CN116829596A - Plasma kallikrein antibodies and uses thereof - Google Patents

Plasma kallikrein antibodies and uses thereof Download PDF

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Publication number
CN116829596A
CN116829596A CN202280011600.0A CN202280011600A CN116829596A CN 116829596 A CN116829596 A CN 116829596A CN 202280011600 A CN202280011600 A CN 202280011600A CN 116829596 A CN116829596 A CN 116829596A
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seq
amino acid
antibody
acid sequence
set forth
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瓦埃·比戴安
普拉迪普·比斯塔
彼得·埃文·哈尔温
托马斯·基塞拉克
强纳森·梵尔林
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Ostya Therapy GmbH
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Ostya Therapy GmbH
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Priority claimed from PCT/US2022/014242 external-priority patent/WO2022165130A1/en
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Abstract

Provided herein are antibodies that bind plasma kallikrein and compositions containing such antibodies or antigen binding fragments thereof. Methods of making and using such antibodies, or antigen-binding fragments thereof, are also provided, e.g., for the treatment of hereditary angioedema or bradykinin-dependent edema.

Description

Plasma kallikrein antibodies and uses thereof
Cross Reference to Related Applications
The present application claims U.S. provisional patent application No. 63/142,748 filed on day 28 of month 1 of 2021; U.S. provisional patent application Ser. No. 63/159,323 filed on day 3 and 10 of 2021; U.S. provisional patent application Ser. No. 63/220,194, filed on 7/9 of 2021; and U.S. provisional patent application No. 63/262,838, filed on 21, 10, 2021, the contents of each of which are hereby incorporated by reference in their entirety.
Technical Field
The field of the application are immunology, specifically therapeutic antibodies, and the treatment of diseases with those antibodies.
Background
Plasma kallikrein is a serine protease and is a potential drug target for various inflammatory, cardiovascular, infectious (sepsis) and neoplastic diseases (Sainz i.m. et al, thromb Haemost 98,77-83,2007). Activation of plasma kallikrein amplifies intrinsic clotting via its feedback activation of factor XII and enhances inflammation by producing the pro-inflammatory nonapeptide bradykinin. As the major kininogenase in the circulation, plasma kallikrein is mainly responsible for bradykinin production in the vasculature. Genetic defects in the major natural inhibitor of plasma kallikrein, the C1 inhibitor protein (C1-INH), lead to Hereditary Angioedema (HAE). HAE patients suffer from an acute episode of painful edema usually induced by an unknown trigger (Zuraw b.l. et al, N Engl J Med 359,1027-1036,2008). The plasma kallikrein-kinin system (plasma KKS) is associated with various diseases by using agents or genetic studies in animal models. Plasma kallikrein binding proteins (e.g., antibodies, such as inhibitory antibodies) are useful therapeutic agents for a variety of diseases and conditions, such as those associated with plasma kallikrein activity levels.
Despite efforts to date, there remains a need for therapeutic antibodies that target plasma kallikrein.
Disclosure of Invention
The invention described herein is based in part on the discovery of high affinity binding proteins, such as antibodies and antigen binding fragments, that bind to plasma kallikrein, such as human plasma kallikrein. The antibodies have a high specificity for plasma kallikrein, e.g., human plasma kallikrein. The antibodies are highly selective for plasma kallikrein relative to prekallikrein and lack significant off-target binding to trypsin and other serine proteases. These binding proteins may also have an extended half-life, for example in blood. Because of these and other features, antibodies may be useful in treating a variety of plasma kallikrein mediated diseases, such as hereditary angioedema and bradykinin dependent edema, and also to facilitate treatment of these diseases at lower doses and/or to extend dosing intervals as compared to other antibodies.
In one aspect of the invention, disclosed herein are antibodies, including antigen binding fragments, that bind plasma kallikrein. The plasma kallikrein may be a human plasma kallikrein, e.g. a human plasma kallikrein having the amino acid sequence as shown in SEQ ID No. 32.
Antibodies and antigen-binding fragments disclosed herein may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14, 23, 29 or 43; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17, 19, 24, 30 or 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16, 25 or 31; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11, 20 or 27; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12, 18, 21 or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13, 22 or 28.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17 or 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 23; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 24; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 20; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 21 or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 22;
for example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 29; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 30; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 31; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 27; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 21 or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 43; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17 or 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 15; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 17; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 23; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 24; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 20; comprising the amino acid sequence shown in SEQ ID NO. 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 22.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 29; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 30; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 31; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 27; CDR2 comprises the amino acid sequence shown in SEQ ID NO. 26; and the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO. 28.
For example, the antibody or binding fragment thereof may have a VH wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 43; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28.
For example, the antibody or binding fragment thereof may have a VH, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14 having up to 1, 2 or 3 amino acid substitutions for the amino acid sequence; CDR2 comprises the amino acid sequence shown in SEQ ID No. 15, 17 or 19, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid substitutions thereto; and HCDR3 comprises the amino acid sequence shown in SEQ ID No. 16 having up to 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions for said amino acid sequence; and a VL wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11 having up to 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12 or 18, with up to 1, 2, 3 or 4 amino acid substitutions to said amino acid sequence; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 13, with up to 1, 2, 3 or 4 amino acid substitutions to said amino acid sequence. In some antibodies or binding fragments, each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprises at most one amino acid substitution, while in other antibodies or binding fragments, at most one CDR selected from HCDR1, HCDR2, or HCDR3 comprises a substitution and/or at most one CDR selected from LCDR1, LCDR2, and/or LCDR3 comprises a substitution.
The VL of an antibody or binding fragment disclosed herein may comprise the amino acid sequence shown in SEQ ID NO. 2, 4 or 6, e.g., SEQ ID NO. 6. The VH of an antibody or binding fragment disclosed herein may comprise SEQ ID NO:1, 3 or 5, e.g., SEQ ID NO:5, and a polypeptide comprising the amino acid sequence shown in seq id no. VH may comprise SEQ ID NO:5 and VL may comprise the amino acid sequence shown in SEQ ID No. 6. VH may comprise the amino acid sequence shown in SEQ ID NO. 1, 3 or 5, and VL may comprise the amino acid sequence shown in SEQ ID NO. 2, 4 or 6.
The light chain of the antibodies or binding fragments disclosed herein may comprise the amino acid sequence shown in SEQ ID NO. 8. The heavy chain of an antibody or binding fragment disclosed herein may comprise an amino acid sequence as set forth in SEQ ID NO. 10, 9 or 7. For example, the light chain may comprise the amino acid sequence of SEQ ID NO. 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO. 7. For example, the light chain may comprise the amino acid sequence of SEQ ID NO. 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO. 9. For example, the light chain may comprise the amino acid sequence of SEQ ID NO. 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO. 10.
The antibody or antigen binding fragment may have a VH region whose amino acid sequence is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid set forth in SEQ ID No. 5, 3 or 1, and/or a VL region whose amino acid sequence is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid set forth in SEQ ID No. 2, 4 or 6.
The antibody or antigen binding fragment may have a heavy chain amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% percent homology to the sequence set forth in SEQ ID NO. 10, 9 or 7, and/or a light chain sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% percent homology to the sequence set forth in SEQ ID NO. 8.
CDR sequences of antibodies and binding fragments disclosed herein can be inserted between human or humanized framework sequences. The antibody may be, for example, a full or full length antibody.
The antibodies or antigen binding fragments disclosed herein may bind to at least one of the following residues: human plasma kallikrein having the amino acid sequence shown in SEQ ID No. 32, K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625. For example, it may bind to one or more of the following residues: human plasma kallikrein K550, R551, K585, S597, Y617 or T625 with the amino acid sequence shown in SEQ ID NO. 32.
The antibodies or antigen binding fragments disclosed herein can be less than or equal to about 1, 2, 3, 4, or 5 x 10 -9 K of M D And (as measured by Surface Plasmon Resonance (SPR) analysis) to a human plasma kallikrein, e.g., a human plasma kallikrein having the amino acid sequence shown in SEQ ID NO: 32. It may have a K substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20) and/or K between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and/or K between 0.1nM and 5nM for human plasma kallikrein D And K between 250nM and 2,000nM for prehuman kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
The invention also relates to antibodies and binding fragments thereof that compete with the antibodies or binding fragments disclosed herein for binding to plasma kallikrein. For example, such antibodies may bind to human plasma kinase The same epitope on peptide releasing enzyme as the antibodies disclosed herein. For example, such antibodies may bind to one or more of the following residues: human plasma kallikrein having the amino acid sequence shown in SEQ ID No. 32, K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625. For example, such antibodies may bind to one or more of the following residues: human plasma kallikrein K550, R551, K585, S597, Y617 or T625 with the amino acid sequence shown in SEQ ID NO. 32. Such antibodies may have one or more of the following characteristics: (i) a serum half-life of at least 20 days; (ii) K substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20 in buffer); (iii) K between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and/or (iv) K between 0.1nM and 5nM for human plasma kallikrein D And a K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
The antibodies or antigen binding fragments disclosed herein may be monoclonal antibodies. It may be a humanized antibody. It may have a heavy chain constant region selected from the group consisting of IgG, igA, igD, igE and IgM. If the constant region is of the IgG class, subclasses may be selected from the group consisting of IgG1, igG2, igG3, and IgG4. If the heavy chain constant region is IgG1, it can have the M252Y, S254T and T256E mutations or the M428L and N434S mutations, as numbered according to the EU numbering index. The heavy chain variable region and the light chain variable region may or may not be linked by a linker. If it is linked by a linker, for example in an scFv antibody, the linker may be a peptide linker having the sequence: (GGGGS) n (SEQ ID NO:35)；(GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1-5.
In another aspect, the invention includes isolated nucleic acids comprising a nucleotide sequence encoding a heavy chain or heavy chain variable region (VH) of an antibody or binding fragment disclosed herein, as well as isolated nucleic acids comprising a nucleotide sequence encoding a light chain or light chain variable region (VL) of an antibody or antigen binding fragment disclosed herein. The heavy and light chains may be encoded on the same nucleic acid sequence, or the heavy chain may be encoded on one nucleic acid and the light chain may be encoded separately by another nucleic acid. The VL and VH may be encoded on the same nucleic acid sequence, or the VH may be encoded on one nucleic acid and the VL may be encoded separately by another nucleic acid. The expression vector may contain nucleic acids encoding the heavy and light chains, or the expression vector may contain nucleic acids encoding the VL and VH chains. The host cell may contain an expression vector comprising nucleic acids encoding the heavy and light chains, or the host cell may contain an expression vector comprising nucleic acids encoding the heavy and light chains. The host cell may contain an expression vector comprising nucleic acids encoding VH and VL, or the host cell may contain an expression vector comprising nucleic acids encoding VH and an expression vector comprising nucleic acids encoding VL. The host cell may be grown under conditions such that it expresses heavy and light chains or VH and VL to produce the antibodies or antigen-binding fragments of the invention.
In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment disclosed herein or a nucleic acid encoding the same. For example, the pharmaceutical composition may be injectable or intravenously administrable.
In another aspect, the invention provides a method of treating an individual having a disease or disorder selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post angioplasty), systemic lupus erythematosus nephritis or burn, or a pharmaceutical composition containing the same, by administering a plasma kallikrein antibody or antigen binding fragment thereof as disclosed herein.
The disclosed antibodies or antigen binding fragments are useful in methods of treating plasma kallikrein mediated disorders in an individual, including hereditary angioedema or bradykinin-dependent edema. The disclosed antibodies or antigen binding fragments thereof may also be used in the manufacture of a medicament for the treatment of the above-mentioned disorders, such as hereditary angioedema or bradykinin-dependent edema.
The disclosed antibodies, or binding fragments thereof, may also be used to reduce bradykinin content in an individual to reduce or inhibit bradykinin production in an individual and/or to reduce or inhibit plasma kallikrein activity in an individual.
Drawings
FIG. 1 is a bar graph showing the selectivity of various hybridoma cell lines based on Relative Light Units (RLU) for human plasma kallikrein.
FIG. 2 is a graph showing the inhibitory activity of various antibodies against human plasma kallikrein.
FIG. 3 provides graphs showing the inhibitory activity of various antibodies against human plasma kallikrein, with pKal concentrations of 1nM (upper graph) and 10nM (upper graph).
FIG. 4 provides a graph showing the inhibitory activity of various antibodies against human plasma kallikrein, with a pKal concentration of 1nM.
FIG. 5 provides a graph showing that MAb4-YTE is more effective at inhibiting bradykinin production in vitro than DX-2930.
FIG. 6 provides a Surface Plasmon Resonance (SPR) binding data graph comparing affinities of DX-2930, MAb4-YTE and MAb4-LS for pKal (upper panel) and preKal (lower panel) at pH 7.4 and pH 6.0.
FIG. 7 is a graph showing the plasma kallikrein (pKal) activity plotted against MAb4-YTE concentration in a cross-species pKal binding assay as described in example 2. As shown, MAb4-YTE is not only a potent inhibitor of human pKal, but also of monkey, rat and rabbit pKal.
FIG. 8 provides a graph of pharmacokinetic data showing the concentration of MAb4, MAb4-LS and MAb4-YTE in cynomolgus monkeys over time.
Figure 9 provides a graph showing that MAb4-YTE has a significantly prolonged plasma half-life in cynomolgus monkeys compared to DX-2930. The concentration of DX-2930 over time is shown in the upper panel, the concentration of MAb4-YTE is shown in the middle panel, and the data from the upper and middle panels are shown together in the lower panel.
FIG. 10 is a graph showing that the duration of action of MAb4-YTE in vivo is longer relative to the comparison antibody.
FIG. 11 provides a graph showing IC50 of gabexate mesylate, DX-2930 and MAb4-YTE in trypsin inhibition assays.
FIG. 12 is a graph showing competitive binding of MAb4-LS and MAb4-YTE antibodies to the surface of DX-2930, competitive binding of MAb4-YTE and DX-2930 antibodies to the surface of MAb4-LS, and competitive binding of MAb4-LS and DX-2930 antibodies to the surface of MAb 4-YTE.
FIG. 13 is a graph showing peptide interactions of human pKal and MAb 4-YTE.
FIGS. 14A-J show interactions between human pKal and MAb 4-YTE. FIG. 14A shows a band/surface representation of a front view of a pKal-MAb4-YTE epitope cross-linking mapping. FIG. 14B shows a band/surface representation of a back view of a cross-linking mapping of pKal-MAb4-YTE epitopes. FIG. 14C shows a band/surface representation of side view 1 of a cross-linked mapping of pKal-MAb4-YTE epitopes. FIG. 14D shows a band/surface representation of side view 2 of a cross-linked mapping of pKal-MAb4-YTE epitopes. FIG. 14E shows a strip/surface representation of a top view of a pKal-MAb4-YTE epitope cross-linked mapping. FIG. 14F shows a band representation of a front view of a cross-linked mapping of pKal-MAb4-YTE epitopes. FIG. 14G shows a band representation of the back view of the cross-linking mapping of pKal-MAb4-YTE epitopes. FIG. 14H shows a band representation of side view 1 of a cross-linked mapping of pKal-MAb4-YTE epitopes. FIG. 14I shows a band representation of side view 2 of a cross-linked mapping of pKal-MAb4-YTE epitope. FIG. 14J shows a band representation of a top view of a cross-linked mapping of pKal-MAb4-YTE epitopes.
FIG. 15 is a peptide interaction diagram of human pKal and DX-2930.
FIGS. 16A-J show the interaction between human pKal and DX-2930. FIG. 16A shows a band/surface representation of a front view of a pKal-DX-2930 epitope cross-linked mapping. FIG. 16B shows a band/surface representation of a back view of the cross-linking mapping of the pKal-DX-2930 epitope. FIG. 16C shows a band/surface representation of side view 1 of a cross-linked mapping of pKal-DX-2930 epitope. FIG. 16D shows a band/surface representation of side view 2 of a cross-linked mapping of pKal-DX-2930 epitope. FIG. 16E shows a band/surface representation of a top view of a pKal-DX-2930 epitope cross-linked plot. FIG. 16F shows a band representation of a front view of a pKal-DX-2930 epitope cross-linked mapping. FIG. 16G shows a band representation of the rear view of the cross-linking mapping of the pKal-DX-2930 epitope. FIG. 16H shows a band representation of side view 1 of the cross-linked mapping of pKal-DX-2930 epitope. FIG. 16I shows a band representation of side view 2 of a cross-linked mapping of pKal-DX-2930 epitope. FIG. 16J shows a band representation of a top view of a pKal-DX-2930 epitope cross-linked plot.
FIG. 17 is a diagram showing human pKal residues crosslinked by antibody DX-2930 as compared to antibody MAb4-YTE, as described in example 5 below, and showing that these antibodies bind different epitopes on pKal.
FIGS. 18A-C show the nucleic acid sequence (top row; SEQ ID NO: 48) and amino acid sequence (bottom row; SEQ ID NO: 10) of the heavy chain of MAb 4-YTE. The sequence starts in fig. 18A, continues to fig. 18B and ends in fig. 18C.
FIGS. 19A-B show the nucleic acid sequence (top row; SEQ ID NO: 49) and amino acid sequence (bottom row; SEQ ID NO: 8) of the light chain of MAb 4-YTE. The sequence starts in fig. 19A and ends in fig. 19B.
Detailed Description
The invention described herein is based in part on the discovery of high affinity binding proteins, such as antibodies and antigen binding fragments, that bind to plasma kallikrein, such as human plasma kallikrein. The antibodies have a high specificity for plasma kallikrein, e.g., human plasma kallikrein. The antibodies are highly selective for plasma kallikrein relative to prekallikrein and lack significant off-target binding to trypsin and other serine proteases. These binding proteins may also have an extended half-life, for example in blood. Because of these and other features, antibodies may be useful in treating a variety of plasma kallikrein mediated diseases, such as hereditary angioedema and bradykinin dependent edema, and also to facilitate treatment of these diseases at lower doses and/or to extend dosing intervals as compared to other antibodies.
Antibodies described herein may have one or more of the following characteristics: (i) a serum half-life of at least 20 days; (ii) K substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20 in buffer); (iii) K between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and/or (iv) K between 0.1nM and 5nM for human plasma kallikrein D And a K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
These binding proteins may also be used in compositions, e.g., pharmaceutical compositions, for the treatment of a variety of plasma kallikrein-mediated disorders, such as hereditary angioedema and bradykinin-dependent edema.
I. Plasma kallikrein antibodies
As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies. Antibodies described herein include full length or intact antibodies that contain two immunoglobulin heavy chains associated with two immunoglobulin light chains.
Antibody light chains are typically composed of a variable region (V L ) And a constant region (C L ) Composition is prepared. Heavy chains are usually composed of a variable region (V H ) And at least three constant regions (CH 1 、CH 2 And CH (CH) 3 ) (or more, depending on isotype) composition. The variable region of each light/heavy chain pair is typically found in the amino-terminal portion of each chain, forming an antibody binding site that is primarily responsible for antigen recognition and determines the binding specificity of the antibody. Thus, in general, whole or full length antibodies have two binding sites, i.e., are bivalent. The two binding sites may be identical and target the same antigen; alternatively, as in a bifunctional or bispecific antibody, the two binding sites may be different, e.g., each binding site targeting a different antigen, or a different epitope of the same antigen.
The carboxy-terminal portion of an immunoglobulin heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are divided into kappa and lambda light chains. Furthermore, human heavy chains are generally classified as μ, δ, γ, α, or ε, and isotypes of antibodies are defined as IgM, igD, igG (e.g., igG1, igG2, igG3, igG 4), igA, and IgE, respectively.
Typically, the variable domains of both the heavy and light chains of an antibody comprise three hypervariable regions, also known as Complementarity Determining Regions (CDRs), located within four relatively conserved Framework Regions (FR). CDRs are typically arranged by framework regions so as to be able to bind to a particular epitope. Typically, from the N-terminus to the C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. As used herein, "HCDR1", "HCDR2" and "HCDR3" refer to CDR1, 2 or 3, respectively, of the heavy chain variable region, and "LCDR1", "LCDR2" and "LCDR3" refer to CDR1, 2 or 3, respectively, of the light chain variable region.
Typically, the allocation of amino acids for each CDR is in accordance with the definition of Kabat (see, e.g., sequences of Proteins of Immunological Interest, kabat et al; national Institutes of Health, bethesda, md.; 5 th edition; NIH publication No. 91-3242 (1991); kabat (1978) Adv. Prot. Chem.32:1-75; kabat et al, (1977) J. Biol. Chem. 252:6609-6616), or Chothia (see, e.g., chothia et al, (1987) J mol. Biol.196:901-917, or Chothia et al, (1989) Nature 342:878-883), although alternative definitions are known in the art, e.g., IMGT (see, e.g., lefranc (2005), nucl. Acids Res.,33, D593-D597; lefranc et al, (2003), dev. Comp. Company, 27,55-77; lefrance et al, (1987) J. Mol. 252; lepr. Etv. 185; lepr. 2005-172; see, e.g., 1987) J. Mol. Biol.196:901-917, or Chothia et al, (1989) Nature 342:878-883), although alternatives are known in the art, e.g., IMGT (see, e.g., lefrance (2005), nucl. Acids Res.,33, D593-D597; lefrance, lefrance et al, (1997), lefrance. Lefrance et al. (1997).
As used herein, the term "parent antibody" is used to refer to an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for the intended use, e.g., humanization of the antibody for use as a human therapeutic antibody.
As used herein, unless otherwise indicated, the term "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full length or intact antibody, e.g., a fragment that retains one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, fab ', F (ab') 2 And Fv fragments; a double body; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments; a single domain antibody; recombinant heavy chain-only antibodies (VHH); and shark heavy chain only antibodies (VNAR).
As used herein, the term "Fab fragment" is understood to mean a C comprising one light chain and one heavy chain H 1 and variable region.
The term "Fc" region, as used herein, refers to a region comprising two cs each comprising at least an antibody H 2 and C H 3 domain heavy chain fragments.
The term "Fab' fragment" as used herein is understood to mean an antibody fragment comprising a light chain and a part or fragment of a heavy chain comprising V H Domain and C H 1 domain and C H 1 and C H 2 regions between the domains. The interchain disulfide bond formed between the two heavy chains of the two Fab 'fragments can form F (ab') 2 Fragments.
As used herein, the term "Fv region" refers to an antibody region comprising variable regions from the heavy and light chains but lacking constant regions.
The term "single chain Fv" or "scFv" refers to a V comprising an antibody H And V L Antibody fragments of domains, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide comprises V H And V is equal to L Polypeptide linkers between the domains that enable the scFv to form the desired structure for antigen binding. For reviews of scFv see, e.g., pluckaphun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, volume 113, rosenburg and Moore, springer-Verlag, new York, pages 269-315. See also International patent application publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.
As used herein, the term "monoclonal antibody" refers to a population of antibodies that is substantially homogeneous, i.e., the antibody molecules that make up the population are identical in amino acid sequence, except for minor natural mutations that may be present. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, particularly in their CDRs, which are typically specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by the hybridoma method described first by Kohler et al (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in Clackson et al (1991) Nature 352:624-628 Marks et al (1991) J.mol.biol.222:581-597. See also Presta (2005) J.allergy Clin.Immunol.116:731.
As used herein, the term "chimeric antibody" is understood to mean an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species, see, e.g., U.S. Pat. nos. 4,816,567; and Morrison et al, (1984) Proc.Natl. Acad. Sci. USA 81:6851-6855).
As used herein, the term "humanized antibody" refers to a form of antibody that contains sequences from human and non-human (e.g., murine, rat) antibodies. For example, a humanized antibody may comprise variable regions in which the CDRs or portions thereof are derived from or correspond to sequences of a non-human immunoglobulin and all or substantially all of the FR or portions thereof are derived from or correspond to sequences of a human immunoglobulin. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (e.g., a human light chain constant region and/or a human heavy chain constant region, such as a human Fc, or human CH1, CH2, and/or CH3 domains). The CDR sequences described herein may be interposed between human or humanized framework sequences.
Humanization may be performed to reduce the immunogenicity of antibodies, e.g., non-human antibodies, when administered to humans. In one method of humanization, a chimeric protein is produced in which a mouse immunoglobulin constant region is replaced with a human immunoglobulin constant region. See, e.g., morrison et al, 1984, PROC.NAT.ACAD.SCI.81:6851-6855, neuberger et al, 1984,NATURE 312:604-608; U.S. patent No. 6,893,625 (Robinson); no. 5,500,362 (Robinson); and No. 4,816,567 (cabin).
In one approach, known as CDR grafting, CDRs of light and heavy chain variable regions are grafted into a framework of another species. For example, murine CDRs can be grafted into human FR. In some embodiments, CDRs of antibody light and heavy chain variable regions are grafted into human FR or a common human FR. To generate a common human FR, FR from several human heavy or light chain amino acid sequences are aligned to identify the common amino acid sequence. CDR grafting is described in the following: U.S. patent No. 7,022,500 (Queen); 6,982,321 (Winter); no. 6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); no. 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); no. 5,565,332 (Hoogenboom); no. 5,585,089 (Queen); no. 5,530,101 (Queen); jones et al (1986) NATURE 321:522-525; riechmann et al (1988) NATURE 332:323-327; verhoeyen et al (1988) SCIENCE 239 1534-1536; and Winter (1998) FEBS LETT 430:92-94.
In a type called "SUPERRHUMANIZATION TM In the method of "the human CDR sequences are selected from human germline genes based on the structural similarity of the human CDR to the mouse antibody to be humanized. See, for example, U.S. patent No. 6,881,557 (Foote); and Tan et al, 2002, J.IMMUNOL.169:1119-1125.
Other methods of reducing immunogenicity include "remodeling", "superchimerism" and "veneering/resurfacing". See, e.g., vaswami et al 1998,ANNALS OF ALLERGY,ASTHMA, & IMMUNOL.81:105; roguska et al, 1996,PROT.ENGINEER 9:895-904; and U.S. patent No. 6,072,035 (Hardman). In the veneering/resurfacing method, surface accessible amino acid residues in murine antibodies are substituted with more common amino acid residues at the same positions in human antibodies. Antibody resurfacing of this type is described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).
Another method of converting mouse antibodies into a form suitable for human medical use is known as ACTIVMAB TM Technology (Vaccinex, inc., rochester, NY) involves expression of antibodies in mammalian cells based on vaccinia virus vectors. High levels of IgG heavy and light chain combinatorial diversity can be generated. See, for example, U.S. patent No. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). Another method of converting mouse antibodies into a form suitable for use in humans is the technique commercially practiced by KaloBios Pharmaceuticals, inc (Palo Alto, CA). This technique involves the use of proprietary human "receptor" libraries to generate "epitope-focused" libraries for antibody selection. Another method of modifying mouse antibodies into a form suitable for human medical use is HUMAN ENGINEERING TM Techniques that are commercially practiced by XOMA (US) LLC. See, for example, international (PCT) publication WO 93/11794 and U.S. Pat. No. 5,766,886 (Studnica); 5,770,196 (Studnic ka); 5,821,123 (Studnic ka); and 5,869,619 (Studnic ka).
Antibodies disclosed herein can also be converted to different types, e.g., to human IgG, and the like. By converting antibodies to human antibodies, a human individual should not recognize the antibodies as foreign antibodies. The conversion of non-human IgG antibodies to human IgG antibodies is well known and can be routinely performed after the natural sequences are known. As discussed herein, antibodies can be modified according to known methods. Such methods are described, for example, in: riechmann L, clark M, waldmann H, winter G (1988). Reshaping human antibodies for therapy ". Nature 332 (6162): 332-323; tsurushita N, park M, pakabunto K, ong K, avdalovic a, fu H, jia, V squez M, kumar s. (2004), which is incorporated herein by reference in its entirety. Thus, the antibodies of the invention may be humanized antibodies.
As used herein, the term "fully human antibody" refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine carbohydrate chains if produced in mice, mouse cells, or hybridomas derived from mouse cells.
As used herein, the terms "specifically bind," "immunospecific bind," "immunospecifically bind," or "specifically bind" refer to the binding of an antibody to a predetermined antigen (e.g., plasma kallikrein) or an epitope present on an antigen. In some embodiments, the dissociation constant (K) of the antibody for binding to a predetermined antigen (e.g., plasma kallikrein) D ) Is 10 -7 M or less, and/or K binding to a predetermined antigen D K being bound to a non-specific antigen other than the predetermined antigen (e.g. BSA, casein or another non-specific polypeptide) D At least twice as small. The phrases "antibody that recognizes plasma kallikrein" and "antibody specific for plasma kallikrein" are used interchangeably herein with the term "antibody that immunospecifically binds to plasma kallikrein". In some embodiments, the antibody specifically or preferentially binds plasma kallikrein over other proteins, such as, but not limited to, human prekallikrein. In some embodiments, the antibody or antigen binding fragment of the antibody binds to its antigen (plasma kallikrein) with an affinity that is at least 2-fold greater, at least 10-fold greater, up to 20 times less or at least 100 times greater.
Methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow et al, antibodies: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1988), colligan et al, current Protocols in Immunology, greene Publishing assoc.and Wiley Interscience, n.y. (1992,1993), and Muller, meth. Enzymol.92:589 601 (1983), which are incorporated herein by reference in their entirety.
As used herein, the term "homolog" refers to a protein sequence that has at least 40% but less than 100% sequence homology or identity to a reference sequence. The percent identity between two peptide chains can be determined by a preset setting of the AlignX module using Vector NTI v.9.0.0 (Invitrogen corp., carlsbad, calif.) or by a pairwise alignment using other suitable alignment software (e.g., BLAST). In some embodiments, the antibody or antigen binding fragment thereof has at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology or identity to a sequence described herein. In some embodiments, the antibody has conservative substitutions compared to the sequences described herein. Exemplary conservative substitutions are illustrated in table 1 and are encompassed within the scope of the disclosed subject matter. Conservative substitutions may be present in the framework region or antigen binding site, so long as it does not adversely affect the properties of the antibody. Substitutions may be made to improve antibody properties, such as stability or affinity. Conservative substitutions will result in molecules having similar functional and chemical characteristics as those molecules that undergo such modifications. Exemplary amino acid substitutions are set forth below
Shown in table 1.
In some embodiments, variants of the proteins and peptides provided herein are provided. In some embodiments, the variant comprises a substitution, deletion, or insertion. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) substitutions. As described herein, a substitution may be a conservative substitution. In some embodiments, the substitutions are non-conservative. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) deletions. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) insertions. In some embodiments, substitutions, deletions, or insertions are present in CDRs provided herein. In some embodiments, the substitution, deletion, or insertion is not present in a CDR provided herein.
Typically, a variant antibody or antigen-binding fragment of an antibody provided herein retains at least 10% of its plasma kallikrein binding activity when activity is expressed on a molar basis (when compared to an unmodified or reference antibody, which may be a parent antibody). In some embodiments, a variant antibody (or antigen fragment thereof) or antigen binding fragment of an antibody provided herein retains at least 20%, 50%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or more of the binding affinity of plasma kallikrein as compared to an unmodified or reference antibody, which may be a parent antibody. As described herein, it is also contemplated that an antibody or antigen binding fragment of the invention may include conservative or non-conservative amino acid substitutions, which may also be referred to as "conservative variants" or "functional conservative variants" of the antibody, without substantially altering its biological activity.
As used herein, the term "epitope" refers to a portion of any molecule capable of being recognized by an antibody and bound to one or more antigen binding regions of the antibody. Epitopes are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural features as well as specific charge features. Examples of epitopes include, but are not limited to, residues to which an antibody provided herein binds. The epitope may be a linear or a continuous epitope, i.e.a linear sequence of amino acids of the primary structure of the antigen, such as human plasma kallikrein (SEQ ID NO: 32). Alternatively, in other embodiments, when the antigen assumes its secondary structure, the epitope may be a conformational epitope having a specific three-dimensional shape. For example, a conformational epitope may comprise non-linearities, i.e. non-contiguous amino acids of an antigen.
The antibodies disclosed herein can be inhibitory antibodies, e.g., having high potency, specificity, and prolonged serum residence time, wherein the high potency translates into increased potency at low drug doses, and the high specificity reduces side effects due to inhibition of the associated off-target serine protease.
An antibody or antigen-binding fragment thereof that binds to plasma kallikrein, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence as provided herein or a variant thereof, may comprise one or more of the following features: serum half-life of at least 20 days; k substantially independent of pH between pH 6.0 and 7.4 D (e.g., as determined by SPR in a buffer containing 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca2+, 1mg/mL BSA, 0.02% Tween-20) at 37 ℃; k between 0.1nM and 5nM for human plasma kallikrein D (e.g., as determined by SPR at 37℃in a buffer containing 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca2+, 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and/or a KD of between 0.1nM and 5nM for human plasma kallikrein and a K of between 250nM and 2,000nM for human prekallikrein D (e.g., as determined by SPR at 37℃in a buffer containing 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca2+, 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
In some embodiments, the antibody is a monoclonal antibody that binds to plasma kallikrein. In some embodiments, the antibody binds to an amino acid of an epitope of plasma kallikrein. In some embodiments, the plasma kallikrein is human plasma kallikrein (UNIPROT P03952, KLKB 1) having the following amino acid sequence:
in some embodiments, the plasma kallikrein is cynomolgus monkey plasma kallikrein (UNIPAT A0A2K5VTJ9-1, SEQ ID NO: 45), rabbit plasma kallikrein (UNIPAT G1T127, SEQ ID NO: 46), or rat plasma kallikrein (UNIPAT P14272, SEQ ID NO: 47). For example, the antibody or antigen binding fragment thereof may bind to rabbit, rat or monkey plasma kallikrein.
The human plasma kallikrein sequence shown above includes a signal peptide sequence of amino acid residues 1-19 that can be cleaved during its post-translational processing. In some embodiments, the antibody binds to a human plasma kallikrein that lacks a signal peptide sequence, e.g., mature human plasma kallikrein.
In some embodiments, the antibody comprises an amino acid sequence as provided herein.
The sequence of the antibody may be modified to produce a human IgG antibody. The transformation of the sequences provided herein may be modified to produce other types of antibodies. CDRs may also be linked to other antibodies, proteins or molecules to produce antibody fragments that bind to plasma kallikrein. This may be in the form of an antibody drug conjugate ("ADC") or a multispecific molecule. The sequences may also be made into chimeric antibodies as described herein.
In some embodiments, the antibody comprises an amino acid sequence comprising a sequence provided herein or a fragment thereof. In some embodiments, the antibody comprises one or more amino acid sequences as provided herein, antigen binding fragments thereof, or human IgG variants thereof. "human IgG variant thereof" refers to an antibody that has been modified to human IgG when the starting antibody is not a human IgG antibody.
Antibodies may also be modified to chimeric or human antibodies. Antibodies may also be used in injectable pharmaceutical compositions. As also described herein, the antibody may be an isolated antibody or an engineered antibody.
In some embodiments, there is provided "derivatives" of antibodies, fragments, regions, or derivatives thereof, including those proteins encoded by truncated or modified genes to produce molecular species functionally similar to immunoglobulin fragments. Modifications include, but are not limited to, the addition of gene sequences encoding cytotoxic proteins, such as plant and bacterial toxins. Modifications may also include reporter proteins, such as fluorescent or chemiluminescent tags. Fragments and derivatives may be produced in any manner.
The identification of these antigen binding regions and/or epitopes recognized by the antibodies described herein provides information necessary to generate additional monoclonal antibodies with similar binding characteristics and therapeutic or diagnostic utility in parallel with embodiments of the application.
The variable regions described herein can be combined with any type of constant region, including human constant regions or murine constant regions. Human genes encoding the constant (C) region of antibodies, fragments and regions can be obtained from a library of human fetal livers by known methods. The human C region gene may be derived from any human cell, including those expressing and producing human immunoglobulins. Human C H The regions may be derived from any known class or isotype of the human H chain, including γ, μ, α, δ, or ε, and subtypes thereof, such as G1, G2, G3, and G4. Since the H chain isotype is responsible for various effector functions of antibodies, C H The selection of the region will be guided by the desired effector function, such as complement fixation or activity in Antibody Dependent Cellular Cytotoxicity (ADCC). Preferably C H The region is derived from γ1 (IgG 1), γ3 (IgG 3), γ4 (IgG 4) or μ (IgM). Human C L The region may be derived from human L chain isotypes, kappa or lambda.
In some embodiments, the antibody comprises an Fc domain. In some embodiments, the Fc domain comprises a mutation to extend the half-life of the antibody. In some embodiments, the Fc domain comprises mutations, such as those described in U.S. patent No. 7,670,600, which is incorporated by reference herein in its entirety. In some embodiments, the constant region comprises a mutation at amino acid residue 428 numbered according to the EU numbering index of Kabat relative to the wild-type human IgG constant domain. Without being bound by any particular theory, antibodies comprising mutations corresponding to residue 428 may have increased half-lives compared to the half-life of IgG having a wild-type human IgG constant domain. In some embodiments, the mutation is to replace a natural residue with threonine, leucine, phenylalanine, or serine. In some embodiments, the antibody further comprises one or more amino acid substitutions at one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 429-436, numbered according to the Kabat EU numbering index, relative to the corresponding wild-type human IgG constant domain. Specific mutations or substitutions at these positions are described in U.S. patent No. 7,670,600, which is incorporated herein by reference in its entirety.
Other mutations may be used in the Fc domain, such as those provided in U.S. patent No. 8,394,925, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region is a variant Fc region comprising amino acid substitutions at positions 428 and 434, wherein the amino acid substitutions are leucine that is not a wild-type amino acid at position 428 and serine that is not a wild-type amino acid at position 434, wherein the polypeptide is an antibody and wherein numbering is according to the EU index of Kabat et al. In some embodiments, the Fc region comprises a S228P, L235E, M428L or N434S substitution. In some embodiments, the Fc region comprises an M428L substitution. In some embodiments, the Fc region comprises an N434S substitution. In some embodiments, the Fc region comprises M428L and N434S substitutions, wherein numbering is according to the EU index of Kabat et al. In some embodiments, the Fc region comprises an M252Y, S T and/or T256E substitution or the Fc region comprises an M252Y, S T and T256E substitution, wherein numbering is according to the EU index of Kabat et al.
In some embodiments, the antibodies described herein are used to detect the presence of an antigen. The antibodies of the invention may be used in any device or method to detect the presence of an antigen.
I.A. antibody CDR sequences
The antibodies or antigen binding fragments disclosed herein may be defined by their CDR sequences. For example, an antibody or antigen binding fragment may include one or more of the amino acid sequences provided in table 2, which are CDRs of the antibody or antigen binding fragment defined by Kabat numbering.
The antibody or antigen binding fragment may include one or more of the amino acid sequences provided in table 3, which are CDRs of the antibody or antigen binding fragment defined by Chothia numbering.
The antibody or antigen binding fragment may comprise one or more amino acid sequences provided in table 4, which are CDRs of the antibody or antigen binding fragment defined by IMGT numbering.
The antibody or antigen binding fragment may include one or more of the amino acid sequences provided in table 5, which are CDRs of the antibody or antigen binding fragment defined by AbM numbering.
The antibody or antigen binding fragment thereof may comprise a heavy or light chain CDR having the sequence of SEQ ID No. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31. For example, the light chain CDRs of an antibody or antigen binding fragment thereof may have the sequence of SEQ ID NO. 11, 12, 13, 18, 20, 21, 22, 26, 27 or 28. For example, the heavy chain CDRs of an antibody or antigen binding fragment thereof may have the sequence of SEQ ID NO:14, 15, 16, 17, 19, 23, 24, 25, 29, 30 or 31.
xx
The CDRs described herein can be interchanged with CDRs shown in the above tables that are characterized by different definitions (e.g., chothia and IMGT). For example, LCDR1 defined by Kabat may be interchanged with LCDR1 defined by Chothia, IMGT, or AbM; LCDR2 defined by Kabat is interchangeable with LCDR2 defined by Chothia, IMGT or AbM, for example. For example, in some embodiments, an antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:11, 20, or 27; LCDR2 has the sequence of SEQ ID NO. 12, 18, 21 or 26; and LCDR3 has the sequence of SEQ ID NO. 13, 22 or 28; in some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2 and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO:14, 23, 29 or 43; HCDR2 has the sequence of SEQ ID NO:15, 17, 19, 24, 30 or 44; and HCDR3 has the sequence of SEQ ID NO:16, 25 or 31.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID No. 11, LCDR2 has the sequence of SEQ ID No. 12, and LCDR3 has the sequence of SEQ ID No. 13.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:11, LCDR2 has the sequence of SEQ ID NO:18, and LCDR3 has the sequence of SEQ ID NO: 13.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:20, LCDR2 has the sequence of SEQ ID NO:21, and LCDR3 has the sequence of SEQ ID NO: 22.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:20, LCDR2 has the sequence of SEQ ID NO:26, and LCDR3 has the sequence of SEQ ID NO: 22.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID No. 27, LCDR2 has the sequence of SEQ ID No. 21, and LCDR3 has the sequence of SEQ ID No. 28.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID No. 27, LCDR2 has the sequence of SEQ ID No. 26, and LCDR3 has the sequence of SEQ ID No. 28.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID No. 11, LCDR2 has the sequence of SEQ ID No. 12, and LCDR3 has the sequence of SEQ ID No. 28.
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:11, LCDR2 has the sequence of SEQ ID NO:18, and LCDR3 has the sequence of SEQ ID NO: 28.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 14, HCDR2 has the sequence of SEQ ID No. 15, and HCDR3 has the sequence of SEQ ID No. 16.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 14, HCDR2 has the sequence of SEQ ID No. 17, and HCDR3 has the sequence of SEQ ID No. 16.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 14, HCDR2 has the sequence of SEQ ID No. 19, and HCDR3 has the sequence of SEQ ID No. 16.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 23, HCDR2 has the sequence of SEQ ID No. 24, and HCDR3 has the sequence of SEQ ID No. 25.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 29, HCDR2 has the sequence of SEQ ID No. 30, and HCDR3 has the sequence of SEQ ID No. 31.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 43, HCDR2 has the sequence of SEQ ID No. 44, and HCDR3 has the sequence of SEQ ID No. 25.
The different CDR motifs may be combined in any combination, including those combinations not depicted in the table above. For example, the following embodiments are provided as non-limiting examples of such combinations.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12 or 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 15, 17 or 19; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 15; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 17; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 19; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 15; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 17; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 19; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 16; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO. 21 or 26; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 23; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 24; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO. 21; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 23; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 24; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO. 26; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 23; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 24; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 27; LCDR2 has the amino acid sequence of SEQ ID NO. 21 or 26; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 29; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 30; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 31; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 27; LCDR2 has the amino acid sequence of SEQ ID NO. 21; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 29; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 30; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 31; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 27; LCDR2 has the amino acid sequence of SEQ ID NO. 26; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 29; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 30; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 31; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12 or 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 43; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 44; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 12; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 43; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 44; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO. 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO. 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO. 43; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 44; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 25; or a variant of any of the foregoing.
In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14, having up to 1, 2, 3, or 4 amino acid substitutions thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID No. 15, 17 or 19 having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid substitutions for said amino acid sequence; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16, having up to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions for said amino acid sequence. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12 or 18, with up to 1, 2, 3 or 4 amino acid substitutions to said amino acid sequence; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 13, with up to 1, 2, 3 or 4 amino acid substitutions to said amino acid sequence. Amino acid substitutions may be conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". These variants may be derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, these variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14, 23, 29, or 43, having up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID No. 15, 17, 19, 24, 30 or 44 having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid substitutions for said amino acid sequence; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16, 25 or 31, having up to 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions thereto. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11, 20, or 27 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12, 18, 21 or 26, with up to 1, 2, 3 or 4 amino acid substitutions thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13, 22 or 28 with up to 1, 2, 3, 4, 5 or 6 amino acid substitutions thereto. Amino acid substitutions may be conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". These variants may be derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, these variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region having LCDR1, LCDR2 and LCDR3, wherein LCDR1 has a sequence selected from the group consisting of SEQ ID NO:11, 20 or 27, wherein LCDR1 comprises at most one conservative amino acid substitution, LCDR2 has a sequence selected from the group consisting of SEQ ID NO:12, 18, 21 and 26, wherein LCDR2 comprises at most one conservative amino acid substitution, and LCDR3 has a sequence selected from the group consisting of SEQ ID NO:13, 22 and 28, wherein LCDR3 comprises at most one conservative amino acid substitution, and (ii) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 has a sequence selected from the group consisting of SEQ ID NO:14, 23 or 29, wherein HCDR1 comprises at most one conservative amino acid substitution, HCDR2 has a sequence selected from the group consisting of SEQ ID NO:15, 19, 24 or 30, wherein HCDR2 comprises at most one conservative amino acid substitution, and HCDR3 has a sequence selected from the group consisting of SEQ ID NO:16, 25 or 31, wherein HCDR3 comprises at most one conservative amino acid substitution.
In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region having LCDR1, LCDR2 and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO:11, wherein LCDR1 comprises at most one conservative amino acid substitution, LCDR2 has the sequence of SEQ ID NO:18, wherein LCDR2 comprises at most one conservative amino acid substitution, and LCDR3 has a sequence selected from the group consisting of SEQ ID NO:13, wherein LCDR3 comprises at most one conservative amino acid substitution, and (ii) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO:14, wherein HCDR1 comprises at most one conservative amino acid substitution, HCDR2 has the sequence of SEQ ID NO:19, wherein HCDR2 comprises at most one conservative amino acid substitution, and HCDR3 has the sequence of SEQ ID NO:16, wherein HCDR3 comprises at most one conservative amino acid substitution.
In some embodiments, the light chain variable region CDR1 is replaced with any other light chain CDR1 sequence disclosed herein. In some embodiments, the light chain variable region CDR2 is replaced with any other light chain CDR2 sequence disclosed herein. In some embodiments, the light chain variable region CDR3 is replaced with any other light chain CDR3 sequence disclosed herein. In some embodiments, the heavy chain variable region CDR1 is replaced with any other heavy chain CDR1 sequence disclosed herein. In some embodiments, the heavy chain variable region CDR2 is replaced with any other heavy chain CDR2 sequence disclosed herein. In some embodiments, the heavy chain variable region CDR3 is replaced with any other heavy chain CDR3 sequence disclosed herein.
The antibody or binding fragment thereof of the present invention may have a sequence of HCDR1, HCDR2 and/or HCDR3 corresponding to the sequence of HCDR1, HCDR2 and/or HCDR3 of the heavy chain variable region sequence of SEQ ID No. 1, 3 or 5; and/or LCDR1, LCDR2 and/or LCDR3, the sequence of which corresponds to the sequence of LCDR1, LCDR2 and/or LCDR3 of the light chain variable region sequence of SEQ ID NO. 2, 4 or 6. For example, an antibody or binding fragment thereof of the invention may have HCDR1, HCDR2 and HCDR3, the sequences of which correspond to the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence of SEQ id no 3 or 5; and LCDR1, LCDR2 and LCDR3, the sequences of which correspond to the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region sequences of SEQ ID NO. 4 or 6. For example, an antibody or binding fragment thereof of the invention may have HCDR1, HCDR2 and HCDR3, the sequences of which correspond to the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 5; and LCDR1, LCDR2 and LCDR3, the sequences of which correspond to the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of SEQ ID NO. 6. CDRs can be defined by Kabat, chothia, IMGT or AbM. For example, the CDRs are defined by Kabat. For example, the CDRs are defined by Chothia.
Variable and full-length sequences of i.b. antibodies
In some embodiments, the antibody comprises one or more peptides having the following sequences as provided in table 6, or variants thereof:
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in some embodiments, the antibody or antigen binding fragment comprises a sequence selected from one or more of the following: SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, or variants thereof.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 1, 3 or 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 2, 4 or 6.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 2, 4 or 6.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 2, 4 or 6.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 2, 4 or 6.
In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 2, or the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 1 and a light chain comprising the amino acid sequence of SEQ ID NO. 2.
In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 4, or the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 3 and a light chain comprising the amino acid sequence of SEQ ID NO. 4.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 5, and a light chain comprising the amino acid sequence of SEQ ID NO. 6.
In some embodiments, the antibody or antigen binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5, or the antibody contains a heavy chain comprising the amino acid sequence of SEQ ID NO. 5.
In some embodiments, the antibody or antigen binding fragment contains a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6, or the antibody contains a light chain comprising the amino acid sequence of SEQ ID NO. 6.
In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 1, 3, 5, 7, 9 or 10, and a light chain comprising the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8.
In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 7, 9 or 10, and a light chain comprising the amino acid sequence of SEQ ID NO. 8.
In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 7 and a light chain comprising the amino acid sequence of SEQ ID NO. 8.
In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 9, and a light chain comprising the amino acid sequence of SEQ ID NO. 8.
In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 10, and a light chain comprising the amino acid sequence of SEQ ID NO. 8.
In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID No. 1, 3 or 5.
The antibodies or antigen binding fragments provided herein may comprise a heavy chain variable region comprising the amino acid sequence provided in SEQ ID NOs 1, 3 or 5, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid substitutions. For example, one or more substitutions may be found in only the CDR regions, only the framework regions, or in both the CDR and framework regions. For example, the heavy chain variable region may have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 1, 3 or 5, wherein any variation from SEQ ID NO. 1, 3 or 5 does not occur in HCDR1, HCDR2 or HCDR 3. For example, variations are found in one or more framework regions. In some examples, the amino acid substitution may be (a) a conservative amino acid substitution. The antibodies described in this paragraph are referred to herein as "variants". Such variants may be derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, such variants may not be derived from the sequences provided herein, and may be isolated de novo, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibody or antigen binding fragment comprises a light chain variable region comprising an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
The antibodies or antigen binding fragments provided herein may comprise a light chain variable region comprising the amino acid sequence provided in SEQ ID NOs 2, 4 or 6, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid substitutions. For example, one or more substitutions may be found in only the CDR regions, only the framework regions, or in both the CDR and framework regions. For example, the light chain variable region may have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 2, 4 or 6, wherein any variation from SEQ ID No. 2, 4 or 6 does not occur in LCDR1, LCDR2 or LCDR 3. For example, variations are found in one or more framework regions. In some examples, the amino acid substitution may be (a) a conservative amino acid substitution. The antibodies described in this paragraph are referred to herein as "variants". Such variants may be derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, such variants may not be derived from the sequences provided herein, and may be isolated de novo, e.g., according to the methods provided herein for obtaining antibodies.
The heavy chain variable region (VH) and light chain variable region (VL) sequences may be in any form, including but not limited to scFv forms wherein the VH and VL regions are linked to a peptide linker. Examples of peptide linkers that can be used to link the various peptides provided herein include, but are not limited to: (GGGGS) n (SEQ ID NO:35)；(GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1-5. In some embodiments, the variable regions, e.g., heavy chain variable region and light chain variable region, are not linked to a linker, e.g., a peptide linker, while in other embodiments, the variable regions, e.g., heavy chain variable region and light chain variable region, are linked to a linker, e.g., a peptide linker
In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of IgG, igA, igD, igE and IgM. In some embodiments, the antibody comprises an IgG class and a heavy chain constant region selected from the subclasses IgG1, igG2, igG3, and IgG 4.
In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region comprises a wild-type (WT) sequence. In some embodiments, the IgG1 heavy chain constant region comprises the M252Y, S254T and T256E mutations or the M428L and N434S mutations numbered according to the EU numbering index. In some embodiments, the IgG1 heavy chain constant region comprises M252Y, S T and T256E mutations numbered according to the Kabat EU numbering index. In the heavy chain amino acid sequence disclosed herein as SEQ ID NO. 9, the sequence contains M428L and N434S mutations, the M428L mutation occurring at residue 432 and the N434S mutation occurring at residue 438 of the heavy chain sequence. In the heavy chain amino acid sequence disclosed herein as SEQ ID NO. 10, the sequence contains the M252Y, S T and T256E mutations, the M252Y mutation occurring at residue 256 of the heavy chain sequence, the S254T occurring at residue 258 of the heavy chain sequence, and the T256E occurring at residue 260 of the heavy chain sequence.
In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 10, 9 or 7.
In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 10.
In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 9.
In some embodiments, the antibody or antigen binding fragment thereof comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs 1-10 and 11-19.
In some embodiments, the antibody or antigen binding fragment thereof comprises a sequence or variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID No. 1, 3, 5, 7, 9, or 10, or a variant of any of the foregoing. In some embodiments, the antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID No. 2, 4, 6, or 8, or a variant of any of the foregoing.
In some embodiments, the antibody or antigen binding fragment thereof comprises V having the amino acid sequence of SEQ ID NO. 2, 4, 6 or 8, or any combination thereof L A peptide. V (V) L The peptide may comprise a variant of any of these sequences as provided herein.
In some embodiments, the antibody or antigen binding fragment thereof comprises V having the amino acid sequence of SEQ ID NO. 1, 3, 5, 7, 9 or 10 or any combination thereof H A peptide. V (V) H The peptide may comprise a variant of any of these sequences as provided herein.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 1, 3, 5, 7, 9 or 10, and V L The peptide comprises the sequence of SEQ ID NO 2, 4, 6 or 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises a sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 1, 3, 5, 7, 9 or 10, and V L The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID No. 2, 4, 6 or 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 1, 3, 5, 7, 9 or 10, and V L The peptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 2, 4, 6 or 8, with the proviso that V L The peptide comprises a light chain CDR having the sequence of SEQ ID NO 11, 12, 13, 18, 20, 21, 22, 26, 27 or 28 and V H The peptide comprises a heavy chain CDR having the sequence of SEQ ID NO 14, 15, 16, 17, 19, 23, 24, 25, 29, 30, 31, 43 or 44. In some embodiments, V H Or V L CDRs in the chain are as shown in the combinations provided herein.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 1, 3, 5, 7, 9 or 10, and V L The peptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 2, 4, 6 or 8, with the proviso that V L The peptide comprises LCDR1 having a sequence selected from SEQ ID NO. 11, 20 or 27; LCDR2 having a sequence selected from SEQ ID NOS 12, 18, 21 and 26; and LCDR3 having a sequence selected from SEQ ID NOS 13, 22 and 28; and V is H The peptide comprises HCDR1 having a sequence selected from SEQ ID NOs 14, 23, 29 or 43; HCDR2 having a sequence selected from SEQ ID NOs 15, 17, 19, 24, 30 or 44; and HCDR3 having a sequence selected from SEQ ID NO. 16, 25 or 31.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises a sequence identical to SEQ ID NO1, 3, 5, 7, 9 or 10, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and V L The peptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 2, 4, 6 or 8, with the proviso that V L The peptide comprises an LCDR1 having a sequence selected from the group consisting of SEQ ID NOs 11, 20 or 27 wherein LCDR1 comprises at most one conservative amino acid substitution; LCDR2 having a sequence selected from the group consisting of SEQ ID NOs 12, 18, 21 and 26 wherein LCDR2 comprises at most one conservative amino acid substitution; and LCDR3 having a sequence selected from the group consisting of SEQ ID NOs 13, 22 and 28, wherein LCDR3 comprises at most one conservative amino acid substitution; and V is H The peptide comprises an HCDR1 having a sequence selected from SEQ ID NOs 14, 23, 29 or 43 wherein HCDR1 comprises at most one conservative amino acid substitution; HCDR2 having a sequence selected from SEQ ID NOs 15, 17, 19, 24, 30 or 44 wherein HCDR2 comprises at most one conservative amino acid substitution; and HCDR3 having a sequence selected from SEQ ID NO. 16, 25 or 31, wherein HCDR3 comprises at most one conservative amino acid substitution.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 1, and V L The peptide comprises the sequence of SEQ ID NO. 2.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 1, and V L The peptide comprises the sequence of SEQ ID NO. 4, 6 or 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 3, and V L The peptide comprises the sequence of SEQ ID NO. 4.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 3, and V L The peptide comprises SEQ ID NO:2. 6 or 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 5, and V L The peptide comprises the sequence of SEQ ID NO. 6.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 5, and V L The peptide comprises the sequence of SEQ ID NO. 2, 4 or 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 7, and V L The peptide comprises the sequence of SEQ ID NO. 6.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 7, and V L The peptide comprises the sequence of SEQ ID NO. 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO. 7, and V L The peptide comprises the sequence of SEQ ID NO. 2 or 4.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 9, and V L The peptide comprises the sequence of SEQ ID NO. 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 9, and V L The peptide comprises the sequence of SEQ ID NO. 6.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 9, and V L The peptide comprises the sequence of SEQ ID NO. 2 or 4.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 10, and V L The peptide comprises the sequence of SEQ ID NO. 8.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 10, and V L The peptide comprises the sequence of SEQ ID NO. 6.
In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Peptides and V L Peptides, wherein V H The peptide comprises the sequence of SEQ ID NO 10, and V L The peptide comprises the sequence of SEQ ID NO. 2 or 4.
In addition to these specific combinations, any V disclosed herein H The peptide may be in association with any V disclosed herein L Peptide combinations.
As provided herein, the different peptides (V H Or V L ) May be attached to the peptide linker or not, but rather for a continuous sequence. In some embodiments, the peptide linker comprises (GGGGS) n (SEQ ID NO:35)；(GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1-5. The linked peptide form may be represented by formula V H -Z-V L Or V L -Z-V H And wherein Z is a peptide linker. In some embodiments, Z is (GGGGS) n (SEQ ID NO:35)；(GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1-5.
Antibody epitope binding
In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues K550 or R551 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues K585 or S597 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, or S597 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues Y617 or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues Y617, M618, D619, W620, I621, L622, E623, K624, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues K550, R551, K585, S597, Y617, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that comprises at least one of residues K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least one of the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to at least two of the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to at least three of the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to at least four of the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to at least five of the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to the following residues: the human plasma kallikrein shown in SEQ ID NO. 32, K550, R551, K585, S597, Y617 or T625.
In some embodiments, the antibody or antigen binding fragment thereof binds to at least one of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, ten three, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, or twenty-four of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least two of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least three of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least four of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least five of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least six of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least seven of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least eight of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least nine of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least ten of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least eleven of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least ten of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least ten of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least fourteen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least fifteen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least sixteen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least seventeen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least eighteen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least nineteen of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least twenty of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least twenty-one of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least twenty of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least twenty three of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least twenty-four of the following residues: k550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624 or T625 of wild type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to at least K550 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least R551 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least K585 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least H586 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least N587 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least G588 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least M589 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least W590 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least R591 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least L592 of wild type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least V593 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least G594 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least I595 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least T596 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least S597 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least Y617 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least M618 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least D619 of wild type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least W620 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least I621 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least L622 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least E623 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least K624 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen binding fragment thereof binds to at least T625 of wild-type human plasma kallikrein (SEQ ID NO: 32).
In some embodiments, the antibody or antigen binding fragment thereof binds to a linear epitope of human plasma kallikrein. For example, the linear epitope has the sequence of SEQ ID NO: 37. 38 or 39. The antibody or antigen binding fragment thereof may bind to one, two or three of these linear epitopes. In further embodiments, the antibody or antigen binding fragment does not bind to a linear epitope having the sequence of SEQ ID NO. 40, 41 and/or 42, and/or any amino acid residues of the amino acid sequence of SEQ ID NO. 40, 41 and/or 42.
Antibody conjugates
The antibodies provided herein may also bind to chemical moieties. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor. The chemical moiety may be an MRI detectable label. In some embodiments, this may be referred to as an antibody drug conjugate. In some embodiments, the chemical moiety is a polymer that increases the half-life of the antibody molecule in the individual. Suitable polymers include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2kDa, 5kDa, 10kDa, 12kDa, 20kDa, 30kDa, or 40 kDa), dextran, and monomethoxy polyethylene glycol (mPEG). Lee et al, (1999) (bioconj. Chem. 10:973-981) disclose PEG-conjugated single chain antibodies. Wen et al, (2001) (bioconoj. Chem. 12:545-553) disclose the conjugation of antibodies to PEG linked to a radioactive metal chelator (diethylenetriamine pentaacetic acid (DTPA)). Examples of chemical moieties include, but are not limited to, antimitotic agents such as calicheamicin (e.g., ozagrimocin), monomethyl auristatin E, maytansine, and the like. Other examples include, but are not limited to, bioactive anti-microtubule agents, alkylating agents, and DNA minor groove binding agents. The chemical moiety may be linked to the antibody via a linking group (maleimide), a cleavable linker (e.g., a cathepsin cleavable linker (valine-citrulline)), and in some embodiments via one or more spacers (e.g., p-aminobenzyl carbamate).
The antibodies and antibody fragments of the invention may also be conjugated to a label, such as 99 Tc、 90 Y、 111 In、 32 P、 14 C、 125 I、 3 H、 131 I、 11 C、 15 O、 13 N、 18 F、 35 S、 51 Cr、 57 To、 226 Ra、 60 Co、 59 Fe、 57 Se、 152 Eu、 67 CU、 217 Ci、 211 At、 212 Pb、 47 Sc、 109 Pd、 234 Th and 40 K、 157 Gd、 55 Mn、 52 tr and Tr 56 Fe。
The antibodies and antibody fragments may also be conjugated to fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, luciferin and derivatives thereof, rhodamine and derivatives thereof, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and combinations thereof, 152 Eu, dansyl, umbelliferone, fluorescein, luminal, iso-luminal, aromatic acridinium ester, imidazole, acridinium salt, oxalate, aequorin, 2, 3-dihydrophthalazindione, biotin/avidin, spin-labeling and stable free radicals.
Antibody molecules may also bind to cytotoxic factors such as diphtheria toxin, pseudomonas exotoxin a chain, ricin a chain, abrin a chain, pristimerin a chain, alpha-fumagillin, aleurone and compounds (e.g., fatty acids), caryophyllin protein, pokeweed (Phytoiacca americana) protein PAPI, PAPII and PAP-S, balsam pear inhibitors, jatrophin (curcin), crotonin (crotin), soapbark inhibitors, mitogen (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin) and enomycin (enomycin).
Any method known in the art for binding the antibody molecules of the invention to various moieties may be employed, including those methods described by: hunter et al, (1962) Nature144:945; david et al, (1974) Biochemistry 13:1014; pain et al, (1981) J.Immunol. Meth.40:219; and Nygren, J. (1982) Histochem. And Cytochem.30:407. Methods for binding antibodies are conventional and well known in the art.
In some embodiments, provided herein are antibodies (e.g., anti-plasma kallikrein antibodies). In some embodiments, the antibody is a recombinant antibody that binds to plasma kallikrein. In some embodiments, the plasma kallikrein protein is a human plasma kallikrein protein. In some embodiments, the antibody does not specifically bind to prekallikrein protein. As used herein, the term "recombinant antibody" refers to an antibody that does not occur in nature. In some embodiments, the term "recombinant antibody" refers to an antibody that is not isolated from a human individual.
Nucleic acids and methods of production
The invention also encompasses nucleic acids encoding each antibody or antigen binding fragment disclosed herein, including nucleic acids encoding the heavy or light chain or heavy or light chain variable region of such antibodies disclosed herein. Thus, in some embodiments, nucleic acid molecules encoding antibodies or fragments disclosed herein are provided. In some embodiments, the nucleic acid encodes an amino acid sequence of an antibody or antigen binding fragment thereof, or a heavy or light chain variable region provided herein.
For example, in one embodiment, the nucleic acid encodes the heavy chain variable region of any one of SEQ ID NOs 1, 3 or 5, while in another embodiment, the nucleic acid encodes the light chain variable region of any one of SEQ ID NOs 2, 4 or 6. In certain embodiments, the nucleic acid encodes the heavy chain sequence of SEQ ID NO. 7, 9 or 10, while in another embodiment, the nucleic acid encodes the light chain sequence of SEQ ID NO. 8.
In one embodiment, the nucleic acid encodes a heavy chain or heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO 14, 23, 29 or 43; HCDR2 having the amino acid sequence of SEQ ID NO 15, 17, 19, 24, 30 or 44; and HCDR3 having the amino acid sequence of SEQ ID NO. 16, 25 or 31.
In another embodiment, the nucleic acid encodes a light chain or light chain variable region comprising LCDR1 having the amino acid sequence of SEQ ID No. 11, 20 or 27; LCDR2 having the amino acid sequence of SEQ ID NO 12, 18, 21 or 26; and LCDR3 having the amino acid sequence of SEQ ID NO. 13, 22 or 28.
In another embodiment, an antibody of the invention comprises a heavy chain variable region encoded by the nucleic acid sequence of SEQ ID NO. 33 and a light chain variable region encoded by the nucleic acid sequence of SEQ ID NO. 34. The antibodies of the invention may also comprise a heavy chain encoded by the nucleic acid sequence of SEQ ID NO. 48 and a light chain encoded by the nucleic acid sequence of SEQ ID NO. 49.
The production of antibodies having known sequences, as described herein, is conventional and can be performed by any method. Methods for producing antibodies, such as those disclosed herein, are known in the art. Once the sequence is known, antibodies can also be generated according to known methods, including according to the examples provided herein.
For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be linked to other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. The production of defined genetic constructs is within the skill of the art. Alternatively, the sequences provided herein can be cloned from hybridomas by conventional hybridization techniques or Polymerase Chain Reaction (PCR) techniques using synthetic nucleic acid probes whose sequences are based on the sequence information provided herein, or prior art sequence information on genes encoding the heavy and light chains of murine antibodies in hybridoma cells.
The nucleic acid encoding the desired antibody may be incorporated (linked) into an expression vector which may be introduced into a host cell via conventional transfection or transformation techniques. Exemplary host cells are e.coli cells, chinese Hamster Ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), and myeloma cells that do not otherwise produce IgG proteins. The transformed host cell may be grown under conditions that allow the host cell to express genes encoding immunoglobulin light chain variable regions and/or heavy chain variable regions. Thus, the invention provides vectors comprising nucleic acids encoding antibodies or fragments thereof of the invention, as well as cells comprising such nucleic acids or vectors.
The specific expression and purification conditions will vary depending on the expression system used. For example, if the gene is to be expressed in E.coli, the gene is first cloned into an expression vector by positioning the engineered gene downstream of the appropriate bacterial promoter (e.g.Trp or Tac) and prokaryotic signal sequences. Expressed secreted proteins accumulate in refractile bodies or inclusion bodies and can be collected after disruption of cells by French press or sonication. The refractile bodies are then solubilized and the protein refolded and cleaved by methods known in the art.
If the engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing the appropriate eukaryotic promoter, secretion signal, poly A sequence and stop codon. Optionally, the vector or gene construct may contain enhancers and introns. The expression vector optionally contains sequences encoding all or part of the constant region, enabling expression of all or part of the heavy or light chain. The gene construct may be introduced into eukaryotic host cells using conventional techniques. Host cell expression V L Or V H Fragment, V L -V H Heterodimers, V H -V L Or V L -V H A single chain polypeptide, an intact immunoglobulin heavy or light chain, or a portion thereof, each of which may be linked to a portion having another function (e.g., cytotoxicity). In some embodiments, the host cell is transfected with a single vector expressing a polypeptide that expresses all or part of the heavy chain [ ] For example, a heavy chain variable region) or a light chain (e.g., a light chain variable region). In some embodiments, the host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In some embodiments, the host cell is co-transfected with more than one expression vector (e.g., one expression vector that expresses a polypeptide comprising all or part of a heavy chain or heavy chain variable region, and another expression vector that expresses a polypeptide comprising all or part of a light chain or light chain variable region).
Polypeptides comprising immunoglobulin heavy chain variable or light chain variable regions may be produced by growing (culturing) host cells transfected with expression vectors encoding such variable regions under conditions permitting expression of the polypeptide. After expression, the polypeptide may be collected and purified or isolated using techniques known in the art, such as an affinity tag, e.g., glutathione-S-transferase (GST) or histidine tag.
Monoclonal antibodies that bind to plasma kallikrein or an antigen binding fragment of an antibody can be produced by growing (culturing) host cells transfected with: (a) An expression vector encoding a complete or partial immunoglobulin heavy chain, and a separate expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector encoding both chains (e.g., whole or partial heavy and light chains) under conditions that allow expression of both chains. The whole antibody (or antigen binding fragment) may be collected and purified or isolated using techniques known in the art, such as protein a, protein G, an affinity tag, e.g., glutathione-S-transferase (GST) or a histidine tag. It is within the ordinary skill in the art to express the heavy and light chains from a single expression vector or from two separate expression vectors.
The nucleic acid sequence encoding an antibody described herein may be genomic DNA or cDNA, or RNA (e.g., mRNA) encoding at least one of the variable regions described herein. A convenient alternative to using chromosomal gene segments as a source of DNA encoding the V region antigen binding fragments is to construct chimeric immunoglobulin genes using cDNA, for example, as reported in Liu et al (Proc.Natl.Acad.Sci., USA 84:3439 (1987) and J.immunology 139:3521 (1987), which references are hereby incorporated by reference in their entirety.
For example, a cDNA encoding a V region antigen binding fragment capable of detecting, binding to, or neutralizing plasma kallikrein may be provided using known methods based on the use of the amino acid sequences provided herein. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson et al, infra). Using the genetic code, one or more different oligonucleotides can be identified, each of which will be capable of encoding an amino acid. The probability that a particular oligonucleotide will actually constitute an actual XXX coding sequence can be estimated by taking into account the abnormal base pairing relationships and the frequency with which particular codons (encoding particular amino acids) are actually used in eukaryotic or prokaryotic cells expressing the antibody or fragment. Such "rules of codon usage" are disclosed by Lathes et al, J.molecular.biol. 183:1.12 (1985). Using the "codon usage rule" of Lathes, a single oligonucleotide or set of oligonucleotides containing a theoretical "most likely" nucleotide sequence capable of encoding an antibody variable or constant region sequence was identified.
V. pharmaceutical compositions and administration thereof
The plasma kallikrein binding proteins, including antibodies and antigen binding fragments thereof, and including nucleic acid molecules encoding the same, disclosed herein can be formulated as pharmaceutical compositions, e.g., for administration to an individual.
As used herein, the term "pharmaceutical composition" refers to a combination of one or more active agents (e.g., antibodies or antigen binding fragments as described herein) and a carrier (inert or active) suitable for diagnostic or therapeutic use in vivo or ex vivo.
Pharmaceutical compositions containing antibodies, fragments thereof, or nucleic acids encoding such antibodies as disclosed herein may be presented in dosage unit form and may be prepared by any suitable method. In some embodiments, to prepare a pharmaceutical or sterile composition of an anti-plasma kallikrein antibody or other protein provided herein, the antibody or antigen binding fragment thereof or other protein provided herein is admixed with a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, for example, remington's Pharmaceutical Sciences, 18 th edition (Mack Publishing Company, 1990).
As used herein, the phrases "pharmaceutically acceptable" and "pharmacologically acceptable" refer to compounds, molecular entities, compositions, materials, and/or dosage forms that do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. For human administration, the formulation should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA office of biological standards. "pharmaceutically acceptable" and "pharmacologically acceptable" may mean approved by or by a regulatory agency of the federal or a state government or a corresponding agency in a country other than the united states, or listed in the united states pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
As used herein, "carrier" refers to a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting an agent, such as an antibody or binding fragment thereof, from one organ or portion of the body to another organ or portion of the body.
As used herein, "pharmaceutically acceptable excipient" refers to the following: to aid in the administration of active agents, such as antibodies or binding fragments thereof, and/or to be absorbed by an individual and may be included in the compositions of the present invention without significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, naCl, physiological saline solution (e.g., phosphate buffered saline solution), emulsion (e.g., oil/water or water/oil emulsion), ringer's lactate, common sucrose, common dextrose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, salt solutions (e.g., ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, dextrose, amylose or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments, and the like. Such formulations may be sterilized and, if desired, mixed with adjuvants which do not react deleteriously with the compositions of the invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants and/or fragrance materials, and the like. For examples of excipients see Martin, remington' sPharmaceutical Sciences, 15 th edition, mack publication co., easton, PA (1975).
Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and agents for modulating tonicity, such as sodium chloride or dextrose.
For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor ELTM (BASF, parsippany, N.J.), phosphate Buffered Saline (PBS), or dextrose solution. The carrier should be stable under the conditions of manufacture and storage and should be preserved from microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
The pharmaceutical formulation is preferably sterile. Sterilization may be accomplished, for example, by filtration through sterile filtration membranes. In the case where the composition is lyophilized, filter sterilization may be performed either before or after lyophilization and reconstitution.
Formulations of antibodies provided herein can be prepared by mixing with an acceptable carrier, excipient, or stabilizer in the form of, for example, a lyophilized powder, slurry, aqueous solution, or suspension (see, e.g., hardman et al (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, mcGraw-Hill, new York, N.Y., gennaro (2000) Remington: the Science and Practice of Pharmacy, lippincott, williams, and Wilkins, new York, N.Y., avis et al (edit) (1993) Pharmaceutical Dosage Forms: parenteral Medications, marcel Dekker, N.Y., lieberman et al (edit) (1990) Pharmaceutical Dosage Forms:Tablets, marcel Dekker, N.Y., lieberman et al (edit) (1990) Pharmaceutical Dosage Forms:perse Systems, marcel Dekker, N.Y., iner and Kotkoskie (2000) Excipient Toxicity and Safety, ink, new York, N.Y.). In some embodiments, the antibody is diluted to an appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added to enhance tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.
Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD 50 (dose lethal to 50% of the population) and ED 50 (a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 /ED 50 ). In particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably in the circulating concentration range, including ED 50 And has little or no toxicity. The dosage may vary within this range depending upon the dosage form used and the route of administration.
The antibody compositions of the invention may be administered to an individual, for example, according to Physics' Desk Reference 2003 (Thomson Healthcare; 57 th edition (11/1/2002)).
The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal or intraarterial.
In some embodiments, the antibody or antigen binding fragment thereof may be administered by an invasive route, e.g., by injection. In some embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra-articular (e.g., in an arthritic joint), intratumorally, or by inhalation, aerosol delivery. Administration by a non-invasive route (e.g., orally; e.g., in a pill, capsule, or tablet) is also within the scope of embodiments of the present invention. Administration by intravenous infusion is one way in which antibodies may be administered to an individual.
In some embodiments, the anti-plasma kallikrein antibody, or antigen binding fragment thereof, is administered in combination with at least one additional therapeutic agent, such as, but not limited to, any therapeutic agent for treating the disorders provided herein. In some embodiments, the antibody is administered in combination with another treatment for a disorder provided herein. For example, the anti-plasma kallikrein antibodies of the invention may be administered with one or more additional therapies for treating HAE, e.g.(C1 esterase inhibitor),>(atibant) injection, (Ai Kala peptide) and +.>(C1 esterase inhibitor, contestat alfa) and->(C1 esterase inhibitor),>(C1 esterase inhibitor subcutaneously),>(ranadlumab-flyback)), ORLADEYO TM (Bei Luosi he (berotralstat)), garacimab (garadalimab), doridazosen (donidarsosen), synthetic 17-alpha-alkylated androgens, such as danazol (danazol) and sudazol (stanozolol), and/or antifibrinolytic agents, such as aminocaproic acid. The anti-plasma kallikrein antibody may be administered concurrently with one or more of the aforementioned additional therapies, e.g., it may be co-administered. The anti-plasma kallikrein antibody may be administered sequentially before or after one of the foregoing therapies, e.g., on a different day or a different week or even a different month than any of the foregoing additional therapies.
The composition may be administered with medical devices known in the art. For example, the pharmaceutical compositions of the present invention may be administered by injection with a hypodermic needle (including, for example, a pre-filled syringe or an auto-injector).
The pharmaceutical composition may also be administered with a needleless subcutaneous injection device; for example, U.S. patent No. 6,620,135; no. 6,096,002; no. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; devices disclosed in 4,790,824 or 4,596,556.
The pharmaceutical composition may also be administered by infusion. Examples of well known implants and modules for administration of pharmaceutical compositions include: U.S. patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196 discloses an osmotic drug delivery system having multiple compartment compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art. Infusion may also be performed by intravenous delivery of the antibody in a pharmaceutically acceptable carrier (e.g., physiological saline) over a prescribed period of time. Intravenous delivery of the pharmaceutical composition may be via a port implanted in a vein or artery of the individual.
Alternatively, the antibody may be administered locally rather than systemically, e.g., via injection or in a depot or sustained release formulation. Furthermore, the antibodies may be administered in a targeted drug delivery system, for example, in liposomes coated with tissue specific antibodies.
The administration regimen will depend on several factors including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the dosing regimen delivers sufficient therapeutic antibody to achieve an improvement in the targeted disease state while minimizing undesirable side effects. Thus, the amount of biologic delivered will depend in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance for the selection of appropriate doses of therapeutic antibodies is available (see, e.g., wawrzynczak (1996) anti-body Therapy, bio Scientific Pub. Ltd, oxfordshire, UK; kresina (eds.) (1991) Monoclonal Antibodies, cytokines and Arthritis, marcel Dekker, new York, NY; bach (eds.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, marcel Dekker, new York, NY; baert et al (2003) New Engl. J. Med.348:601-608; milgom et al (1999) New Engl. J. Med.341:1966-1973; slamon et al (2001) New Engl. J. Med.344:783-792; beninaminovitz et al (2000) New Engl. J. 613: ggl-343-35; med. 348:348-46).
The appropriate dosage may be determined by a clinician, for example, using parameters or factors known or suspected in the art to affect treatment. Typically, the dose starts at a slightly smaller amount than the optimal dose and then increases in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic indicators include, for example, inflammatory symptoms or the levels of inflammatory cytokines produced. In general, it is desirable to use a biological agent from the same species as the target animal being treated, thereby minimizing any immune response to the agent. For example, in the case of human individuals, chimeric, humanized and fully human antibodies may be required.
The antibody or antigen binding fragment thereof may be provided by continuous infusion or by a dose administered, for example, daily, 1-7 times per week, weekly, biweekly, monthly, bi-monthly, or quarterly. In some embodiments, the total weekly dose is typically at least 0.05 μg/kg body weight, more typically at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25mg/kg, 1.0mg/kg, 2.0mg/kg, 5.0mg/ml, 10mg/kg, 25mg/kg, 50mg/kg or more (see, e.g., yang et al (2003) New Engl.J. Med.349:427-434; herld et al (2002) New Engl.J. Med.346:1692-1698; liu et al (1999) J. Neurol. Neurosurg. Psych.67:451-456; portilji et al (2003) Cancer cell. Immunol. 52:133-144). Dosages may also be provided to achieve a predetermined target concentration of antibodies in the serum of an individual, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, the antibody is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, 1500, 2000, or 2500 milligrams per individual per week, every two weeks, "every 4 weeks," monthly, every two months, or quarterly.
VI therapeutic methods and medical uses
The plasma kallikrein binding proteins, including antibodies and antigen binding fragments thereof, disclosed herein are useful for treating a disease in an individual.
As used herein, the term "treatment" or "treatment" includes inhibiting or delaying the progression of symptoms associated with a disorder and/or lessening the severity of symptoms of such a disorder. The term further encompasses ameliorating existing uncontrolled or undesired symptoms, preventing additional symptoms, and ameliorating or preventing the root cause of such symptoms.
As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody or antigen-binding fragment thereof that, when administered alone or in combination with additional therapeutic agents to a cell, tissue, or individual, is effective to cause a measurable improvement in one or more symptoms of a disease or disorder or progression of such disease or disorder, such as HAE or a known plasma kallikrein causing a disorder of the observed pathology. A therapeutically effective dose further refers to an amount of antibody or binding fragment thereof sufficient to at least partially ameliorate symptoms, e.g., treat, cure, prevent or ameliorate a related medical condition, e.g., a condition in which HAE or known plasma kallikrein causes an observed pathology, or increase the rate at which such condition is treated, cured, prevented or ameliorated. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to the ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of the therapeutic agent will increase the diagnostic index or parameter by at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. In cases where subjective measures are used to assess disease severity, an effective amount may also result in an improvement in the subjective measures. In some embodiments, the amount is a therapeutically effective amount if the amount is an amount useful for treating or preventing HAE symptoms or conditions where plasma kallikrein is known to cause observed pathology. For example, a therapeutically effective amount of an antibody or binding fragment thereof of the invention may reduce the frequency of HAE episodes, or may reduce the severity of HAE episodes, such as the extent of edema experienced by an individual during an episode.
As used herein, the term "individual" refers to any organism, such as an animal, including mammals (e.g., rats, mice, dogs, cats, rabbits) and, for example, humans. In one embodiment, the subject is a human. An individual may also be referred to as a patient. In some embodiments, the subject is in need thereof. An "individual in need" refers to an individual who has been identified as in need of treatment for the disorder to be treated and who is treated with a particular intent to treat such disorder. The disorder may be, for example, any disorder described herein. "individual" is used interchangeably with "patient".
In some embodiments, the method comprises administering a therapeutically or prophylactically effective amount of one or more antibodies or antigen-binding fragments of antibodies described herein, or a pharmaceutical composition comprising the therapeutically effective amount, to a susceptible individual or an individual exhibiting a condition in which plasma kallikrein is known to cause an observed pathology. Antibodies in any active form may be administered, including but not limited to scFv, fab and F (ab') 2 Fragments and other forms of antibodies provided herein。
As used herein, "plasma kallikrein-related pathology" refers to a disorder caused by the function or abnormal expression of plasma kallikrein. These disorders include, but are not limited to, hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post angioplasty), systemic lupus nephritis, and burns. In some embodiments, the pathology is hereditary angioedema. In some embodiments, the pathology is bradykinin dependent edema.
In some embodiments, the antibodies provided herein are used to treat an individual having or suspected of having a plasma kallikrein-related disorder. The plasma kallikrein related disorder is selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post angioplasty), systemic lupus erythematosus nephritis, and burns.
In some embodiments, the plasma kallikrein binding protein reduces abnormal coagulation associated with a contact activation system (i.e., an intrinsic activation system) by at least 10%, as measured by, for example, an APTT clotting assay (e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no detectable abnormal coagulation)).
In some embodiments, the antibodies provided herein are useful for treating a disorder described herein, such as hereditary angioedema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn; or promote wound healing. In some embodiments, the disorder is hereditary angioedema or bradykinin-dependent edema. In some embodiments, the disorder is hereditary angioedema.
In some embodiments, the antibodies provided herein are used in the manufacture of a medicament for treating a disorder described herein, such as hereditary angioedema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or peri-tumor cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn; or for the manufacture of a medicament for promoting wound healing.
Treatment of an individual may comprise administering a therapeutically effective amount of an antibody or antigen-binding fragment described herein, or a pharmaceutical composition comprising such an antibody or fragment. Antibodies can be provided in kits, such as those provided herein. The antibody may be used or administered alone or in combination with another therapeutic, analgesic, or diagnostic agent (e.g., as provided herein). In providing an antibody or fragment thereof capable of binding to plasma kallikrein, or an antibody capable of protecting a recipient patient from plasma kallikrein, to a patient, the dosage of the administered agent will vary depending on factors such as the patient's age, weight, height, sex, general health, past medical history, and the like.
Antibodies capable of treating a condition associated with plasma kallikrein activity or for treating a plasma kallikrein-related pathology are intended to be provided to an individual in an amount sufficient to affect a reduction, regression or improvement of a plasma kallikrein-related symptom or pathology. Such pathologies include, but are not limited to, hereditary angioedema, bradykinin-dependent edema, hereditary angioedema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, intestinal tract disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, and burns. For example, the pathology is Hereditary Angioedema (HAE).
Thus, in some embodiments, methods of treating an individual having a plasma kallikrein-mediated disorder are provided. In some embodiments, the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof as provided herein. In some embodiments, the disorder is hereditary angioedema, bradykinin-dependent edema, hereditary angioedema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or peri-tumor cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, and burns. In some embodiments, the disorder is hereditary angioedema. In some embodiments, the disorder is bradykinin dependent edema.
Also disclosed herein are methods of reducing or inhibiting plasma kallikrein activity in an individual in need thereof. For example, an antibody or binding fragment thereof disclosed herein can be administered to an individual in a therapeutically effective amount to reduce or inhibit the activity of plasma kallikrein, e.g., human plasma kallikrein, in the individual. The individual may have a plasma kallikrein related disorder as disclosed herein, such as HAE. In some embodiments, plasma kallikrein activity may be reduced or inhibited in an amount sufficient to prevent HAE onset in an individual for at least one month, two months, three months, four months, five months, six months, or more.
Also disclosed herein are methods of reducing or inhibiting bradykinin production or activity in an individual in need thereof. For example, an antibody or binding fragment disclosed herein can be administered to an individual in a therapeutically effective amount to reduce the amount or activity of bradykinin in the individual. The individual may have a plasma kallikrein related disorder as disclosed herein, such as HAE. In some embodiments, bradykinin production or activity may be reduced or inhibited by an amount that prevents HAE onset in an individual for at least one month, two months, three months, four months, five months, six months, or more.
As provided herein, the antibodies or antigen binding fragments thereof can be combined with other therapeutic agentsTogether, the other therapeutic agents may include one or more additional therapies for treating HAE, e.g.(C1 esterase inhibitor),(Aitiabante injection)>(Ai Kala peptide) and->(C1 esterase inhibitor, kanster Alfa)>(C1 esterase inhibitor),>(C1 esterase inhibitor subcutaneously),>(ranaduzumab), ORLADEYO TM (Bei Luosi he), garacimab, doridalosen, synthetic 17-alpha-alkylated androgens, such as danazol and sudole, and/or antifibrinolytic agents, such as aminocaproic acid. The additional therapies may be administered simultaneously or sequentially with the antibodies disclosed herein.
Kits useful for practicing embodiments described herein are also provided. The kit of the invention may comprise a first container containing or packaged with the above-described antibodies. The kit may also comprise another container containing or packaged with a solution necessary or convenient for implementing the embodiment. The container may be made of glass, plastic or foil and may be a vial, bottle, pouch, tube, bag, or the like. The kit may also contain written information, such as a program for practicing the embodiment or analytical information, such as the amount of reagent contained in the first container means. The container may be in another container means, such as a box or bag, together with the written information.
In some embodiments, antibodies that bind to plasma kallikrein are provided. In some embodiments, the antibody is isolated. In some embodiments, the antibody specifically binds to plasma kallikrein.
In some embodiments, the antibody inhibits or neutralizes the function of a plasma kallikrein protein, e.g., human plasma kallikrein. As used herein, the term "neutralizing" means that the activity or function of a protein is inhibited. Inhibition may be complete or partial. In some embodiments, the activity or function of the protein is inhibited by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%. The percent inhibition may be based on the function or activity of the protein in the absence of antibody. In some embodiments, the antibody inhibits a clotting function promoted by plasma kallikrein. Accordingly, the present invention provides methods of modulating plasma kallikrein activity, e.g., contacting plasma kallikrein with a plasma kallikrein antibody disclosed herein or a pharmaceutical composition comprising the antibody, to modulate the activity of plasma kallikrein, e.g., human plasma kallikrein. In some embodiments, the methods comprise administering an antibody provided herein or a pharmaceutical composition comprising the antibody to an individual to modulate plasma kallikrein activity in the individual.
The article "a/an" is used in this disclosure to refer to one or more of the grammatical objects of the article (i.e., at least one) unless the context is inappropriate. For example, "an element" means one element or more than one element.
The term "and/or" is used in the present invention to mean "and" or "unless otherwise indicated. Unless otherwise understood from the context, the expression "and/or" in relation to three or more enumerated objects is to be understood to have the same meaning.
It should be understood that the expression "at least one" includes each recited object as well as various combinations of two or more recited objects after the expression unless otherwise understood from the context and use.
Unless otherwise specifically stated or understood from the context, the terms "include" and "having" and "contain" are generally understood to be open-ended and non-limiting, e.g., not to exclude other non-recited elements or steps.
The invention also includes the specific quantitative values themselves when the term "about" is used before a quantitative value unless explicitly stated otherwise. As used herein, the term "about" refers to a ± 5% change from nominal unless otherwise indicated or inferred from context.
As used herein, the term "combination" means that the agents can be administered to an animal or individual together in a mixture, simultaneously as a single agent, or sequentially in any order as a single agent.
Unless otherwise indicated or understood from the context, where molecular weight is provided instead of, for example, absolute value of the polymer, molecular weight is understood to be average molecular weight.
The use of any and all examples, or exemplary language, such as "e.g." comprising "or" including "is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
It should be understood that the order of steps or order of performing certain actions is immaterial so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.
Throughout the description, where compositions are described as having, comprising, or containing specific components, or where processes and methods are described as having, comprising, or containing specific steps, it is further contemplated that compositions of the present invention consisting essentially of, or consisting of, the recited components are present, and that processes and methods according to the present invention consist essentially of, or consist of, the recited processing steps are present.
In the present application, when an element or component is referred to as being included in and/or selected from a list of enumerated elements or components, it should be understood that the element or component may be any of the enumerated elements or components, or the element or component may be selected from the group consisting of two or more of the enumerated elements or components.
Furthermore, it is to be understood that the elements and/or features of the compositions and methods described herein may be combined in various ways without departing from the spirit and scope of the application, whether explicit or implicit herein. For example, where a particular compound is mentioned, the compound may be used in various embodiments of the compositions of the application and/or in the methods of the application unless otherwise understood from the context. In other words, in the present application, the embodiments have been described and depicted in a manner that enables writing and drawing of clear and concise applications, but it is contemplated and understood that the embodiments may be combined or separated in various ways without departing from the present teachings and application. For example, it is to be understood that all features described and depicted herein are applicable to all aspects of the application described and depicted herein.
The subject matter will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the claims should in no way be construed as limited to these examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein. Those skilled in the art will readily recognize a number of non-critical parameters that may be changed or modified to produce substantially similar results.
Examples
Example 1: production of novel plasma kallikrein antibodies.
Five prekal-release (prekal) knockout mice were immunized via the fly-festival pathway with active human plasma kal-release (pkal, enzyme Research Labs, south bond, IN). When serum titers indicate a strong immune response, draining lymph nodes are collected and lymphocytes are fused with myeloma lines using electrofusion. The resulting hybridomas were plated in thirty 384-well plates and screened for binding to human pkal and prekal by ELISA. The hybridoma cell line that preferentially binds to pkal was subcloned and expanded.
The monoclonal IgG was retested for pkal and prekal binding by ELISA and the inhibition of pkal (1 nM enzyme) enzyme activity was screened in a fluorescent peptide substrate assay (Kenniston et al J Biol chem.2014, 22, 289 (34): 23596-608). As shown in fig. 1, three monoclonal lineages including 24L13 were confirmed to be selective for pkal. FIG. 2 shows that one lineage (24L 13, the antibody designated MAb 1) was also inhibitory in enzyme assays (Kenniston, 2014) compared to the positive control C1inh and another antibody DX-2930 (described in U.S. Pat. No. 8,816,055). The hybridomas were sequenced to determine the heavy and light chain variable domains (SEQ ID NO:1 and SEQ ID NO:2; MAb 1). As shown in fig. 3, the recombinantly expressed MAb1 retained the inhibitory activity of hybridoma-derived IgG. The nucleic acid sequence of the heavy chain variable domain of MAb1 is provided as SEQ ID NO. 33 and the nucleic acid sequence of the light chain variable domain of MAb1 is provided as SEQ ID NO. 34.
The closest human germline sequence of the murine variable domain was identified by comparison with the human germline sequence listed in IMGT (IMGT. Org). A nested set of framework modifications was designed for heavy and light chains to evolve murine sequences into the human germline, expressed in pairs, and tested for binding affinity to pkal, as well as for inhibition efficacy of pkal enzymatic activity. Variant VH5_VL4 (MAb 2) maintained binding affinity and inhibitory potency and was optimally humanized (SEQ ID NO:3, SEQ ID NO: 4). Furthermore, the NG structure in heavy chain CDR2 tends to be successfully modified to NA to reduce the risk of deamidation (SEQ ID NO: 5).
MAb1 light chain CDR sequences were varied and Fab phage display libraries were constructed. Continuous selection with pkal and deselection with prekal were used to enrich for pkal-selective clones. Selective clones were expressed as IgG and tested for inhibition of the pkal enzyme activity (FIG. 4). The light chain CDR2 variant clone VL 4-213T.05 (MAb 3-VL; SEQ ID NO: 6) was identified as a selective variant with high pkal inhibitory potency. This light chain CDR2 grafted onto a humanized VL4 variant (SEQ ID NO:6, MAb 3-VL) retains selectivity and activity when expressed with a humanized, prone to fixed heavy chain variable region (SEQ ID NO:5, MAb 3-VH). When expressed as full length human IgG1 kappa form, the humanized and optimized constructs for MAb3 VL and VH sequences were designated MAb4 (heavy chain SEQ ID NO:7, light chain SEQ ID NO: 8).
To further optimize the therapeutic potential of MAb4, the heavy chain constant domains were modified by introducing LS (corresponding to M428L and N434S according to EU numbering) or YTE mutations (corresponding to M252Y, S T and T256E according to EU numbering) (SEQ ID 9 and SEQ ID 10, respectively). When expressed with light chain SEQ ID NO. 8, these humanized IgG constructs were designated MAb4-LS and MAb4-YTE, respectively.
Example 2: in vitro characterization of plasma kallikrein antibodies
In vitro potency of MAb4 and its Fc variants MAb4-YTE and MAb4-LS compared to DX-2930 was tested in a physiologically relevant functional assay, DX-2930 being a commercially available monoclonal antibody known to inhibit plasma kallikrein and a clinical study object for the treatment of HAE. Specifically, each antibody was observed for its ability to inhibit the cleavage of the pkal of its natural substrate, high Molecular Weight Kininogen (HMWK). In these experiments, 10 or 30nM pkal was pre-incubated with inhibitory antibodies for 1 hour at room temperature, followed by addition of 600nM HMWK and incubation for 20 minutes, and the reaction quenched by addition of 0.5. Mu.M AEBSF. The concentration of Bradykinin (BK) released from pKal catalyzed HMWK was measured in a commercial ELISA kit (enco, farm dale, NY) using 1/100 dilution of the reaction product. A comparison of the newly generated antibody in the pKal inhibition assay with reference antibody DX-2930 is shown in FIG. 5. The Mab4 and Mab4-YTE and Mab4-LS constructs showed more potent pKal inhibition and steeper Hill slopes compared to DX-2930. In this case, MAb4 and its Fc variant are potent inhibitors of pkal cleavage of HMWK and exhibit a Hill slope of about 2 (see, e.g., fig. 5), consistent with the assumption that MAb4 and its Fc variant may be an ectopic inhibitor of pkal. Furthermore, the introduction of YTE or LS modifications into the Mab4 construct did not significantly affect the efficacy of the construct in inhibition assays.
The HMWK analysis parameters are physiologically relevant because the concentration of high molecular weight kininogen used is that circulating in the human body and the concentration of plasma kallikrein used is within the range of estimated concentrations in plasma from Hereditary Angioedema (HAE) patients during the onset. Both Mab4-YTE and DX-2930 showed a dose-dependent inhibition of bradykinin production, indicating a decrease in plasma kallikrein activity, and Mab4-YTE exhibited a stronger inhibitory potency against plasma kallikrein activity and bradykinin release than DX-2930, as shown in figure 5.
IC for further determination of antibodies 90 Values to provide a relative efficacy index. In HAE, the level of treatment-related inhibition to prevent HAE onset is considered to be about 90% plasma kallikrein inhibition. This corresponds to a measurement called IC90, which is the concentration of agent that produces 90% inhibition. In the HMWK functional assay, as shown in fig. 5, the IC 90 The value is the concentration of antibody that produces 90% inhibition of kallikrein activity and is considered a therapeutically relevant level of inhibition. DX-2930 IC 90 IC with a value of 300nM, and MAb4 and its Fc variants 90 The value was 30nM. Thus, this in vitro HMWK functional assay showed that MAb4-YTE was about 10-fold more potent than DX-2930. The results of this analysis are consistent with the clinical efficacy observed for DX-2930, as clinical trials of DX-2930 have shown that a steady state plasma content of DX-2930 of 200-300nM is required to optimally reduce HAE seizure rate and maximize seizure free duration.
The kinetics and selectivity of the lead pkal antibodies were examined by SPR at 37 ℃ and pH 7.4 or pH 6.0. MAb4-LS (SEQ ID NOS: 9 and 8) and MAb4-YTE (SEQ ID NOS: 10 and 8) exhibited low single digit nanomolar affinity for pkal with significant selectivity compared to prekal (FIG. 6). In fact, MAb4-LS and MAb4-YTE and DX-2930 have more than 1000 times the affinity for plasma kallikrein for prekallikrein. (FIG. 6) K of MAb4-YTE relative to human plasma kallikrein D 1.1nM at pH 7.4 and 0.9nM at pH 6.0, whereas K of MAb4-LS D 2.2nM at pH 7.4 and 1.2nM at pH 6.0. These antibodies do not exhibit any significant pH dependence in the target bound to human plasma kallikrein, especially in the range of pH 6.0 to pH 7.4. In contrast, reference antibody DX-2930 exhibited K at pH 7.4 D 18nM and 236nM at pH 6.0 (FIG. 6). Thus, the Mab-YTE and Mab4-LS antibodies bind plasma kallikrein more strongly (more than 5-fold, e.g., 8-16-fold) than do the reference antibody DX-2930, and Mab4-YT in a functional plasma kallikrein inhibition assayE is about 10 times as potent as DX-2930 and identical. Furthermore, the DX-2930 antibody shows considerable pH sensitivity under the same conditions. For example, the DX-2930 antibody shows K when measured at pH 6 and pH 7.4 D The reduction is more than 10 times. Table 7 shows K based on SPR-bound MAb4-YTE and DX-2930 at pH 7.4, 37℃as described above a 、K d And K D Values.
In summary, the data presented in FIG. 6 shows that the antibody Mab4-YTE or Mab4-LS has one or more of the following features:
(a) K substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20 in buffer);
(b) K between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and
(c) K between 0.1nM and 5nM for human plasma kallikrein D And a K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4.
The plasma kallikrein antibodies (MAb 4-LS and MAb 4-YTE) with Fc mutations were also assessed for hFcRn binding. Two dilution series of MAb4-LS and MAb4-YTE were prepared: 2000nM to 31.25nM MAb4-LS and MAb4-YTE, pH 6.0 and 2000nM to 500nM, pH 7.4 in PBS containing 0.05% P20 (polysorbate 20). IgG1 was used as a control antibody. Antibody samples were assessed for FcRn binding via SPR using Biacore T200. The Biacore chip is CM5 (Sino Biological, catalog number CT 009-H08H) coupled to human FcRn. Running buffer used PBS 0.05% P20 and pH 6 or pH 7.4. The hFcRn binding at pH 6.0 is shown in table 8. MAb4-LS and MAb4-YTE compared to unmodified MAb4 (3.10X10) 2 And 2.77×10 2 K d Compared with 2.29×10 1 K d ) And compared with DX-2930 (1.67×10) 1 K d ) All have increased pH-dependent hFcRn binding because the rate of dissociation at pH 6.0 decreases (10-fold slower). At pH7.4, there was no evidence that all three antibodies bound, indicating that FcRn binding of MAb4, MAb4-LS and MAb4-YTE was pH dependent [ data not shown]. Thus, without wishing to be bound by theory, both MAb4-LS and MAb4-YTE antibodies bind better to FcRn in endosomes (pH 6) and weaker to FcRn in blood (pH 7.4) than the parent MAb4 antibody, such that the antibodies are recycled back into the blood via the IgG-FcRn recycling pathway, resulting in a circulatory half-life of MAb4-LS and MAb4-YTE of about 3 to 4 times that of MAb4 and DX-2930 (see table 10 in example 3).
MAb4-YTE was also shown to be effective in inhibiting not only human but also cynomolgus monkey, rat and rabbit plasma kallikrein activity as assessed using the small fluorogenic substrate Pro-Phe-Arg-AMC (PFR-AMC) of pKal. MAb4-YTE antibodies were incubated with 1nM pKal from each species at 37℃for 1 hr in three replicate wells at the indicated concentrations as shown in the graph of FIG. 7. 10. Mu.M of the fluorogenic reporter substrate PFR-AMC was added and pKal activity was measured in kinetic mode in a fluorescence plate reader for 60 minutes. Linear regression analysis was performed on the first 40 minutes of substrate release data from each reaction well to obtain the initial reaction rate (RFU/min) for each inhibitor concentration. Fitting (4 parameter fitting) the initial reaction rates to obtain IC 50 Values, and normalized to obtain percentages of enzyme activity within the upper and lower limits of the fitted curve. IC (integrated circuit) 50 The data are shown in table 9. The pKal activity levels plotted against MAb4-YTE concentration (in nM) are shown in figure 7. This data shows that MAb4-YTE is not only a potent inhibitor of humans, but is also a potent pKal in monkeys, rabbits, and ratsAn inhibitor.
Example 3: in vivo pharmacokinetic characterization of plasma kallikrein antibodies
MAb4 (SEQ ID NOS: 7 and 8), MAb4-LS (SEQ ID NOS: 9 and 8) and MAb4-YTE (SEQ ID NOS: 10 and 8) were further characterized in cynomolgus monkey pharmacokinetic studies along with another plasma kallikrein antibody DX-2930. These studies of these antibodies were performed simultaneously, but were independent studies, rather than direct comparisons face-to-face. Cynomolgus monkey, LM-prime or prime, 3.0-4.0kg, male, n=18 purchased from Hainan Jingang Biotech co. Antibodies were prepared in 20mM histidine, 150mM NaCl, 0.01% Tween80, pH 6.5. In these studies, all antibodies were administered to male cynomolgus monkeys (n=3) at 5mg/kg over 10 minutes via IV infusion. Blood was collected from the head and saphenous veins into pre-chilled citrate tubes at each time point. Samples were taken once a week for 42 days (DX-2930 and MAb 4) or 84 days (MAb 4-YTE and MAb 4-LS). The blood samples were placed on wet ice and centrifuged at 4 ℃ to obtain plasma within 15 minutes after sample collection. The plasma samples were stored at approximately-70 ℃. The presence of each anti-plasma kallikrein antibody in the plasma samples was determined by ELISA using plates coated with anti-human antibodies as capture agents and human IgG heavy and light chain monkey-adsorbed antibodies as detection reagents (Southern Biotech, catalog number: 2049-01 and Bethy, catalog number A80-319P). ELISA plates were read using SpectraMax M2.
MAb4 and DX-2930 (MAb 4 half-life is shown in FIG. 8 and DX-2930 half-life is shown in FIG. 9) have a terminal half-life of 10-12 days, which is the same report as USFDA review documents and publications on DX-2930 in non-human primates. In contrast, MAb4-LS exhibited a terminal half-life of about 23 days and MAb4-YTE exhibited a terminal half-life of about 34 days (see FIG. 8). The terminal half-life of MAb4-YTE was about 3 to 4 times the terminal half-life observed for DX-2930 and MAb 4. These results demonstrate the superiority of MAb4 antibodies with extended half-life. This result is surprising, as it was found that introducing an LS or YTE mutation into another plasma kallikrein antibody did not increase half-life (data not shown). In addition, the increased inhibitory potency and half-life of the newly generated antibodies suggests that an effective amount of MAb4-YTE or MAb4-LS can be maintained in vivo for a longer period of time than DX-2930. FIG. 9 shows PK half-lives of MAb4-YTE (middle panel) and DX-2930 (upper panel) compared to pKal IC90 of each antibody. (the data in the upper and middle panels are superimposed on each other in the lower panel to show the plasma levels of both MAb4-YTE and DX-2930). By about day 10, the plasma levels of DX-2930 were below the predicted minimum therapeutic concentration (IC 90), while MAb4-YTE was still above the predicted minimum therapeutic concentration (IC 90) for >84 days. Table 10 provides a summary of PK data from cynomolgus monkey studies. Without wishing to be bound by theory, the pH-dependent increase in FcRn binding of the Fc modified antibodies described in example 2 results in slower clearance and an extended half-life in cynomolgus monkeys.
Evaluation of in vitro potency data and half-life results from non-human primate studies indicate potential potency duration of antibodies. In particular, the antibody plasma concentrations measured at each time point of a cynomolgus monkey study can be used to predict the expected level of plasma kallikrein inhibition at that time point, based on the plasma kallikrein inhibition observed for that antibody concentration in an in vitro potency assay.
FIG. 10 provides a predictive model based on plasma concentrations from cynomolgus monkey PK studies and pKal inhibition determined in an in vitro functional assay based on HMWK assay data and in vivo PK data compared to Mab4-YTE and DX-2930 in percent in vivo pKal inhibition. In cynomolgus monkey studies, DX-2930 plasma levels were reduced to below the minimum therapeutic concentration predicted by in vitro potency analysis or IC90 by about day 10, and levels predicted to produce about 50% plasma kallikrein inhibition by day 20 to in vitro potency analysis. In contrast, in cynomolgus monkey studies, MAb4-YTE plasma levels remained higher than IC90 predicted by in vitro efficacy analysis for 84 days, which is the duration of the entire experiment.
These preclinical data indicate that at the same dose, MAb4-YTE has a half-life of months in humans and thus has a significantly longer duration of action than DX-2930. This may allow lower doses of MAb4-YTE to have longer duration of action than DX-2930. Thus, MAb4-YTE may be an effective prophylactic therapy for HAE patients by inhibiting the pathological activity of plasma kallikrein for a long period of time. Furthermore, antibodies may be administered less frequently while maintaining therapeutically effective plasma levels necessary to prevent HAE attacks. Thus, antibodies such as MAb4-YTE or MAb4-LS may be administered infrequently, for example every three months, or even for longer intervals. This would be a significant advantage over currently available HAE therapies (e.g., DX-2930). Thus, MAb4-YTE may combine the advantages of infrequent dosing, lower doses, and long term inhibition of HAE attacks.
Example 4 in vitro characterization of off-target binding of plasma kallikrein antibodies
Membrane proteome array analysis was performed to identify any non-specific binding of MAb 4. Off-target binding was not observed for MAb4 (data not shown).
In addition, antibodies are tested to determine if they contain effector function. Antibodies were found to have no effector function (data not shown).
The specificity of an antibody is also determined by assessing whether the antibody binds to an unrelated serine protease. Antibodies were screened against a panel of serine proteases (about 20) and no significant cross-reactive binding was observed (data not shown).
Trypsin inhibition assays were developed and performed to determine if MAb4 exhibited any cross-reactivity. A dilution series of 0.25nM trypsin, human pancreatin (catalogue number 16-19-032000,Athens Research and Technology Inc) and MAb4-YTE starting at 1. Mu.M was incubated with PFR-AMC (fluorogenic substrate, bachem, catalogue number 4004023) in buffer for 2 hours. Fluorescence intensity was measured every 5 minutes. Gabexate mesylate was used as a positive control in a dilution series starting at 1 μm. The analysis was performed in duplicate. The results are provided in fig. 11 and table 11. MAb4-YTE did not inhibit trypsin activity and IC50 could not be calculated. In contrast, antibody DX-2930 inhibited trypsin activity with an IC50 (243 nM to 376 nM) within the potency range of DX-2930 to inhibit plasma kallikrein functional activity (about 300 nM). Thus, mab4-YTE exhibited a better protease selectivity profile relative to DX-2930.
Additional serine protease inhibition assays were developed and performed to determine whether MAb4 exhibited any cross-reactivity. MAb4-YTE and DX-2930 were tested in duplicate in a 10 dose assay, 3-fold serial dilutions were made for 16 proteases starting at 1. Mu.M. Control compounds were tested in a 10 dose assay, starting with 10 μm or 100 μm (tPA, proteolytic enzyme 2, kallikrein 5, kallikrein 2, kallikrein 1, granzyme B and APC) and performing 3-fold serial dilutions. Protease activity was monitored as a time course measurement of the increase in fluorescent signal from the fluorescently labeled peptide substrate and the initial linear portion of the signal slope (signal/min) was analyzed. In the presence of PBS buffer, the% enzyme activity of the antibody titration was calculated as 100% activity relative to vehicle control. The IC50 values for protease inhibition by MAb4-YTE and DX-2930 are summarized in Table 12 below.
Empty cells indicate no inhibition or inability of compound activity to fit to the IC50 curve. IC50 values above 1 μm were estimated from the best curve fit available.
The IC50 of MAb4-YTE against any serine protease tested in this assay could not be calculated. DX-2930 was estimated to have an IC50 of greater than 1. Mu.M (1.00E-06) for serine transmembrane protease and thrombin A. The difference in serine protease inhibition profile was consistent with higher protease selectivity for Mab4-YTE compared to DX-2930.
In summary, MAb4-YTE does not inhibit trypsin activity or any serine protease tested, and therefore does not exhibit off-target inhibition activity. In contrast, DX-2930 plasma kallikrein antibodies exhibit significant trypsin inhibitory activity and off-target inhibitory activity against serine transmembrane protease and thrombin a, demonstrating the superior properties of MAb4-YTE in specificity and selectivity for its target due to lack of off-target activity.
Example 5: epitope characterization of plasma kallikrein antibodies
Competitive binding assay
SPR-based competition experiments (Biosensor Tools, salt Lake City, UT) showed that MAb4 bound to an epitope different from DX-2930 (FIG. 12), DX-2930 has been demonstrated to be an active site binding, competitive inhibitor (Kenniston, 2014). DX-2930, MAb4-LS and MAb4-YTE were amine coupled to Xantec 30M sensor chips using standard NHS/ECD coupled to 10mM NaAc pH 5.0. For capture and stacking studies, the running buffer contained HBS containing 1.3mM Ca++ with 1mg/ml BSA and 0.02% p20, pH 7.4. Human plasma kallikrein was captured onto different antibody surfaces (time 0-180 seconds, 1 μm) and then each mAb was tested at 1 μm on each surface binding complex.
No binding was observed when each antibody was tested against itself (fig. 12). However, both MAb4-LS and MAb4-YTE bound to kallikrein captured by the DX-2930 antibody surface (FIG. 12). In addition, DX-2930 binds to kallikrein captured by MAb4-LS and MAb4-YTE antibodies (FIG. 12). These results indicate that MAb4-LS and MAb4-YTE bind to different epitopes than DX-2930.
Cross-linking and Mass fingerprinting of pKal-antibody complexes
First, human pKal was characterized via peptide mass fingerprinting. Human plasma kallikrein protein was subjected to trypsin, chymotrypsin, asp-N, elastase and thermolysin proteolysis, followed by nLC-QExactive+orbitrap MS/MS analysis. An nLC Ultimate 3000-RSLC system was used inline with QExactive+ mass spectrometer (Thermo Scientific). 7. Mu.M human pKal was incubated with 1. Mu.L DSS for d0/d12 (2 mg/mL; DMF) followed by 180 minutes at room temperature. Control samples were prepared without the addition of crosslinking reagents. The samples were incubated for 180 minutes at room temperature. After incubation, the crosslinking reaction was terminated by adding 1 μl ammonium bicarbonate (20 mM final concentration) and incubated for 60 minutes at room temperature. Then, both tubes were dried using speedvac before 8M urea was resuspended (10. Mu.L). After mixing, DTT (1 μl,500 mM) was added to both tubes. The mixture was then incubated at 37℃for 60 minutes. After incubation, iodoacetamide (1 μl, 1M) was added to each tube, followed by incubation in a dark room at room temperature for an additional 60 minutes. After incubation, 100 μl of proteolytic buffer was added to both tubes. Trypsin buffer contained 50mM Ambic pH 8.5,5% acetonitrile; chymotrypsin buffer contains Tris HCl100mM, caCl2 10mM pH 7.8; ASP-N buffer contains phosphate buffer 50mM pH 7.8; the elastase buffer contained Tris HCl 50mM pH 8.0, and the thermolysin buffer contained Tris HCl 50mM, caCl2 0.5mM pH 9.0.
Trypsin hydrolysis
100. Mu.L of reduced/alkylated human pKal was mixed with 1. Mu.L of trypsin (Promega) at a ratio of 1/100. The proteolytic mixture was incubated overnight at 37 ℃.
Chymotrypsin proteolysis
100. Mu.L of reduced/alkylated human pKal was mixed with 0.5. Mu.L chymotrypsin (Promega) at a ratio of 1/200. The proteolytic mixture was incubated at 25℃overnight.
ASP-N proteolysis
100. Mu.L of reduced/alkylated human pKal was mixed with 0.5. Mu.L ASP-N (Promega) at a ratio of 1/200. The proteolytic mixture was incubated overnight at 37 ℃.
Elastase proteolysis
100. Mu.L of reduced/alkylated human pKal was mixed with 1. Mu.L of elastase (Promega) at a ratio of 1/100. The proteolytic mixture was incubated overnight at 37 ℃.
Thermophilic protease proteolysis
100. Mu.L of reduced/alkylated human pKal was mixed with 2. Mu.L of thermolysin (Promega) at a 1/50 ratio. The proteolytic mixture was incubated overnight at 70 ℃.
After digestion, 1% final formic acid was added to the solution. After proteolysis, 1. Mu.L of the peptide solution resulting from proteolysis was loaded onto a nano liquid chromatography system (Ultimate 3000-RSLC). Quarrupole-Orbitrap MS analysis was performed according to the manufacturer's instructions. An overlap map of trypsin, chymotrypsin, ASP-N, elastase and thermolysin peptides was obtained based on proteolytic MS results, covering 99.05% of the human pKal sequence.
Next, after incubation with deuterated cross-linking agents, the epitopes of the human pKal/MAb4-YTE and human pKal/DX-2930 complexes were determined as described above. After enrichment of the cross-linked peptides, the samples were analyzed by high resolution mass spectrometry (nLC-Quadrupole-Orbitrap MS) and the resulting data was analyzed using XQuest and Stavrox software.
mu.L of the prepared mixture of human pKal/MAb4-YTE (1.4. Mu.M and 0.8. Mu.M) and human pKal/DX-2930 (1.4. Mu.M and 0.4. Mu.M) was mixed with 2. Mu.L of DSS d0/d12 (2 mg/mL; DMF) and incubated for 180 min at room temperature. After incubation, the reaction was stopped by adding 1 μl ammonium bicarbonate (20 mM final concentration), followed by incubation for 1 hour at room temperature. The solution was then dried using speedvac prior to H2O 8M urea suspension (20 μl). After mixing, 2. Mu.L of DTT (500 mM) was added to the solution. The mixture was then incubated at 37℃for 1 hour. After incubation, 2 μl iodoacetamide (1M) was added followed by incubation in a dark room at room temperature for 1 hour. After incubation, 80. Mu.L of proteolytic buffer was added. Trypsin buffer contained 50mM Ambic pH 8.5,5% acetonitrile; chymotrypsin buffer contains Tris HCl 100mM, caCl2 10mM pH 7.8; ASP-N buffer contains phosphate buffer 50MM pH 7.8; the elastase buffer contained Tris HCl 50mM pH 8.0, and the thermolysin buffer contained Tris HCl 50mM, caCl2 0.5mM pH 9.0. The proteolytic hydrolysis of trypsin, chymotrypsin, ASP-N, elastase and thermolysin was performed using the conditions described above for pKal protein. Cross-linked peptides were analyzed using Xquest version 2.0 and Stavrox 3.6 software.
The molecular interface between human-PKAL and MAb4-YTE was characterized using chemical cross-linking, high-mass MALDI mass spectrometry and nLC-Orbitrap mass spectrometry. After trypsin, chymotrypsin, ASP-N, elastase and thermolysin proteolytic treatment of the deuterated d0d 12-containing protein complex human-PKAL/MAb 4-YTE, nLC-orbitrap MS/MS analysis detected ten cross-linked peptides between human-PKAL and MAb 4-YTE.
Analysis of the crosslinking data showed that MAB4-YTE interactions at human pKal include the following amino acids: 550. 551, 585, 591, 597, 617, 625. FIG. 13 provides a peptide interaction diagram of human pKal and MAB 4-YTE. The human pKal sequence is shown at the bottom and the cross-linked epitopes of Mab4-YTE are indicated at residues 550, 551, 585, 597, 617 and 625. FIGS. 14A-J show the band/surface and band model of the epitope site of the antibody on human pKal. The MAb4-YTE antibody epitope site was modeled on the PDB structure 6O1G, which uses UNIPOTP 03952 as a reference sequence. Amino acids indicated in the model in darker color correspond to residues 550-551 (KR (SEQ ID NO: 37)), residues 585-597 (KHNGMWRLVGITS (SEQ ID NO: 38)) and residues 617-625 (YMDWILEKT (SEQ ID NO: 39) of the human pKal sequence.
Analysis of the cross-linking data showed that the interaction of DX-2930 at human pKal included the following amino acids: 434. 446, 475, 482, 555, 558, 560. FIG. 15 provides a peptide interaction diagram of human pKal and DX-2930. The human pKal sequence is shown at the bottom, and the cross-linked epitopes of DX-2930 are indicated at residues 434, 446, 475, 482, 555, 558 and 560. FIGS. 16A-J show the band/surface and band model of the epitope site of an antibody on human pKal. The DX-2930 antibody epitope site is modeled by the PDB structure 6O 1G. Amino acids indicated in the model in darker color correspond to residues 434-446 (HCFDGLPLQDVWR (SEQ ID NO: 40)), residues 475-482 (YKVSEGNH (SEQ ID NO: 41)) and residues 555-560 (YKITQR (SEQ ID NO: 42)) of the human pKal sequence. FIG. 17 provides a map showing human pKal residues cross-linked by antibody DX-2930 compared to antibody MAb4-YTE
Cross-linking mass spectrometry data demonstrated that MAB4-YTE bound to a different epitope on human pKal than DX-2930. Without wishing to be bound by theory, the different binding sites of Mab4-YTE on plasma kallikrein compared to DX-2930 may explain the improved protease selectivity profile, lack of pH sensitivity of plasma kallikrein binding, and overall superior profile of Mab4-YTE compared to DX-2930.
Incorporated by reference
The entire disclosure of each patent and scientific document cited herein is incorporated by reference for all purposes.
Equivalent(s)
The scope of embodiments of the invention is not limited to the specific embodiments described herein. Indeed, various modifications other than those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the embodiments and any appended claims.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.
Sequence(s)
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Sequence listing
<110> austiya treatment Co., ltd (ASTRIA THERAPEUTICS, INC.)
<120> plasma kallikrein antibodies and uses thereof
<130> P231607CN1-P
<140>
<141>
<150> 63/262,838
<151> 2021-10-21
<150> 63/220,194
<151> 2021-07-09
<150> 63/159,323
<151> 2021-03-10
<150> 63/142,748
<151> 2021-01-28
<160> 49
<170> patent In version 3.5
<210> 1
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic polypeptides
<400> 1
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 2
<211> 107
<212> PRT
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<400> 2
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Phe Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Arg Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 3
<211> 121
<212> PRT
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<220>
<223> description of artificial sequence: synthetic polypeptides
<400> 3
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 4
<211> 107
<212> PRT
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<223> description of artificial sequence: synthetic polypeptides
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Arg Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 5
<211> 121
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<400> 5
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Ala Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 6
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<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Tyr Ala Ser His Arg Gly Trp Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Arg Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 7
<211> 450
<212> PRT
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<220>
<223> description of artificial sequence: synthetic polypeptides
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Ala Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 8
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<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Tyr Ala Ser His Arg Gly Trp Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Arg Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 450
<212> PRT
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<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Ala Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu
420 425 430
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 10
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic polypeptides
<400> 10
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Ala Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr
245 250 255
Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 11
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<400> 11
Lys Ala Ser Gln Asp Val Gly Ile Ala Val Ala
1 5 10
<210> 12
<211> 7
<212> PRT
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<400> 12
Trp Ala Ser Thr Arg His Thr
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 13
Gln Gln Tyr Arg Ser Tyr Pro Leu Thr
1 5
<210> 14
<211> 5
<212> PRT
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<400> 14
Ser Tyr Trp Met Asn
1 5
<210> 15
<211> 17
<212> PRT
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<220>
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<400> 15
Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 16
<211> 12
<212> PRT
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<220>
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<400> 16
Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr
1 5 10
<210> 17
<211> 17
<212> PRT
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<400> 17
Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Gly Lys Phe Gln
1 5 10 15
Gly
<210> 18
<211> 7
<212> PRT
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<400> 18
Tyr Ala Ser His Arg Gly Trp
1 5
<210> 19
<211> 17
<212> PRT
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<220>
<223> description of artificial sequence: synthetic peptides
<400> 19
Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn Tyr Asn Ala Lys Phe Gln
1 5 10 15
Gly
<210> 20
<211> 7
<212> PRT
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<220>
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<400> 20
Ser Gln Asp Val Gly Ile Ala
1 5
<210> 21
<211> 3
<212> PRT
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<400> 21
Trp Ala Ser
1
<210> 22
<211> 7
<212> PRT
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<220>
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<400> 22
Tyr Arg Ser Tyr Pro Leu Thr
1 5
<210> 23
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 23
Gly Tyr Ala Phe Ser Ser Tyr
1 5
<210> 24
<211> 6
<212> PRT
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<400> 24
Tyr Pro Gly Asp Asp Asp
1 5
<210> 25
<211> 12
<212> PRT
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<400> 25
Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr
1 5 10
<210> 26
<211> 3
<212> PRT
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<400> 26
Tyr Ala Ser
1
<210> 27
<211> 6
<212> PRT
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<400> 27
Gln Asp Val Gly Ile Ala
1 5
<210> 28
<211> 9
<212> PRT
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<220>
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<400> 28
Gln Gln Tyr Arg Ser Tyr Pro Leu Thr
1 5
<210> 29
<211> 8
<212> PRT
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<400> 29
Gly Tyr Ala Phe Ser Ser Tyr Trp
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 30
Ile Tyr Pro Gly Asp Asp Asp Thr
1 5
<210> 31
<211> 14
<212> PRT
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<400> 31
Ala Gly Ser Leu Met Val Thr Thr Gly Ala Pro Phe Asp Tyr
1 5 10
<210> 32
<211> 638
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 32
Met Ile Leu Phe Lys Gln Ala Thr Tyr Phe Ile Ser Leu Phe Ala Thr
1 5 10 15
Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr Glu Asn Ala Phe Phe Arg
20 25 30
Gly Gly Asp Val Ala Ser Met Tyr Thr Pro Asn Ala Gln Tyr Cys Gln
35 40 45
Met Arg Cys Thr Phe His Pro Arg Cys Leu Leu Phe Ser Phe Leu Pro
50 55 60
Ala Ser Ser Ile Asn Asp Met Glu Lys Arg Phe Gly Cys Phe Leu Lys
65 70 75 80
Asp Ser Val Thr Gly Thr Leu Pro Lys Val His Arg Thr Gly Ala Val
85 90 95
Ser Gly His Ser Leu Lys Gln Cys Gly His Gln Ile Ser Ala Cys His
100 105 110
Arg Asp Ile Tyr Lys Gly Val Asp Met Arg Gly Val Asn Phe Asn Val
115 120 125
Ser Lys Val Ser Ser Val Glu Glu Cys Gln Lys Arg Cys Thr Asn Asn
130 135 140
Ile Arg Cys Gln Phe Phe Ser Tyr Ala Thr Gln Thr Phe His Lys Ala
145 150 155 160
Glu Tyr Arg Asn Asn Cys Leu Leu Lys Tyr Ser Pro Gly Gly Thr Pro
165 170 175
Thr Ala Ile Lys Val Leu Ser Asn Val Glu Ser Gly Phe Ser Leu Lys
180 185 190
Pro Cys Ala Leu Ser Glu Ile Gly Cys His Met Asn Ile Phe Gln His
195 200 205
Leu Ala Phe Ser Asp Val Asp Val Ala Arg Val Leu Thr Pro Asp Ala
210 215 220
Phe Val Cys Arg Thr Ile Cys Thr Tyr His Pro Asn Cys Leu Phe Phe
225 230 235 240
Thr Phe Tyr Thr Asn Val Trp Lys Ile Glu Ser Gln Arg Asn Val Cys
245 250 255
Leu Leu Lys Thr Ser Glu Ser Gly Thr Pro Ser Ser Ser Thr Pro Gln
260 265 270
Glu Asn Thr Ile Ser Gly Tyr Ser Leu Leu Thr Cys Lys Arg Thr Leu
275 280 285
Pro Glu Pro Cys His Ser Lys Ile Tyr Pro Gly Val Asp Phe Gly Gly
290 295 300
Glu Glu Leu Asn Val Thr Phe Val Lys Gly Val Asn Val Cys Gln Glu
305 310 315 320
Thr Cys Thr Lys Met Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu
325 330 335
Pro Glu Asp Cys Lys Glu Glu Lys Cys Lys Cys Phe Leu Arg Leu Ser
340 345 350
Met Asp Gly Ser Pro Thr Arg Ile Ala Tyr Gly Thr Gln Gly Ser Ser
355 360 365
Gly Tyr Ser Leu Arg Leu Cys Asn Thr Gly Asp Asn Ser Val Cys Thr
370 375 380
Thr Lys Thr Ser Thr Arg Ile Val Gly Gly Thr Asn Ser Ser Trp Gly
385 390 395 400
Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Thr Ala Gln Arg
405 410 415
His Leu Cys Gly Gly Ser Leu Ile Gly His Gln Trp Val Leu Thr Ala
420 425 430
Ala His Cys Phe Asp Gly Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr
435 440 445
Ser Gly Ile Leu Asn Leu Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser
450 455 460
Gln Ile Lys Glu Ile Ile Ile His Gln Asn Tyr Lys Val Ser Glu Gly
465 470 475 480
Asn His Asp Ile Ala Leu Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr
485 490 495
Glu Phe Gln Lys Pro Ile Cys Leu Pro Ser Lys Gly Asp Thr Ser Thr
500 505 510
Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys
515 520 525
Gly Glu Ile Gln Asn Ile Leu Gln Lys Val Asn Ile Pro Leu Val Thr
530 535 540
Asn Glu Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg
545 550 555 560
Met Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly
565 570 575
Asp Ser Gly Gly Pro Leu Val Cys Lys His Asn Gly Met Trp Arg Leu
580 585 590
Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro
595 600 605
Gly Val Tyr Thr Lys Val Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys
610 615 620
Thr Gln Ser Ser Asp Gly Lys Ala Gln Met Gln Ser Pro Ala
625 630 635
<210> 33
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthesis of polynucleotides
<400> 33
caggttcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatt 60
tcctgcaaag cttctggcta cgcattcagt agctactgga tgaactgggt gaagcagagg 120
cctggagagg gtcttgagtg gattggacag atttatcctg gagatgatga tactaactac 180
aacggaaagt tcaagggcaa ggccacactg actgtagaca aatcctccac cacagcctac 240
atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aggatctttg 300
atggttacga cgggggcccc ctttgactac tggggccaag gcaccactct cacagtctcc 360
tca 363
<210> 34
<211> 321
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthesis of polynucleotides
<400> 34
gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60
atcacctgca aggccagtca ggatgtgggt attgctgttg cctggtatca acagaaacca 120
gggcaatctc ctaaatttct gatttactgg gcatccaccc ggcacactgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccattaggaa tgtgcagtct 240
gaagacttgt cagattattt ctgtcagcaa tataggagct atcctctcac gttcggctcg 300
gggacaaagt tggaactaaa a 321
<210> 35
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 35
Gly Gly Gly Gly Ser
1 5
<210> 36
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 36
Gly Gly Gly Gly Ala
1 5
<210> 37
<211> 2
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: MAb4-YTE epitope 1 sequence
<400> 37
Lys Arg
1
<210> 38
<211> 13
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: MAb4-YTE epitope 2 sequences
<400> 38
Lys His Asn Gly Met Trp Arg Leu Val Gly Ile Thr Ser
1 5 10
<210> 39
<211> 9
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: MAb4-YTE epitope 3 sequences
<400> 39
Tyr Met Asp Trp Ile Leu Glu Lys Thr
1 5
<210> 40
<211> 13
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: DX2930 epitope 1 sequence
<400> 40
His Cys Phe Asp Gly Leu Pro Leu Gln Asp Val Trp Arg
1 5 10
<210> 41
<211> 8
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: DX2930 epitope 2 sequence
<400> 41
Tyr Lys Val Ser Glu Gly Asn His
1 5
<210> 42
<211> 6
<212> PRT
<213> Unknown (Unknown)
<220>
<223> unknown description: DX2930 epitope 3 sequence
<400> 42
Tyr Lys Ile Thr Gln Arg
1 5
<210> 43
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 43
Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn
1 5 10
<210> 44
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthetic peptides
<400> 44
Gln Ile Tyr Pro Gly Asp Asp Asp Thr Asn
1 5 10
<210> 45
<211> 638
<212> PRT
<213> crab-eating macaque (Macaca fascicularis)
<400> 45
Met Ile Leu Phe Lys Gln Ala Thr Tyr Phe Ile Ser Leu Phe Ala Thr
1 5 10 15
Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr Glu Asn Ala Phe Phe Arg
20 25 30
Gly Gly Asp Val Ala Ser Met Tyr Thr Pro Thr Ala Gln His Cys Gln
35 40 45
Met Met Cys Thr Phe His Pro Arg Cys Leu Leu Phe Ser Phe Leu Pro
50 55 60
Ala Ser Ser Ile Asn Asp Met Glu Lys Arg Phe Gly Cys Phe Leu Lys
65 70 75 80
Asp Ser Val Thr Gly Thr Leu Pro Lys Val Arg Arg Ala Gly Ala Ile
85 90 95
Ser Gly His Ser Leu Lys Gln Cys Gly His Gln Ile Ser Ala Cys His
100 105 110
Arg Asp Ile Tyr Lys Gly Ile Asp Met Arg Gly Val Asn Phe Asn Val
115 120 125
Ser Lys Val Ser Ser Val Glu Glu Cys Gln Lys Arg Cys Thr Asn Asn
130 135 140
Ile Arg Cys Gln Phe Phe Ser Tyr Ala Thr Gln Thr Phe His Asn Ala
145 150 155 160
Glu Tyr Arg Asn Thr Cys Leu Leu Lys His Ser Pro Gly Gly Thr Pro
165 170 175
Thr Thr Ile Lys Val Leu Asn Asn Val Glu Ser Gly Phe Ser Leu Lys
180 185 190
Pro Cys Ala Leu Ser Glu Ile Gly Cys His Met Asn Ile Phe Gln His
195 200 205
Leu Ala Phe Ser Asp Val Asp Val Ala Arg Val Leu Ala Pro Asp Ala
210 215 220
Phe Val Cys Arg Thr Ile Cys Thr Tyr His Pro Ser Cys Leu Phe Phe
225 230 235 240
Thr Phe Tyr Thr Asn Ala Trp Lys Ile Glu Ser Gln Arg Asn Val Cys
245 250 255
Phe Leu Lys Thr Ser Glu Ser Gly Thr Pro Ser Ser Ser Thr Pro Gln
260 265 270
Glu Asn Thr Thr Ser Gly Tyr Ser Leu Leu Thr Cys Lys Arg Thr Leu
275 280 285
Pro Glu Pro Cys His Ser Lys Ile Tyr Pro Gly Val Asp Phe Gly Gly
290 295 300
Glu Glu Leu Asn Val Thr Phe Val Lys Gly Val Asn Val Cys Gln Glu
305 310 315 320
Thr Cys Thr Lys Met Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu
325 330 335
Pro Glu Asp Cys Lys Glu Glu Lys Cys Lys Cys Phe Leu Arg Leu Ser
340 345 350
Ser Asp Gly Ser Pro Thr Arg Ile Thr Tyr Gly Thr Gln Gly Ser Ser
355 360 365
Gly Tyr Ser Leu Arg Leu Cys Asn Thr Gly Asp Ser Ser Val Cys Thr
370 375 380
Thr Lys Thr Ser Ser Arg Ile Val Gly Gly Thr Asn Ser Ser Trp Gly
385 390 395 400
Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Met Ala Gln Arg
405 410 415
His Leu Cys Gly Gly Ser Leu Ile Gly His Gln Trp Val Leu Thr Ala
420 425 430
Ala His Cys Phe Asp Gly Leu Pro Leu Pro Asp Val Trp Arg Ile Tyr
435 440 445
Ser Gly Ile Leu Asn Leu Ser Asp Ile Thr Lys Glu Thr Pro Phe Ser
450 455 460
Gln Ile Lys Glu Ile Ile Ile His Gln Asn Tyr Arg Ile Ser Glu Gly
465 470 475 480
Asn His Asp Ile Ala Leu Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr
485 490 495
Glu Phe Gln Lys Pro Ile Cys Leu Pro Ser Lys Gly Asp Thr Asn Thr
500 505 510
Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys
515 520 525
Gly Glu Ile Gln Asp Ile Leu Gln Lys Val Asn Ile Pro Leu Val Thr
530 535 540
Asn Glu Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg
545 550 555 560
Met Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly
565 570 575
Asp Ser Gly Gly Pro Leu Ala Cys Lys His Asn Gly Met Trp Arg Leu
580 585 590
Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro
595 600 605
Gly Val Tyr Thr Lys Val Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys
610 615 620
Thr Gln Ser Ser Asp Gly Asn Ala Arg Met Gln Ala Pro Ala
625 630 635
<210> 46
<211> 629
<212> PRT
<213> Rabbit (Oryctolagus cuniculus)
<400> 46
Met Ile Pro Phe Arg Gln Ala Ala Tyr Phe Val Cys Leu Phe Thr Thr
1 5 10 15
Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr Lys Asn Ala Phe Phe Arg
20 25 30
Gly Gly Asp Val Thr Ser Met Tyr Ser Pro Asn Ala Gln His Cys Gln
35 40 45
Met Met Cys Thr Phe His Pro Arg Cys Leu Phe Phe Ser Phe Leu Pro
50 55 60
Ala Ser Ser Thr Asn Asp Ala Gln Lys Arg Phe Gly Cys Phe Leu Lys
65 70 75 80
Asp Ser Val Thr Gly Thr Leu Pro Arg Val His Gln Thr Asn Ala Ile
85 90 95
Ser Gly His Ser Leu Lys Gln Cys Gly His Gln Ile Asn Ala Cys His
100 105 110
Arg Ala Thr Tyr Glu Gly Ile Asp Met Arg Gly Thr Asn Phe Asn Ile
115 120 125
Ser Lys Val Gln Ser Val Glu Glu Cys Gln Lys Gln Cys Thr Asp Asn
130 135 140
Ile His Cys Gln Phe Phe Thr Tyr Ala Thr Gln Thr Phe Tyr Asn Ala
145 150 155 160
Glu His Leu Asn Asn Cys Ile Leu Lys Lys Ser Ala Thr Gly Thr Pro
165 170 175
Ser Ser Ile Lys Leu Leu Glu Asn Val Val Ser Gly Phe Ser Leu Lys
180 185 190
Pro Cys Ala Leu Ser Glu Ser Gly Cys His Met Asn Leu Phe Gln His
195 200 205
Leu Ala Phe Ser Asp Val Asp Val Ala Arg Val Leu Thr Pro Asp Ala
210 215 220
Phe Val Cys Arg Thr Ile Cys Thr Tyr His Ser Asn Cys Leu Phe Phe
225 230 235 240
Thr Phe Tyr Thr Asn Glu Trp Asp Thr Glu Ser Gln Arg Asn Ala Cys
245 250 255
Phe Leu Lys Thr Ser Glu Ser Gly Thr Pro Ser Phe Pro Thr Pro Gln
260 265 270
Glu Asn Thr Ile Ser Gly Tyr Ser Leu Leu Thr Cys Lys Lys Thr Leu
275 280 285
Pro Glu Pro Cys His Phe Lys Ile Tyr Ser Gly Val Asp Phe Gly Gly
290 295 300
Glu Glu Leu Asn Val Thr Phe Val Lys Gly Val Thr Leu Cys Gln Glu
305 310 315 320
Thr Cys Thr Lys Thr Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu
325 330 335
Pro Glu Asp Cys Lys Gly Glu Lys Cys Arg Cys Ser Leu Arg Leu Ser
340 345 350
Leu Asp Gly Ser Pro Thr Ser Ile Thr His Gly Thr Gln Lys Ser Ser
355 360 365
Gly Tyr Ser Leu Arg Leu Cys Lys Thr Glu Asn Gly Ser Val Cys Thr
370 375 380
Thr Lys Ile Asn Ala Arg Ile Val Gly Gly Ser Asn Ser Ser Arg Gly
385 390 395 400
Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Ala Ala Gln Ser
405 410 415
His Val Cys Gly Gly Ser Ile Ile Gly His Gln Trp Val Leu Thr Ala
420 425 430
Ala His Cys Phe Asp Gly Leu Pro Phe Pro Glu Ile Trp Arg Ile Tyr
435 440 445
Gly Gly Ile Leu Tyr Leu Ser Glu Val Thr Lys Glu Thr Ala Phe Ser
450 455 460
Gln Ile Lys Glu Ile Ile Ile His Pro Lys Tyr Lys Ile Ser Glu Thr
465 470 475 480
Gly His Asp Ile Ala Leu Ile Gln Leu Gln Ala Pro Leu Asn Asp Thr
485 490 495
Asp Ile Gln Lys Pro Ile Cys Leu Pro Ser Lys Asp Asp Thr Asn Ala
500 505 510
Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe Thr Lys Glu Lys
515 520 525
Gly Glu Ile Gln Asn Ile Leu Gln Lys Ala Asn Ile Pro Leu Val Thr
530 535 540
Asn Glu Glu Cys Gln Lys Ser Tyr Arg Asp His Ala Ile Thr Lys Gln
545 550 555 560
Met Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly
565 570 575
Asp Ser Gly Gly Pro Leu Val Cys Lys His Asn Asn Ile Trp Leu Leu
580 585 590
Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro
595 600 605
Gly Val Tyr Thr Lys Val Ala Glu Tyr Val Asp Trp Ile Leu Gln Lys
610 615 620
Met Gln Glu Lys Asp
625
<210> 47
<211> 638
<212> PRT
<213> brown mouse (Rattus norvegicus)
<400> 47
Met Ile Leu Phe Lys Gln Val Gly Tyr Phe Val Ser Leu Phe Ala Thr
1 5 10 15
Val Ser Cys Gly Cys Leu Ser Gln Leu Tyr Ala Asn Thr Phe Phe Arg
20 25 30
Gly Gly Asp Leu Ala Ala Ile Tyr Thr Pro Asp Ala Gln His Cys Gln
35 40 45
Lys Met Cys Thr Phe His Pro Arg Cys Leu Leu Phe Ser Phe Leu Ala
50 55 60
Val Ser Pro Thr Lys Glu Thr Asp Lys Arg Phe Gly Cys Phe Met Lys
65 70 75 80
Glu Ser Ile Thr Gly Thr Leu Pro Arg Ile His Arg Thr Gly Ala Ile
85 90 95
Ser Gly His Ser Leu Lys Gln Cys Gly His Gln Leu Ser Ala Cys His
100 105 110
Gln Asp Ile Tyr Glu Gly Leu Asp Met Arg Gly Ser Asn Phe Asn Ile
115 120 125
Ser Lys Thr Asp Ser Ile Glu Glu Cys Gln Lys Leu Cys Thr Asn Asn
130 135 140
Ile His Cys Gln Phe Phe Thr Tyr Ala Thr Lys Ala Phe His Arg Pro
145 150 155 160
Glu Tyr Arg Lys Ser Cys Leu Leu Lys Arg Ser Ser Ser Gly Thr Pro
165 170 175
Thr Ser Ile Lys Pro Val Asp Asn Leu Val Ser Gly Phe Ser Leu Lys
180 185 190
Ser Cys Ala Leu Ser Glu Ile Gly Cys Pro Met Asp Ile Phe Gln His
195 200 205
Phe Ala Phe Ala Asp Leu Asn Val Ser Gln Val Val Thr Pro Asp Ala
210 215 220
Phe Val Cys Arg Thr Val Cys Thr Phe His Pro Asn Cys Leu Phe Phe
225 230 235 240
Thr Phe Tyr Thr Asn Glu Trp Glu Thr Glu Ser Gln Arg Asn Val Cys
245 250 255
Phe Leu Lys Thr Ser Lys Ser Gly Arg Pro Ser Pro Pro Ile Ile Gln
260 265 270
Glu Asn Ala Val Ser Gly Tyr Ser Leu Phe Thr Cys Arg Lys Ala Arg
275 280 285
Pro Glu Pro Cys His Phe Lys Ile Tyr Ser Gly Val Ala Phe Glu Gly
290 295 300
Glu Glu Leu Asn Ala Thr Phe Val Gln Gly Ala Asp Ala Cys Gln Glu
305 310 315 320
Thr Cys Thr Lys Thr Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu
325 330 335
Pro Gln Asp Cys Lys Ala Glu Gly Cys Lys Cys Ser Leu Arg Leu Ser
340 345 350
Thr Asp Gly Ser Pro Thr Arg Ile Thr Tyr Glu Ala Gln Gly Ser Ser
355 360 365
Gly Tyr Ser Leu Arg Leu Cys Lys Val Val Glu Ser Ser Asp Cys Thr
370 375 380
Thr Lys Ile Asn Ala Arg Ile Val Gly Gly Thr Asn Ser Ser Leu Gly
385 390 395 400
Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Val Ser Gln Asn
405 410 415
His Met Cys Gly Gly Ser Ile Ile Gly Arg Gln Trp Ile Leu Thr Ala
420 425 430
Ala His Cys Phe Asp Gly Ile Pro Tyr Pro Asp Val Trp Arg Ile Tyr
435 440 445
Gly Gly Ile Leu Asn Leu Ser Glu Ile Thr Asn Lys Thr Pro Phe Ser
450 455 460
Ser Ile Lys Glu Leu Ile Ile His Gln Lys Tyr Lys Met Ser Glu Gly
465 470 475 480
Ser Tyr Asp Ile Ala Leu Ile Lys Leu Gln Thr Pro Leu Asn Tyr Thr
485 490 495
Glu Phe Gln Lys Pro Ile Cys Leu Pro Ser Lys Ala Asp Thr Asn Thr
500 505 510
Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Tyr Thr Lys Glu Arg
515 520 525
Gly Glu Thr Gln Asn Ile Leu Gln Lys Ala Thr Ile Pro Leu Val Pro
530 535 540
Asn Glu Glu Cys Gln Lys Lys Tyr Arg Asp Tyr Val Ile Thr Lys Gln
545 550 555 560
Met Ile Cys Ala Gly Tyr Lys Glu Gly Gly Ile Asp Ala Cys Lys Gly
565 570 575
Asp Ser Gly Gly Pro Leu Val Cys Lys His Ser Gly Arg Trp Gln Leu
580 585 590
Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Lys Glu Gln Pro
595 600 605
Gly Val Tyr Thr Lys Val Ala Glu Tyr Ile Asp Trp Ile Leu Glu Lys
610 615 620
Ile Gln Ser Ser Lys Glu Arg Ala Leu Glu Thr Ser Pro Ala
625 630 635
<210> 48
<211> 1353
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthesis of polynucleotides
<400> 48
caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggctcctc cgtgaaggtg 60
tcctgcaagg cctccggcta tgcattcagt agctactgga tgaactgggt gaggcaggct 120
cctggacagg gtcttgagtg gattggacag atttatcctg gagatgacga tactaactac 180
aatgccaagt tccaggggag ggtcaccatc accgtggaca aatccacaac taccgcctac 240
atggaactca gcagcctacg ctctgaggac acagccgtct atttctgtgc tggctccctg 300
atggttacta ccggcgcccc ttttgactac tggggccagg gaaccacggt caccgtctcc 360
tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcttctaa gagcacctct 420
gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480
tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540
tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600
acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 660
cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctctacat cacccgggaa 780
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgagaga ggagcagtac 900
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960
aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1080
gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200
gtgctggact ccgacggctc cttcttcctc tattccaagc tcaccgtgga caagagcagg 1260
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320
acgcagaaga gcctctccct gtctcccggg tga 1353
<210> 49
<211> 645
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> description of artificial sequence: synthesis of polynucleotides
<400> 49
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgca aagcaagtca ggacgtggga atcgctgtcg cctggtatca gcagaaacca 120
gggaaagccc ctaagttcct gatctattac gcatcccacc gcggatgggg cgtgcctgac 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttattt ctgtcaacag taccggtctt atcccctgac ctttggccaa 300
gggaccaagc tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agcttaagtc cggaactgct agcgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg aaactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

Claims (81)

1. An antibody or antigen-binding fragment thereof that binds to plasma kallikrein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and a light chain variable region (VL) sequence comprising LCDR1, LCDR2 and LCDR3, wherein:
a. (i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14, 23, 29 or 43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17, 19, 24, 30 or 44; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16, 25 or 31; and is also provided with
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11, 20 or 27; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12, 18, 21 or 26; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13, 22 or 28;
b. (i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17 or 19; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and is also provided with
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12 or 18; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13;
c. (i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 24; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25, an
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 20; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 21 or 26; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 22;
d. (i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 30; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 31 and
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 27; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 21 or 26; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28; or (b)
e. (i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 44; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25, an
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12 or 18; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15, 17 or 19; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12 or 18; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 19; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 18; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 15; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 17; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 12; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13.
6. The antibody or antigen-binding fragment thereof of claim 1, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 24; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 20; the amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO. 26; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 22.
7. The antibody or antigen-binding fragment thereof of claim 1, wherein:
the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 30; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 31; and is also provided with
The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 27; the CDR2 comprises the amino acid sequence shown in SEQ ID NO. 26; and the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO. 28.
8. The antibody or antigen-binding fragment thereof of claim 1, wherein:
(i) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 44; and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 25, an
(ii) The LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 18; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 28.
9. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3; and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, wherein:
a. the HCDR1 comprises the amino acid sequence shown in SEQ ID No. 14 having at most 1, 2 or 3 amino acid substitutions for the amino acid sequence; the CDR2 comprises the amino acid sequence set forth in SEQ ID No. 15, 17 or 19, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid substitutions thereto; and the HCDR3 comprises the amino acid sequence shown in SEQ ID No. 16 having at most 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions for said amino acid sequence; and is also provided with
b. The LCDR1 comprises the amino acid sequence shown in SEQ ID No. 11 having at most 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions for said amino acid sequence; the LCDR2 comprises an amino acid sequence shown in SEQ ID No. 12 or 18 having at most 1, 2, 3 or 4 amino acid substitutions for said amino acid sequence; and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 13 with up to 1, 2, 3 or 4 amino acid substitutions thereto.
10. The antibody or antigen-binding fragment of claim 9, wherein each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprises at most one amino acid substitution.
11. The antibody or antigen-binding fragment of claim 9 or 10, wherein at most one CDR selected from HCDR1, HCDR2 or HCDR3 comprises a substitution.
12. The antibody or antigen-binding fragment of claim 9 or 10, wherein at most one CDR selected from LCDR1, LCDR2, and/or LCDR3 comprises a substitution.
13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VL region comprises the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13 or 14, wherein the VL region comprises the amino acid sequence set forth in SEQ ID No. 6.
15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 1, 3 or 5.
16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 15, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 5.
17. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 5 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 6.
18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 5 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
19. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 1 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
20. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 3 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
21. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 1, 3 or 5 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 2.
22. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 1, 3 or 5 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 4.
23. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence set forth in SEQ ID No. 1, 3 or 5 and the VL region comprises the amino acid sequence set forth in SEQ ID No. 6.
24. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein the light chain comprises the amino acid sequence set forth in SEQ ID No. 8.
25. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18 or 24, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 7, 9 or 10.
26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 10.
27. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 9.
28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 10, 9 or 7 and the light chain comprises the amino acid sequence set forth in SEQ ID No. 8.
29. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24 to 26, or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 10 and the light chain comprises the amino acid sequence set forth in SEQ ID No. 8.
30. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24, 25, 27 or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 9 and the light chain comprises the amino acid sequence set forth in SEQ ID No. 8.
31. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, 24, 25 or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 7 and the light chain comprises the amino acid sequence set forth in SEQ ID No. 8.
32. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VH region comprises a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID No. 1, 3 or 5.
33. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the VL region comprises a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID No. 2, 4 or 6.
34. The antibody or antigen-binding fragment thereof of any one of claims 32 or 33, wherein the VL region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID No. 6, 4, or 2, and the VH region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID No. 1, 3, or 5.
35. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the heavy chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% percent homology to the sequence set forth in SEQ ID No. 7, 9 or 10.
36. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or 35, wherein the light chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID No. 8.
37. The antibody or antigen-binding fragment thereof of any one of claims 1 to 36, wherein the CDR sequences are interposed between human or humanized framework sequences.
38. The antibody of any one of claims 1 to 37, wherein the antibody is a full or full length antibody.
39. The antibody or antigen-binding fragment thereof of any one of claims 1 to 38, wherein the plasma kallikrein is human plasma kallikrein having the amino acid sequence set forth in SEQ ID No. 32.
40. The antibody or antigen-binding fragment thereof of any one of claims 1 to 39, wherein the antibody or antigen-binding fragment thereof binds to at least one of the following residues: human plasma kallikrein having the amino acid sequence shown in SEQ ID No. 32, K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.
41. The antibody or antigen-binding fragment thereof of claims 1 to 40, wherein the antibody or antigen-binding fragment thereof binds to at least one, two, three, four, five, or six of the following residues: human plasma kallikrein K550, R551, K585, S597, Y617 or T625 with the amino acid sequence shown in SEQ ID NO. 32.
42. The method of any one of claims 1 to 41, wherein the antibody or antigen binding fragment thereof is administered in an amount of less than or equal to about 1, 2, 3, 4, or 5 x 10 as measured by Surface Plasmon Resonance (SPR) analysis -9 K of M D Binds to human plasma kallikrein (SEQ ID NO: 32).
43. As claimed in claim 1 to Error-! Reference source not the antibody or antigen binding fragment thereof of any one of claims, wherein the antibody has one or more of the following characteristics:
a. k substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Assay in buffer of 1mg/mL BSA, 0.02% Tween-20)
b. K between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃in the presence of 50mM HEPES, 150mM NaCl, as by SPR), 0.1 or 1.3mM Ca 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and
c. k between 0.1nM and 5nM for human plasma kallikrein D And K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
44. An isolated antibody or binding fragment thereof that competes with the antibody of any one of claims 1 to 43 for binding to plasma kallikrein.
45. An isolated antibody or binding fragment thereof that binds to the same epitope on human plasma kallikrein as the antibody of any one of claims 1 to 44.
46. A recombinant antibody or antigen-binding fragment thereof that binds to plasma kallikrein, wherein the antibody or antigen-binding fragment thereof optionally comprises one or more of the following:
a. serum half-life of at least 20 days;
b. k substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Assay in buffer of 1mg/mL BSA, 0.02% Tween-20);
c. k between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and
d. k between 0.1nM and 5nM for human plasma kallikrein D And K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃in the presence of 50mM HEPES, as by SPR),150mM NaCl, 0.1 or 1.3mM Ca 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
47. An antibody or antigen-binding fragment thereof that binds to one or more of the following residues: human plasma kallikrein having the amino acid sequence shown in SEQ ID No. 32, K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.
48. The antibody or antigen-binding fragment thereof of claim 47, which binds to one or more of the following residues: human plasma kallikrein K550, R551, K585, S597, Y617 or T625 with the amino acid sequence shown in SEQ ID NO. 32.
49. The antibody or antigen-binding fragment thereof of any one of claims 47 or 48, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics:
a. k substantially independent of pH between pH 6.0 and 7.4 D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Assay in buffer of 1mg/mL BSA, 0.02% Tween-20);
b. k between 0.1nM and 5nM for human plasma kallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and
c. k between 0.1nM and 5nM for human plasma kallikrein D And K between 250nM and 2,000nM for human prekallikrein D (e.g., at 37℃with 50mM HEPES, 150mM NaCl, 0.1 or 1.3mM Ca as by SPR) 2+ Measured in 1mg/mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).
50. The antibody or antigen-binding fragment thereof of any one of claims 1 to 49, wherein the antibody is a monoclonal antibody.
51. The antibody or antigen-binding fragment thereof of any one of claims 1 to 50, wherein the antibody is a humanized antibody.
52. The antibody or antigen-binding fragment thereof of any one of claims 1 to 51, wherein the antibody comprises a heavy chain constant region selected from the group consisting of IgG, igA, igD, igE and IgM.
53. The antibody or antigen-binding fragment thereof of any one of claims 1 to 52, wherein the antibody comprises an IgG class and a heavy chain constant region selected from the subclasses IgG1, igG2, igG3, and IgG 4.
54. The antibody or antigen-binding fragment thereof of any one of claims 1 to 53, wherein the antibody comprises an IgG1 heavy chain constant region.
55. The antibody or antigen-binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises wild-type (WT) sequences.
56. The antibody or antigen binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises M252Y, S254T and T256E mutations or M428L and N434S mutations as numbered according to the EU numbering index.
57. The antibody or antigen binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises M252Y, S254T and T256E mutations as numbered according to the EU numbering index.
58. The antibody or antigen-binding fragment thereof of any one of claims 1 to 57, wherein the heavy chain variable region and the light chain variable region are not connected by a linker.
59. The antibody or antigen-binding fragment thereof of any one of claims 1 to 57, wherein the antibody-binding fragment is a scFv antibody.
60. The antibody or antigen-binding fragment thereof of any one of claims 1 to 57 or 59, wherein the heavy chain variable region and the light chain variable region are linked by a peptide linker.
61. The antibody or antigen-binding fragment thereof of claim 60, wherein the peptide linker comprises the sequence (GGGGS) n (SEQ ID NO:35)；(GGGGA) n (SEQ ID NO: 36) or any combination thereof, wherein each n is independently 1-5.
62. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain or heavy chain variable region (VH) of any one of claims 1 to 61.
63. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain or light chain variable region (VL) of any one of claims 1 to 61.
64. An expression vector comprising the nucleic acid of claim 62.
65. An expression vector comprising the nucleic acid of claim 63.
66. The expression vector of claim 65, further comprising the nucleic acid of claim 62.
67. A host cell comprising the expression vector of claim 64 and the expression vector of claim 65.
68. A host cell comprising the expression vector of claim 66.
69. A method of producing an antibody that binds to plasma kallikrein, or an antigen binding fragment thereof, the method comprising:
(a) Growing the host cell of claim 67 or 68 under conditions such that the host cell expresses one or more polypeptides comprising a heavy chain or heavy chain variable region and a light chain variable region or light chain, thereby producing the antibody or antigen-binding fragment of the antibody; and
(b) Purifying the antibody or antigen binding fragment of the antibody.
70. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 61 or the nucleic acid of any one of claims 62 to 63.
71. The pharmaceutical composition of claim 70, wherein the composition is an injectable pharmaceutical composition.
72. A method of treating a subject having a disease or condition selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or peritumor cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post angioplasty), systemic lupus erythematosus nephritis or burn, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-61 or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof.
73. A method of treating a plasma kallikrein mediated disorder in a subject, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1 to 61 or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof.
74. The method of claim 73, wherein the disorder is hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic events (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn.
75. The antibody or antigen-binding fragment thereof of any one of claims 1 to 611, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for use in the treatment of a disease or disorder selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn.
76. The antibody or binding fragment thereof of any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, for use in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn.
77. Use of an antibody or binding fragment thereof of any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, for the treatment of a disease or disorder selected from the group consisting of: hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-related cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or perineoplastic cerebral edema, sepsis, acute Middle Cerebral Artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn.
78. A method of reducing bradykinin content or inhibiting bradykinin production in an individual in need thereof comprising administering to said individual a therapeutically effective amount of the antibody or binding fragment thereof of any one of claims 1 to 61 or a pharmaceutical composition comprising said antibody or antigen binding fragment thereof.
79. A method of reducing or inhibiting plasma kallikrein activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or binding fragment thereof of any one of claims 1 to 61 or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof.
80. The method of any one of claims 72-79, wherein the disease or disorder is hereditary angioedema.
81. The method of any one of claims 72-79, wherein the disease or disorder is bradykinin dependent edema.
CN202280011600.0A 2021-01-28 2022-01-28 Plasma kallikrein antibodies and uses thereof Pending CN116829596A (en)

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US63/159,323 2021-03-10
US63/220,194 2021-07-09
US202163262838P 2021-10-21 2021-10-21
US63/262,838 2021-10-21
PCT/US2022/014242 WO2022165130A1 (en) 2021-01-28 2022-01-28 Plasma kallikrein antibodies and uses thereof

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