986). |
| Evans et al. “Design of Nonpeptidal Ligands for a Peptide Receptor: Cholecystokinin Antagonists”, J. Med. Chem., vol. 30, p. 1229-1239 (1987). |
| LaPlanche et al. “Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscoptc studies of the Rp-Rp, Sp-Sp, and Rp-Sp duplexes, [dCGGsAATTCC)]2, derived from diastereomeric 0-ethyl phosphorothioates”, Nucleic Acids Research, vol. 14, No. 22, p. 9081-9093 (1986). |
| Lefranc, M-P., “Nomenclature of the Human Immunoglobulin Genes”, Current Protocols in Immunology, supplement 40, A1.P.1-A.IP.37 (2000). |
| Malmqvist, “Biospecific interaction analysis using biosensor technology”, Nature, vol. 361, p. 186-187 (1993). |
| Marasco et al., “Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody”, Proc. Natl. Acad. Sci. USA, vol. 90, p. 7889-7893 (1993). |
| Needleman and Wunsch “A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins”, J. Mol. Biol., vol. 48, p. 443-453 (1970). |
| Nijhuis O. et al., “Endothelial Cells Are Main Producers of Interleukin 8 through Toll-Like Receptor 2 and 4 Signaling during Bacterial Infection in Leukopenic Cancer Patients”, Clinical and Diagnostic Laboratory Immunology, vol. 10, No. 4, p. 558-563 (2003). |
| Pearson and Lipman “Improved tools for biological sequence comparison”, Proc. Natl. Acad. Sci. USA, vol. 85, p. 2444-2448 (1988). |
| Pivarcsi et al., “expression and function of Toll-like receptor 2 and 4 in human keratinocytes”, International Immunology, vol. 15, No. 6, p. 721-730 (2003). |
| Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998). |
| Rizo and Gierasch “Constrained Peptides: Models of Biocative Peptides and Protein Substructures”, Ann. Rev. Biochem., vol. 61, p. 387-418 (1992). |
| Shimazu R. et al., “MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4”, J. Exp. Med., vol. 189, p. 1777-1782 (1999). |
| Sivula J. et al. “Toll-Like Receptor Gene Polymorphisms Confer Susceptibility to Graft-Versus-Host Disease in Allogenic Hematopoietic Stem Cell Transplantation”, Scandinavian Journal of Immunology, vol. 76, pp. 336-341 (2012). |
| Smith an Waterman “Comparison of Biosequences”, Advances in applied Mathematics, vol. 2, p. 482-489 (1981). |
| Stec et al. “Automated Solid-Phase Synthesis, Separation, and Stereochemistry of Phosphorothioate Analogues of Oligodeoxyribonucleotides”, J. Am. Chem. Soc., vol. 106, No. 20, p. 6077-6079 (1984). |
| Stein et al. “Physicochemical properties of Phosphorothioate oligodeoxynucleotides”, Nucl. Acids Res., vol. 16, No. 8, p. 3209-3221 (1988). |
| Thornton et al. “Prediction of progress at last”, Nature, vol. 354, p. 105-106 (1991). |
| Uhlmann and Peyman “Antisense Oligonucleotides: A New Therapeutic Principle”, Chemical Reviews, vol. 90, No. 4, p. 544-584 (1990). |
| Veber and Freidinger “The design of metabolically-stable peptide analogs”, TINS, p. 392-396 (1985). |
| Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. vol. 203, p. 1-60 (2000). |
| Zon et al. “Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions”, Anti Cancer Drug Design vol. 6, p. 539-568 (1991). |
| Zon et al. Oligonucleotides and Analogues: A Practical Approach, p. 87-108, (1991). |
| Gale, R.P. and Y. Reisner (Jun. 28, 1986) “Graft Rejection and Graft-Versus-Host Disease: Minor Images” The Lancet, 327(8496): 1468-1470. |
| Number | Date | Country | |
|---|---|---|---|
| 20150158946 A1 | Jun 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61912617 | Dec 2013 | US |
