binding portion that binds to a formed conformational epitope formed by amino acid sequences, antibo

专利摘要:
BINDING PORTION THAT CONNECTS TO A FORMED CONFORMATIONAL EPYPE FORMED BY AMINO ACID SEQUENCES, ANTIBODY OR FRAGMENT OF THE SAME, PHARMACEUTICAL COMPOSITION, AND USE OF A PORTION OF AN ANTIBODY OR FRAGMENT OF THE SAME ANTIGEN BINDING. The present invention relates to binding moieties that specifically bind to a conformational epitope of C5a, in particular human C5a. Preferred binding moieties are anti-C5a antibodies that bind to this conformational epitope. The binding portions described herein are used as active agents in pharmaceutical compositions for the treatment and prevention of various acute and chronic diseases, in particular acute inflammatory diseases, such as systemic inflammatory response syndrome (SIRS), and varying degrees of sepsis including sepsis, severe sepsis and septic shock.
公开号:BR112012012514A2
申请号:R112012012514-1
申请日:2010-11-26
公开日:2020-07-28
发明作者:Renfeng Guo；Niels Christoph Riedemann；Yan Li；Beifen Shen
申请人:Inflarx Gmbh；
IPC主号:

专利说明:

“CONNECTION PORTION THAT CONNECTS TO A CONFORMATIONAL EPYPE“ FORMED BY AMINO ACID SEQUENCES, ANTIBODY OR FRAGMENT OF THE SAME, PHARMACEUTICAL COMPOSITION, AND, USE OF A PORTION OF
OR AN ANTIBODY OR FRAGMENT OF THE SAME ANTIGEN BINDING ”The present invention relates to binding moieties that specifically bind to a conformational epitope of C5a, in particular human C5a. Preferred binding moieties are anti-C5a antibodies that —select this conformational epitope. The binding portions described herein are used as active agents in pharmaceutical compositions for the treatment and prevention of various acute and chronic diseases, in particular acute inflammatory diseases, such as systemic inflammatory response syndrome (SIRS), and different degrees of sepsis, including sepsis, severe sepsis and septic shock.
BACKGROUND OF THE INVENTION C5a is cleaved from C5 upon activation of the complement. Among complement activation products, C5a is one of the most potent inflammatory peptides, with a broad spectrum of functions (Guoe Ward 2005). C5a is a glycoprotein present in the blood of healthy humans with a molecular weight of 11.2 kDa. The C5a polypeptide portion contains 74 amino acids, which explains a molecular weight of 8.2 kDa, while the carbohydrate portion explains approximately 3 kDa. C5a exerts its effects through high affinity C5a receptors (C5aR and C5L2) (Ward 2009). C5aR belongs to the rhodopsin-like family of G protein-coupled receptors with seven transmembrane segments; C5L2 is similar, but is not coupled to the protein. Currently, it is believed that C5a exerts its biological functions mainly through the C5a-C5aR interaction, since few biological responses have been observed for the C5a-C5L2 interaction. C5aR is widely expressed in myeloid cells, including neutrophils, eosinophils, basophils and monocytes, and non-myeloid cells in many organs, especially in the lung and liver, indicative of the importance of C5a / C5aR signaling.
C5a has a variety of biological functions (Guo and Ward 2005). C5a is a strong chemoattractant for neutrophils and also has chemotactic activity for monocytes and macrophages.
C5a causes oxidative stress (consumption of O;) in neutrophils and improves phagocytosis and release of granular enzymes.
It was also observed that C5a is a vasodilator.
C5Sa has been shown to be involved in the modulation of cytokine expression of various cell types to improve the expression of adhesion molecules in neutrophils.
It is observed that C5a becomes very harmful when it is produced excessively in the establishments of the disease, since it is a strong inducer and enhancer for inflammatory responses that works upstream of the inflammatory reaction chain.
High doses of C5a can lead to non-specific chemotactic “desensitization” with respect to neutrophils, thereby causing a lot of dysfunction (Huber-
Lang et al. 2001a). C5a has been reported to exert various pro-inflammatory responses, and it is reported to be harmful during sepsis.
Inhibition of C5a or the —deC5a (C5aR) receptor by antibodies has been shown to greatly improve survival in various models of sepsis in mice and rats (Czermak et al. 1999; Guo et al. 2000; Huber-Lang et al. 2001b; Riedemann et al. 2002a). In addition, several reports have demonstrated harmful effects of C5a on intact organ and immune system functions during experimental sepsis (Guo et al. 2000; Guo et al. 2002; Huber-Lang et al. 2001a; Huber-Lang et al. 2002 ; Laudes et al. 2002; Riedemann et al. 2003; Riedemann et al. 2004a; Riedemann et al. 2004b). C5a acts like an anaphylaxis and has been reported to have several pro-inflammatory effects.
In humans, high levels of C5a sepsis are reported to be associated with significantly worse results in several studies (Bengtson and Heideman 1988; Nakae et al. 1994; Nakae et al. 1996). In the experimental establishment of sepsis, the exposure of neutrophils to C5a can lead to neutrophil dysfunction and paralysis of signaling pathways, leading to deficient concentration of NADPH oxidase, paralysis of MAPK signaling cascades, greatly reduced oxidative stress, phagocytosis and chemotaxis (Guo et al. 2006a; Huber-Lang et al. 2002). Thymocyte apoptosis and delayed neutrophil apoptosis are two important pathogenic events for the development of sepsis, which are dependent on the presence of C5a (Guo et al. 2000; Guo et al. 2006b). During experimental sepsis, C5a regulates B2-integrin expression in neutrophils by positive regulation to promote cell migration in organs (Guo et al. 2002), one of the main causes for multiple organ failure (MOF). It is also observed that C5a is attributable to the activation of the coagulation pathway that occurs in experimental sepsis (Laudes et al. 2002). C5a stimulates the synthesis and release of human leukocytes from pro-inflammatory cytokines such as TNF-a, IL-B, IL-6, IL-8 and macrophage migration inhibitory factor (MIF) (Hopken et al. 1996; Riedemann et al. 2004a; Strieter et al. 1992). C5a produces a strong synergistic effect with LPS in the production of TNF-o, macrophage inflammatory protein (MIP) -2, cytokine-induced neutrophil chemo (CINC) -I, and IL-IB in alveolar epithelial cells (Riedemann et al. 2002b; Rittirsch et al. 2008). Because the activation of the complement is an event that occurs during the onset of sepsis, C5a can take action before the emergence of the “inflammatory cytokine storm”. It appears that C5a plays a key role in programming - the performance of the cytokine network and the formation of the systemic inflammatory response syndrome (SIRS). The blockade of C5a in the adjustment of the experimental sepsis greatly attenuates MOF and SIRS.
Dispersed positive regulation of C5aR expression occurs during the onset of sepsis, and blocking the interaction of C5a / C5aR by anti-C5a or anti-C5aR antibodies, or CSaR antagonists provides very protective effects in sepsis rodent models (Czermak et al. 1999; Huber-Lang et al. 2001b; Riedemann et al. 2002a).
In addition to the indication of sepsis, C5a blockade is also shown to be protective in many other models of inflammation, such as ischemia / reperfusion injury, kidney disease, graft rejection, malaria, rheumatoid arthritis, infectious intestinal disease, lung disease inflammatory, autoimmune diseases such as lupus, neurodegenerative disease, etc., in several species in the manner partially revised in Klos A. et al (Klos et al. 2009) and Allegretti M. et al (Allegretti et al. 2005). Furthermore, it has recently been discovered that C5a blockade shows a strong therapeutic benefit in a mouse tumor model (Markiewski et al. 2008). TECHNICAL PROBLEMS BASED ON THIS
INVENTION Antibodies that specifically bind to part C5a, but not part C5b, are known from the prior art (Klos et al. (1998) J. Immunol. Meth. 111: 241-252; WO 01/15731; WO 03 / 015819).
However, previously generated anti-C5Sa antibodies exhibit only moderate blocking activities on the biological effects induced by C5a. As a result, the anti-C5a antibodies of the prior art were neither able to achieve a complete blockage of the biological effects induced by C5a, nor were they used in super-stoichiometric amounts, to achieve a reasonably high blockade of C5a activity.
Thus, especially in view of potential clinical use in - patients, there remains a need in the prior art for anti-C5a antibodies or other binding moieties that exhibit stronger blocking activity for C5a-induced biological effects, although they specifically bind to C5a with a lot of affinity. Equally preferably, such antibodies cannot bind to C5b and, consequently, cannot affect the biological activities of C5b.
Surprisingly enough, the inventors of the present invention were able to identify a new conformational binding epitope, with corresponding binding antibodies, that meet the 5 advanced requirements mentioned above and others. In long experiments that support the present invention, two anti-C5a antibodies, out of more than 2000 that can be generated, exhibit an unprecedented blocking activity in the biological effects induced by C5a when used in stoichiometric amounts, that is, 0.5 mol of a bivalent antibody per mol of C5a The above overview does not necessarily describe all the problems solved by the present invention.
SUMMARY OF THE INVENTION In a first aspect, the present invention relates to a linking moiety that binds to a conformational epitope formed by amino acid sequences XX ETCEX; RX, (SEQ ID NO: 18) and XsXsKX7XgXoL (SEQ ID NO: 19 ) of C5a, where X, is selected from the group consisting of N, H, D, F, K, Y, and T; X; is selected from the group consisting of D, L, Y, and H; X; is selected from the group consisting of Q, E, and K; Xa is - selected from the group consisting of A, V, and L; Xs is selected from the group consisting of S, H, P, and N; X; «is selected from the group consisting of H and N; X; is selected from the group consisting of D, N, H, P, and G; Xg is selected from the group consisting of M, L, 1, and V; and X, is selected from the group consisting of Q, L, and L In a second aspect, the present invention relates to an Antibody or antigen-binding fragment thereof comprising: (i) a heavy chain CDR3 sequence as presented in SEQ ID NO: 6; or (11) a heavy chain CDR3 sequence as shown in SEQ ID NO: 7; wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions. In a third aspect, the present invention relates to a pharmaceutical composition comprising (a) the binding portion according to the first aspect, or (b) the antibody or antigen binding fragment thereof according to the second aspect and which further comprises one or more carriers, diluents, excipients, fillers, binders, lubricants, non-stick, disintegrating, adsorbent and / or pharmaceutically acceptable preservatives.
In a fourth aspect, the present invention relates to the use of (a) a binding portion according to the first aspect, or (b) an antibody or antigen binding fragment thereof according to the second aspect, for the preparation of a pharmaceutical composition for the prevention and or treatment of various diseases involving acute inflammation, such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injuries such as ischemic heart disease, injury acute lung disease, pneumonia, acute or chronic graft rejection in transplant patients, graft-versus-host reactions, but also diseases involving a chronic type of inflammation such as renal glomerular diseases, such as glomerulonephritis and other renal failure entities, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, t growth sweat or solid organ cancer.
In a fifth aspect, the present invention relates to a method of treating systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injuries such as ischemic heart disease, acute lung injury, pneumonia, acute or chronic graft rejection in transplant patients, host graft reactions, renal glomerular diseases such as glomerulonephritis and other renal failure entities, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease , Crohn's disease, tumor growth or solid organ cancer in a patient who needs it, the method comprising administering to the patient an effective amount of (a) a binding portion according to the first aspect, or (b) an antibody or fragment of the same antigen binding according to the second aspect. This summary of the invention does not necessarily describe all —features of the invention.
DETAILED DESCRIPTION OF THE INVENTION Definitions Before the present invention is described in detail below, it should be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used here is only for the purpose of describing particular modalities, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning in the manner commonly understood by those skilled in the art.
Preferably, the terms used here are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H.
eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
Throughout this specification and in the claims that follow, unless the context otherwise requires, the word "understand", and variations such as "understands" and "understands", are intended to imply the inclusion of an integer or declared step, or group of whole numbers or steps, but not excluding any other whole number or step, or whole number group or step. Several documents are cited throughout the text of this specification. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, sequence submissions with GenBank access number, etc.), whether above or below, is hereby incorporated by reference in its Integral. Nothing here should be construed as an admission that the invention is not entitled to date such disclosure in advance by virtue of the previous invention.
As used herein, “human C5Sa” refers to the following 74 amino acid peptide:
TLQKKIFEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGR (SEQ ID NO: 1).
The term "binding moiety", as used herein, refers to any molecule, or part of a molecule, that can specifically bind to a target molecule or target epitope. Preferred binding portions in the context of the present application are (a) antibodies or antigen-binding fragments thereof; (b) oligonucleotides; (c) antibody-like proteins or (d) peptidomimetics. The "binding moieties" that can be used to practice the present invention are capable of binding to a mammalian C5a conformational epitope that is formed by the two amino acid sequences, XIX ETCEX; RX, (SEQ ID NO: 18) and XsXsKX; X: XoL (SEQ ID NO: 19), where X, is selected from the group consisting of N, H, D, F, K, Y, and T; X; is selected from the group consisting of D, L, Y, and H; X3 is selected from the group consisting of Q, E, and K; X, is selected from the group consisting of A, V, and L; Xs is selected from the group consisting of S, H, P, and N; X «is selected from the group consisting of H and N; X; is selected from the group consisting of D, N, H, P, and G; X; is selected from the group consisting of M, L, 1, and V; and X, is selected from the group consisting of Q, L, and I. The "binding portions" that are particularly suitable for practicing the present invention are capable of binding to a conformational epitope of human C5a that is formed by the two amino acid sequences, - NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3).
As used herein, a first compound (for example, an antibody) is considered to "bind" to a second compound (for example, an antigen, such as a target protein), if it has a Kg dissociation constant for said second compound of 1 mM or - less, preferably 100 µM or less, preferably 50 µM or less, preferably 30 µM or less, preferably 20 µM or less, preferably 10 µM or less, preferably 5 µM or less, more preferably 1 uM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferable preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, and even more preferably 10 nM or less.
The term "bond" according to the invention preferably refers to a specific bond. "Specific binding" means that a binding portion (for example, an antibody) binds more strongly to a target, such as an epitope for which it is specific, compared to binding to another target. A binding portion binds more strongly to a first target, compared to a second target, if it binds to the first target with a dissociation constant (Ka) that is less than the dissociation constant for the second target. Preferably, the dissociation constant (Ka) for the target to which the binding portion specifically binds is more than 10 times, preferably more than 20 times, more preferably more than 50 times, even more preferably more than 100 times, 200 times , 500 times or 1,000 times less than the dissociation constant (Ka) for the target to which the binding portion does not specifically bind.
As used herein, the term “KJg” (measured in “mol / L”, sometimes abbreviated as “M”) is intended to refer to the dissociation equilibrium constant of the particular interaction between a bonding portion (for example , an antibody or fragment thereof) and a target molecule (for example, an antigen or epitope thereof).
An "epitope", also known as an antigenic determinant, is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells or T cells. As used here, an "epitope" is the part of a macromolecule capable of to bind to a binding moiety (e.g., an antibody or antigen binding fragment thereof) in the manner described herein. In this context, the term "bond" preferably refers to a specific bond. Epitopes generally consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished as the bond to the former, but not to the latter, is lost in the presence of denaturing solvents.
As used herein, a “conformational epitope” refers to an epitope of a linear macromolecule (for example, a polypeptide)
which is formed by the three-dimensional structure of said macromolecule.
In the context of the present application, a "conformational epitope" is a "discontinuous epitope", that is, the conformational epitope in the macromolecule (for example, a polypeptide) that is formed from at least two separate regions in the main sequence of the macromolecule ( for example, the amino acid sequence of a polypeptide). In other words, an epitope is considered a "conformational epitope", in the context of the present invention, if the epitope consists of at least two separate regions in the main sequence in which a binding portion of the invention - (for example, a Antibody or antigen binding fragment) binds simultaneously, in which these at least two separate regions are interrupted by yet another region in the main sequence, in which a binding portion of the invention does not bind.
Preferably, a "conformational epitope" like this is present in a polypeptide, and the two separate regions in the main sequence are two separate amino acid sequences in which a binding portion of the invention (for example, an Antibody or fragment thereof antigen) bind, in which these at least two separate amino acid sequences are interrupted by a few more amino acid sequences in the main sequence, in which a linking portion of the invention does not bind.
Preferably, the interrupting amino acid sequence is a contiguous amino acid sequence comprising two or more amino acids, in which the linking moiety is not linked.
The at least two separate amino acid sequences to which a linker portion of the invention links are not particularly limited in size.
A sequence of separate amino acids such as this can consist of only one amino acid, provided that the total number of amino acids in said at least two separate amino acid sequences is large enough to carry out the specific bond between the linking portion and the conformational epitope.
A "paratope" is the part of an antibody that recognizes the epitope. In the context of the present invention, a "paratope" is the part of a binding portion (for example, an antibody or antigen binding fragment thereof), in the manner described herein, that recognizes the epitope.
The term "antibody" typically refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen-binding portion thereof.
The term "antibody" also includes all recombinant forms of antibodies, in particular the antibodies described herein, for example, antibodies expressed in prokaryotes, non-glycosylated antibodies and any of the antigen binding antibody fragments and derivatives, as described below . Each heavy chain is comprised of a heavy chain variable region (here abbreviated as VH or Vym) and a heavy chain constant region. Each light chain is comprised of a variable light chain region (here abbreviated as VL or V ”) and a constant light chain region. The VH and VL regions can be further subdivided into regions of hypervariability, called regions of - determination of complementarity (CDR), interspersed with regions that are more conserved, called regions of structure (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino termination to the carboxy termination in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a - binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding in tissues or host factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
The term "antigen-binding fragment" of an antibody (or simply "binding moiety"), as used herein, refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen.
It is noted that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
Examples of binding fragments included in the term "antigen binding portion" of an antibody include (1) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (11) F (ab ”) fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546) which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs that can optionally be joined by a synthetic linker.
Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be prepared as a single protein chain, in which the pairs of VL and VH regions form monovalent molecules (known as the single Fv chain (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc.
Natl.
Acad.
Sci.
USA 85: 5879-5883). Single chain antibodies are also intended to be included in the term "antigen binding fragment" of an antibody.
An additional example is a binding domain immunoglobulin fusion protein comprising (i) a polypeptide binding domain that is fused to a polypeptide in the immunoglobulin hinge region, (11) a fused immunoglobulin heavy chain constant region CH2 in the hinge region, and (11) an immunoglobulin heavy chain CH3 constant region fused in the CH2 constant region. The polypeptide of the binding domain can be a heavy chain variable region or a light chain variable region.
Binding domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional technologies known to those skilled in the art, and the fragments are selected for use in the same way as intact antibodies.
Additional examples of "antigen-binding fragments" are called microantibodies, which are derived from simple CDRs.
For example, Heap et al., 2005, describe a microantibody with residues of 17 amino acids derived from the heavy chain CDR3 of an antibody directed against the glycoprotein gp120 of the HIV-1I envelope.
Other examples include small mimetic antibodies that comprise two or more CDR regions, which are fused together, preferably by cognate structure regions.
A small mimetic antibody like this that comprises CDR1 Vu and CDR3 V, linked by FR2 Vu cognata was described by Qiu et al., 2007. Thus, the term “antibody or antigen-binding fragment thereof, as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that binds immunospecifically to an antigen.
They also comprise immunoglobulin-type proteins that are selected using techniques including, for example, phage display to specifically bind to a target molecule or target epitope, for example, the C5a conformational epitope formed by the amino acid sequences XX ETCEX; RX , (SEQ ID NO: 18) and XsXsKX; XgXoL (SEQ ID NO: 19), where X, is selected from the group consisting of N, H, D, F, K, Y, and T; X, is selected from the group consisting of D, L, Y, and H; X; is selected from the group consisting of Q, E, and K; X, is selected from the group consisting of A, V, and L; X; s is selected from the group consisting of S, H, P, and N; X; «is selected from the group consisting of H and N; X; is selected from the group consisting of D, N, H, P, and G; Xg is selected from the group consisting of M, L, 1, and V; and X, is selected from the group consisting of Q, L, and I; or the conformational epitope of human C5a formed by the amino acid sequences according to SEQ ID NO: 2 and SEQ ID NO: 3; or the conformational epitope of human C5a formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and KDM. The immunoglobulin molecules of the invention can be of any type (for example, IgG, IgE, IBM, IgD, IgA and IgY), class (for example, IgG1, IgG2, preferably I2G2a and IZgG2b, IgG3, IgG4, IZA1I and IZA2) or subclass of immunoglobulin molecule.
The antibodies and antigen-binding fragments thereof used in the invention can be of any animal origin, including birds and mammals. Preferably, the antibodies or fragments are of human, chimpanzee, rodent (e.g. mouse, rat, guinea pig or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat or dog. It is particularly preferable that the antibodies are of human or murine origin. The antibodies of the invention also include chimeric molecules in which an antibody constant region derived from one species, preferably human, is combined with the antigen-binding site derived from another species, for example, mouse. Furthermore, the antibodies of the invention include humanized molecules in which the antigen-binding sites of an antibody - derived from a non-human species (for example, mouse) are combined with the constant and structure regions of human origin. In the manner exemplified herein, the antibodies of the invention can be obtained directly from hybridomas that express the antibody, or they can be cloned and expressed recombinantly in a host cell (for example, a CHO cell, or a lymphocytic cell). Additional examples of host cells are microorganisms, such as E. coli and fungi such as yeast. Alternatively, they can be produced recombinantly in a non-human animal or transgenic plant.
The term "chimeric antibody" refers to those antibodies in which a portion of each of the heavy and light chain amino acid sequences is homologous to the corresponding sequences in antibodies derived from a particular species, or belonging to a particular class, while the remainder of the chain segment is homologous to the corresponding sequences in another species or class. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammal, while the constant portions are homologous to the antibody sequences are derived from another species. An evident advantage for such chimeric forms is that the variable region can be conveniently derived from currently known sources, using readily available B cells or hybridomas from non-human host organisms in combination with constant regions derived, for example, from human cell preparations. Although the variable region has the advantage of ease of preparation and the specificity of not being affected by the source, the human constant region is less likely to elicit an immune response from a human subject, when antibodies are injected, than it would be the constant region of a non-human source. However, the definition is not limited to this particular example.
The term "humanized antibody" refers to a molecule with an antigen-binding site that is substantially derived from an immunoglobulin of a non-human species, where the rest of the molecule's immunoglobulin structure is based on the structure and / or following a human immunoglobulin. The antigen binding site can comprise both complete variable domains fused into constant domains and only the complementarity determining regions (CDR) grafted into appropriate structure regions in the variable domains. Antigen binding sites can be wild-type or modified by one or more - amino acid substitutions, for example, modified to resemble human immunoglobulins as closely as possible. Some forms of humanized antibodies retain all of the CDR sequences (for example, a humanized mouse antibody that contains all six CDRs of the mouse antibody). Other forms have one or more CDRs - which are altered from the original antibody.
Different methods for humanizing antibodies are known to those skilled in the art, in the manner reviewed by Almagro & Fransson, 2008, whose content is here incorporated by reference in its entirety. The review article by Almagro & Fransson is summarized below. Almagro & —Fransson distinguish between rational and empirical approaches. Rational approaches are characterized by generating few variants of the genetically modified antibody and evaluating its binding or any other property of interest. If the designated variants do not produce the expected results, a new cycle of determination and evaluation of the connection is initiated. Rational approaches include grafting with CDR, surface recomposition, superhumanization and optimization of human chain content. On the contrary, empirical approaches are based on the generation of large libraries of humanized variants and on the selection of the best clones using enrichment technologies or high-throughput selection. In this way, empirical approaches are dependent on a reliable selection and / or classification system that are capable of searching through a wide range of antibody variants. In vitro display technologies, such as phage display and ribosome display, meet these requirements and are well known to those skilled in the art. Empirical approaches include RF libraries, conducted selection, structure shuffling and modification for humanization.
Grafting with CDR A grafting protocol with CDR typically comprises three decision-making points: (1) definitions of regions that determine the specificity of the donor antibody, that is, the target for the graft, (2) identification of a source of human sequences to be used as RF donors, and (3) selection of residues outside the region that defines specificity, that is, that determines the amino acid positions that are targets for retromutation to repair or improve the affinity of the humanized antibody. (1) Regions that determine the specificity of the antibody The experimental structure of the non-human antibody in complex with the antigen provides a detailed map of residues in contact with the antigen and is therefore responsible for determining its specificity.
The structural information can be complemented with alanine scanning mutagenesis and / or combinatorial mutagenesis to identify residues that contribute more to the binding energy or to the functional parotope.
Since the functional parameter is a subset of the residues in contact, grafting only the functional parameter would reduce the number of non-human residues in the humanized product.
However, only in rare cases are the experimental structure of the antigen-antibody complex and / or the functional parotope available at the beginning of a humanization protocol.
In the absence of a precise definition of residues responsible for a given - antibody specificity, CDRs are often used as regions that define specificity.
It is also possible to use a combination of CDR and HV loop as graft loops.
To reduce the number of residues to be grafted onto human RFs, the SDR graft has been described, that is, the graft of residues that determine specificity (SDRs).
(2) Source of human RFs The second step in a typical CDR graft protocol is to identify human RF donors. Initial work used FRs of human antibodies of known structure, regardless of their homology as a non-human antibody. This approach is known as the “fixed FR method”. The final works used human sequences with the best homology to the non-human antibody. This approach was called “best fit”. While “best-fit” strategies tend to result in antibodies with better affinity, other parameters such as low — immunogenicity and yields must also be taken into account when choosing an RF for humanization. Thus, combinations of “best fit” and “fixed FR” are also possible. For example, part V7 can be humanized according to the fixed FR method and part V4 can be humanized according to the best fit method, or vice versa.
Two sources of human sequences were used: mature and germline sequences. The mature sequences, which are products of immune responses, carry somatic mutations generated by random processes and are not in species selection, resulting in potential — immunogenic residues. Thus, to avoid immunogenic residues, human germline genes are increasingly used as a source of RF donors. The nucleotide sequences of human germline FRs are revealed, for example, in Appendices A and B of the article by Dall Acqua et al, 2005. Furthermore, antibodies based on the gene - of the germline tend to be more flexible compared to mature antibodies. It is known that this greater flexibility best accommodates several CDRs with little or no retromutation in RF to repair the affinity of the humanized antibody.
(3) Retromutations to repair or improve affinity
Commonly, affinity decreases after CDR grafting as a consequence of incompatibilities between non-human CDRs and human RFs.
Therefore, the third step in a typical CDR graft protocol is to define mutations that would repair or prevent loss of affinity.
Retromutations were determined carefully based on the structure or a model of the humanized antibody and tested experimentally.
A website for automated antibody modeling, called WAM, can be found at URL http://antibody.bath.ac.uk.
The software for modeling the structure of the protein can be downloaded from the websites http://salilab.org/modeller/modeller.html (Modeller) and http: // spdbv .vital-it.ch (Swiss PdbViewer). Surface recomposition Surface recomposition is similar to grafting with CDR and shares with the first two points of decision making.
Unlike the CDR graft, the recomposition of the surface maintains the unexposed residues of the non-human antibody.
Only surface residues in the non-human antibody are exchanged for human residues.
Superhumanization While grafting with CDR depends on the comparison of RF between non-human and human sequences, superhumanization is based on a comparison of CDR in such a way that the homology of RF is irrelevant.
The approach includes a comparison of the non-human sequence with the functional human germline gene repertoire.
These genes that encode the same canonical structures, or very close to them, in the murine sequences are then selected.
Next, among the genes that share the canonical structures with the non-human antibody, those with better homology to CDRs are chosen as RF donors.
Finally, non-human CDRs are grafted into these FRs.
Optimization of human chain content This approach is based on a metric system of the “humanity” of the antibody, called human chain content (HSC). In summary, this approach compares the mouse sequence with the repertoire of genes from the human germline. The differences are classified as HSC. The target sequence is humanized by increasing your HSC, other than using a global identity measure, to generate multiple humanized variants.
Structure libraries (abbreviated: RF libraries) In the RF library approach, a collection of residue variants is introduced at specific positions in the RF, followed by mixing the library to select the RF that best supports the grafted CDR. Thus, this approach is similar to grafting with CDR, but instead of creating few retromutations in RF, a combinatorial library of typically more than 100 mutational variants is constructed.
Targeted selection This approach includes combining the Vu or Vr domain of a given specific non-human antibody to a particular antigen with a human Vy and Vi library. Subsequently, specific human V domains are selected against the antigen of interest. For example, a non-human antibody can be humanized by first combining non-human Vy with a library of human light chains. The library is then selected against the target antigen by phage display, and the selected Vr is cloned into a library of human Vx chains and selected against the target antigen. It is also possible to start by combining the non-human Vr with a library of human heavy chains. The library is then selected against the target antigen by phage display, and the selected Vm is cloned into a library of human Vr chains and selected against the target antigen. As a result, a completely human antibody with similar affinity to the non-human antibody can be isolated.
To prevent an epitope from shifting, it is possible to implement a so-called inhibition ELISA that allows the selection of clones that recognize the same epitope as the parental antibody.
Alternatively, conservation of CDR can be applied to prevent an epitope shift.
In the conservation of CDR, one or more non-human CDRs are maintained, preferably the heavy chain CDR3, since this CDR is at the center of the antigen binding site.
Structure shuffling (abbreviated: FR shuffling) In the FR shuffling approach, complete FRs are combined with non-human CDRs.
Using FR shuffling, Dall 'Acqua and colleagues humanized a murine antibody.
All six murine antibody CDRs were cloned into a library containing all FRs of the human germline gene (Dall'Acqua et al., 2005). The libraries were selected for connection in a two-step selection process, first humanizing V ,, followed by Vu.
In a later study, a one-step RF shuffling process was used satisfactorily (Damschroder et al., 2007). The sequences of oligonucleotides that encode all known light-chain structures of the human germline (x) are revealed in Dall'Acqua et al, 2005, as appendix A.
The oligonucleotide sequences encoding known human germline heavy chain structures are disclosed in Dall Acqua et al., 2005, as appendix B.
Modification for humanization Modification for humanization allows the isolation of antibodies that are 91-96% homologous with antibodies with a human germline gene.
The method is based on the experimental identification of essential minimum specificity determinants (MSDs),
and in the sequential replacement of non-human fragments in human FR libraries and evaluation of binding. It starts with CDR3 regions of non-human Vy and V1 chains and progressively replaces other regions of non-human antibody in human FRs, including CDR1 and CDR2 from both Vuquantode V, r The methods for humanizing antibodies explained above are preferred during antibody generation humanized cells that specifically bind to the conformational epitopes described here. Despite this, the present invention is not limited to the methods for humanizing - antibodies previously mentioned.
Some of the previously mentioned humanization methods can be performed without information about the RF sequences in the donor antibody, called the “fixed RF method” (a graft variant with CDR), superhumanization, structure shuffling and modification for humanization. The variations of the “fixed FR method” were performed satisfactorily by Qin et al., 2007 and Chang et al., 2007. In particular, Qin et al. built an antibody fragment that comprises a variable region of human heavy chain in which the three CDR regions were replaced by antigenic peptides, which were derived from the CDR sequences of a murine antibody. Chang et al. these experiments continued and constructed an Fvsc fragment, in which all CDRs in part V7 and CDR3 in part V7 were replaced by antigenic peptides that were derived from the CDR sequences of a murine antibody.
As used herein, "human antibodies" include antibodies with variable and constant regions, derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or specific site in vitro mutation or by in vivo somatic mutation). Human antibodies of the invention include antibodies isolated from human immunoglobulin libraries, or from animals transgenic to one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described, for example, in U.S. Patent 5,939,598 to Kucherlapati & Jakobovits.
The term "monoclonal antibody", as used herein, refers to a preparation of antibody molecules of simple molecular composition. A monoclonal antibody exhibits a unique binding specificity and affinity for a particular epitope. In one embodiment, monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a non-human animal, for example, a mouse, fused to an immortalized cell.
The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, raised or isolated by recombinant means, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transcromosomal with respect to immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome, (c) antibodies isolated from a recombinant antibody library and combinatorial, and (d) antibodies prepared, expressed, created or isolated by any other means that involve the joining of —immunoglobulin gene sequences with other DNA sequences.
The term "transfectoma", as used herein, includes recombinant eukaryotic host cells that express an antibody such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells or fungi, including yeast cells.
As used herein, a "heterologous antibody" is defined with respect to a transgenic organism that produces an antibody like this. This term refers to an antibody with an amino acid sequence or a coding nucleic acid sequence that corresponds to that in an organism that does not consist of the transgenic organism, and which is generally derived from a species other than the transgenic organism.
As used herein, a "hetero-hybrid antibody" refers to an antibody with light and heavy chains from different origins related to organisms. For example, an antibody with a human heavy chain associated with a murine light chain is a hetero-hybrid antibody.
Thus, "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterologous, chimeric, humanized antibodies ( in particular grafted with CDR), de-immunized, or human, Fab fragments, Fab 'fragments, F (ab') 2 fragments, fragments produced by a Fab, Fd, Fv, disulfide-bound Fvs (Fvds) expression library, the single chain antibodies (eg, Fvsc), diabodies or tetribodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), nanobodies (also known as antibodies to single domain), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies in the antibodies of the invention), and epitope binding fragments from any of the foregoing.
The antibodies described herein are preferably isolated. An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to C5a is substantially free of antibodies that specifically bind to antigens other than C5a). An isolated antibody that specifically binds to a human C5a epitope, isoform or variant, however, may cross-react with other related antigens, for example, from other species (for example, homologues of the C5a species, such as rat C5a) . Furthermore, an isolated antibody can be substantially free of other cellular material and / or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies refers to antibodies with different specificities and which are combined into a well-defined composition.
The term “naturally occurring”, as used here, applied to an object, refers to the fact that an object can be found in nature. For example, a sequence of polypeptides or polynucleotides that is present in an organism (including viruses), that can be isolated from a source in nature and that was not intentionally modified by man in the laboratory, is naturally occurring.
As used herein, the term “nucleic acid aptamer” refers to a nucleic acid molecule that has been genetically modified, through repeated cycles of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment), to ligated a target molecule (for a review, see: Brody EN and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J. Biotechnol. 74 (1): 5- 13). The nucleic acid aptamer can be a DNA or RNA molecule. Aptamers may contain modifications, for example, modified nucleotides such as 2-fluorine-substituted pyrimidines.
As used herein, the term "antibody-like protein" refers to a protein that has been genetically modified (for example, by loop mutagenesis) to specifically bind to a target molecule. Typically, an antibody-like protein like this comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural limitation greatly increases the binding affinity of the antibody-like protein to levels comparable to those of an antibody. The variable peptide loop size typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-type proteins include, without limitation, antibodies-like proteins, anticalin-like proteins, and repeated ankyrin-specific proteins (for review, see: Binz HK. Et al. (2005) Engineering novel binding proteins from —nonimmunoglobulin domains. Nat. Biotechnol. 23 (10): 1257-1268). Antibody-like proteins can be derived from large libraries of mutants, for example, obtained from large phage display libraries and can be isolated in analogy to regular antibodies. Likewise, antibody-like binding proteins can be obtained by combinatorial mutagenesis of residues exposed on the surface into globular proteins. Antibody-type proteins are sometimes referred to as "peptide aptamers".
As used here, a “peptidomimetic” is a small protein-like chain determined to mimic a peptide. Peptidomimetics typically originate from the modification of an existing peptide in order to alter the properties of the molecule. For example, they can originate from modifications to alter the stability of the molecule or biological activity. This may play a role in the development of drug-like compounds from existing peptides. These modifications involve changes in the peptide that will not occur naturally (such as the altered main parts and the incorporation of unnatural amino acids). “Conservative substitutions” can be made, for example, based on similarity in polarity, charge, size,
solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the amino acid residues involved. Amino acids can be grouped into the following six standard groups of amino acids: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence the orientation of the chain: Gly, Pro; and (6) aromatics: Trp, Tyr, Phe.
As used herein, "conservative substitutions" are defined as exchanges of one amino acid for another amino acid listed in the same group as the six standard groups of amino acids shown above. For example, the exchange of Asp for Glu maintains a negative charge on the polypeptide thus modified. In addition, glycine and proline can be replaced with one another based on their ability to stop a-propellers. Some preferred conservative substitutions in the previous six groups are replaced by the following subgroups: (1) Ala, Val, Leu and Ile; (11) Ser and Thr; (11) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe.
- By providing the known genetic code and recombinant and synthetic DNA techniques, those skilled in the art can construct DNAs that encode conservative amino acid variants.
As used herein, "non-conservative substitutions" or "non-conservative amino acid exchanges" are defined as exchanges of one - amino acid for another amino acid listed in a group other than the six standard amino acid groups (1) to (6) shown above .
A "biological activity", as used herein, refers to any activity a polypeptide may exhibit, including without limitation: enzyme activity; binding activity with another compound (for example, binding with another polypeptide, in particular binding with a receptor, or binding with a nucleic acid); inhibitory activity (for example, enzyme inhibitory activity); activation activity (for example, enzyme activation activity) or toxic effects.
Regarding variants and
- derived from a polypeptide, the variant or derivative is not required to exhibit such an activity to the same degree as the parent polypeptide.
A variant is considered to be a variant, in the context of this application, if it exhibits adequate activity to a degree of at least 10% of the activity of the parent polypeptide.
Likewise, a derivative is considered a derivative, in the context of the present application, if it exhibits adequate biological activity to a degree of at least 10% of the activity of the parent polypeptide.
The appropriate “biological activity”, in the context of the present invention,
is a binding activity with a C5a conformational epitope formed by amino acid sequences XX ETCEX; RX, (SEQ ID NO: 18) and
155 XsXsKX; X: XoL (SEQ ID NO: 19), where X, is selected from the group consisting of N, H, D, FE, K, Y, and T; X) is selected from the group consisting of
D, L, Y, and H; X; 3 is selected from the group consisting of Q, E, and K; X, is selected from the group consisting of A, V, and L; X; s is selected from the group consisting of S, H, P, and N; X; «is selected from the group consisting of H and
N; X; is selected from the group consisting of D, N, H, P, and G; Xg is selected from the group consisting of M, L, 1, and V; and X, is selected from the group consisting of Q, L, and I.
A particularly suitable "biological activity" in the context of the present invention is a binding activity with the conformational epitope of human C5a formed by the amino acid sequences according to SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the "activity biological "suitable in the context of the present invention is a binding activity with the conformational epitope of human C5a formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and KDM.
Assays for determining binding activity are known to those skilled in the art and include ELISA, such as that described in the examples section.
As used herein, a "patient" means any mammal or bird that may benefit from treatment with the target binding portion described herein.
Preferably, a "patient" is selected from the group consisting of laboratory animals (for example, mouse or rat), domestic animals (including, for example, guinea pig, rabbit, chicken, turkey, pig, sheep, goat, camel , cow, horse, donkey, cat, or dog), or primates, including chimpanzees and humans.
It is particularly preferred that the "patient" is a human being.
According to the American College of Chest Physicians and the Society of Critical Care Medicine (Bone R.C. et al. (1992). "Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.
The ACCP / SCCM Consensus Conference Committee.
American College of Chest Physicians / Society of Critical Care Medicine ". Chest 101 (6): 1644—55), there are different levels of sepsis: Systemic inflammatory response syndrome (SIRS): Defined by the presence of two or more of the following findings : - Body temperature <36ºC (97ºF) or> 38ºC (100ºF) (hypothermia or fever) - Heart rate> 100 beats per minute (tachycardia) - Respiratory rate> 20 breaths per minute or, in the blood gas, a P , CO, less than 32 mm Hg (4.3 kPa) (tachypnea or - hypocapnia due to hyperventilation) - White blood cell count <4,000 cells / mm Or> 12,000 cells / mmº (<4 x 10º or> 12 x 10 th cells / L), or more than 10% in rod form (immature white blood cells) (leukopenia, leukocytosis or bandemia).
Sepsis: Defined as SIRS in response to a confirmed infectious process. The infection may be suspected or proven (by culture, staining, or polymerase chain reaction (PCR)), or a clinical pathognomonic syndrome for infection. Specific evidence for infection includes WBCs in normally sterile fluid (such as urine or cerebrospinal fluid (CSF), evidence of a perforated intestine (open air on abdominal X-ray or CT scan, signs of acute peritonitis), X-ray abnormal chest (CXR) consistent with pneumonia (with focal opacification), or petechia, purple, or fulminating purple.
Severe sepsis: Defined as sepsis with organ dysfunction, hypoperfusion or hypotension.
Septic shock: Defined as sepsis with refractory arterial hypotension or abnormalities of hypoperfusion, despite adequate fluid resuscitation. The signs of systemic hypoperfusion can be either terminal organ dysfunction or serum lactate greater than 4 mmol / dL. Other signs include oliguria and altered mental status. Patients are defined as those with septic shock if they have sepsis and hypotension after aggressive fluid resuscitation (typically above 6 liters or 40 mL / kg of crystalloid).
"Tumor" means an abnormal group of cells or tissue that grows by rapid, uncontrolled cell proliferation, and continues to grow after the stimuli that initiated new growth cease. Tumors show partial or complete loss of structural organization and functional coordination with normal tissue, and in general form a mass - distinct tissue, which can be either benign or malignant.
"Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix,
penetration of the basal endothelial membranes to enter the body cavity and vessels and then, after transport by blood, infiltration into target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the main tumor, because the tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis", which refers to a metastasis that is distant from the main tumor and the regional lymph node system.
As used herein, "treating", "treating" or "treating" a disease or disorder means performing one or more of the following: (a) reducing the severity and / or duration of the disorder, (b) limiting or preventing development of symptoms characteristic of the disorder (s) to be treated, (c) inhibit the worsening of the symptoms characteristic of the disorder (s) to be treated, ( d) limit or prevent the recurrence of the disorder (s) in patients who previously had the disorder (ies), and (e) limit or prevent the recurrence of symptoms in patients who were previously symptomatic for the disorder (s) disorder (s).
As used here, "prevent," which prevents "," prevention "or" prophylaxis "of a disease or disorder means preventing a disorder from occurring in the subject.
As used herein, "administer" includes administration in vivo, as well as administration directly to ex vivo tissue, such as venous grafts.
An "effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal that will receive the therapeutic agent, and the purpose of administration. The effective amount in each individual case can be determined empirically by those skilled in the art according to the methods established in the art. "Pharmaceutically acceptable" means approved by a federal government regulatory agency, or a state government, or listed in the U.S. pharmacopeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.
The term "carrier", as used herein, refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, powdered milk, glycerol, propylene glycol, water , ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, continuous release formulations and the like. The composition can be formulated as a "suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with groups without amino, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those formed with groups without carboxyl, such as those derived from sodium, potassium hydroxide, ammonium, calcium, ferric, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W.
Martin Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with an appropriate carrier amount, in order to provide the form for proper administration to the patient.
The formulation can adapt the mode of administration.
In general, the methods practiced and known in the fields of molecular biology, cell biology, protein and antibody chemistry techniques are described completely in the continuously updated publications “Molecular Cloning: A Laboratory Manual”, (Sambrook et al., Cold Spring Harbor) ; Current Protocols in Molecular Biology (F. M.
Ausubel et al.
Eds., Wiley &Sons); Current Protocols in Protein Science (J. AND.
Colligan et al. eds., Wiley &Sons); Current Protocols in Cell Biology (J. S.
Bonifacino et al., Wiley & Sons) and Current Protocols in Immunology (J. AND.
Colligan et al., Eds., Wiley & Sons). The known techniques that are related to cell culture and media are described in “Large Scale Mammalian Cell Culture (Hu et al., Curr.
Opin., Biotechnol. 8: 148, 1997); “Serum free Media” (K.
Kitano, Biotechnol. 17:73, 1991) and “Suspension Culture of Mammalian Cells” (Birch et al.
Technol Bioprocess. 19: 251, 1990). Modalities of the invention The present invention will now be further described.
In the following passages, different aspects of the invention are defined in more detail.
Each aspect thus defined can be combined with any other aspect or aspects, unless clearly indicated otherwise.
In particular, any characteristic indicated as being preferred or advantageous can be combined with any other characteristic or characteristics indicated as being preferred or advantageous.
In a first aspect, the present invention relates to a binding moiety that binds to a conformational epitope formed by amino acid sequences XX ETCEX; RX, (SEQ ID NO: 18) and XsXsKX7XgXoL (SEQ ID NO: 19) of C5a , where X, is selected from the group consisting of N, H, D, F, K, Y, and T; X; is selected from the group consisting of D, L, Y, and H; X; is selected from the group consisting of Q, E, and K; Xa is selected from the group consisting of A, V, and L; X; s is selected from the group consisting of S, H, P, and N; X; «is selected from the group consisting of H and N; X; is selected from the group consisting of D, N, H, P, and G; Xg is selected from the group consisting of M, L, 1, and V; and X, is selected from the group consisting of Q, L, and L. In other words, a linkage portion according to the first aspect binds at least one amino acid in the amino acid sequence at the same time according to SEQ ID NO: 18, and at least one amino acid in the amino acid sequence according to 15º SEQIDNO: 19.
SEQ ID NO: 18 is a consensual sequence determined by comparing amino acids 30-38 of human C5a with the corresponding amino acid sequences in Pan troglodytes, Macaca mulatta, Sus scrofa, Equus caballus, Bos Taurus, Mus musculus, Rattus norvegicus, Canis lupus, eMonodelphis domestica. SEQ ID NO: 19 is a consensual sequence determined by comparing amino acids 66-72 of human C5a with the corresponding amino acid sequences in Pan troglodytes, Macaca mulatta, Sus scrofa, Equus caballus, Bos Taurus, Mus musculus, Rattus norvegicus, Canis lupus, and Monodelphis domestica.
In preferred embodiments of the first aspect, the linking moiety binds to at least one amino acid of the amino acid sequence XETCEX; R (SEQ ID NO: 20), where X; and X; are defined in the previous way. SEQ ID NO: 20 is a shorter version of the consensus sequence according to SEQ ID NO: 18, and corresponds to human C5a amino acids 31-37.
In preferred embodiments of the first aspect, the linking moiety binds to at least one amino acid of the amino acid sequence XsKX7Xg8Xo (SEQ ID NO: 21), preferably KX; X; g, where Xç, X7, Xg, E XX, are defined in the previous way. SEQ ID NO: 21 is a shorter version of the consensus sequence according to SEQ ID NO: 19 and corresponds to amino acids 67-71 of human C5a. KX; X; it is a shorter version of the consensual sequence according to SEQ ID NO: 21 and corresponds to amino acids 68-70 of human C5a.
In particularly preferred embodiments, the linking portion binds at least one amino acid in the amino acid sequence XETCEX; R (SEQ ID NO: 20), and at least one amino acid in the amino acid sequence KX; X; g, where X ,, X3, X7, and Xg are defined as above.
In preferred embodiments of the first aspect, the conformational epitope is formed by (a) amino acid sequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3) of C5a (sequences of Homo sapiens and Pan troglodytes), (b ) HDETCEQRA (SEQ ID NO: 22) and SHKDLQL (SEQ ID NO: 23) amino acid sequences (Cacaa mulatta sequences), (c) DDETCEERA (SEQ ID NO: 24) and SHKNIQL (SEQ ID NO) amino acid sequences : 25) from C5a (Sus scrofa sequences), (d) DLETCEQRA (SEQ ID NO: 26) and SHKHIQL (SEQ ID NO: 27) amino acid sequences from C5a (Equus caballus sequences), (e) amino acid sequences DDETCEQRA (SEQ ID NO: 28) and HHKNMQL (SEQ ID NO: 29) from C5a (Bos taurus sequences), (f) amino acid sequences FYETCEERV (SEQ ID NO: 30) and PHKPVQL (SEQ ID NO: 31) from C5a (Mus musculus sequences), (g) KYETCEQRV (SEQ ID NO: 32) and HHKGMLL (SEQ ID NO: 33) amino acid sequences (Rattus norvegicus sequences), (h) YDETCEQRA amino acid sequences (SEQ ID
NO: 34) and SNKPLQL (SEQ ID NO: 35) from C5a (Canis lupus sequences), or (1) THETCEKRL (SEQ ID NO: 36) and NHKPVIL (SEQ ID NO: 37) amino acid sequences (sequences Monodelphis domestica). In preferred embodiments of the first aspect, the linking moiety binds to at least one amino acid of an amino acid sequence selected from the group consisting of (a) DETCEQR (SEQ ID NO: 4), (b) DETCEER (SEQ ID NO: 38), (c) LETCEQR (SEQ ID NO: 39), (e) YETCEER (SEQ ID NO: 40), () YETCEQR (SEQ ID NO: 41), and (g) HETCEKR (SEQ ID NO: 42) .
In preferred embodiments of the first aspect, the linking moiety binds to at least one amino acid of an amino acid sequence selected from the group consisting of (a) HKDMQ (SEQ ID NO: 5), preferably KDM; (b) HKDLQ (SEQ ID NO: 43), preferably KDL; (c) HKNIQ (SEQ ID NO: 44), preferably KNI; (d) HKHIQ (SEQ ID NO: 45), preferably KHI; (e) HKNMQ (SEQ ID NO: 46), preferably KNM; (O) HKPVQ (SEQ ID NO: 47), preferably KPV; (g) HKGML (SEQ ID NO: 48), preferably KGM; (h) NKPLQ (SEQ ID NO: 49), preferably KPL; and (1) HKPVI (SEQ ID NO: 50), preferably KPV.
In particularly preferred embodiments of the first aspect, C5a is human C5a. Thus, it is preferable that the linking portion binds to a conformational epitope formed by human amino acids NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3). In other words, a linking portion according to this - preferred embodiment of the first aspect, binds at least one amino acid in the amino acid sequence at the same time according to SEQ ID NO: 2, and at least one amino acid in the sequence of amino acids according to SEQ ID NO: 3. SEQ ID NO: 2 corresponds to amino acids 30-38 of human C5a. SEQ ID NO: 3 corresponds to amino acids 66-72 of
Human C5a.
The human C5a amino acid sequence is represented in SEQ ID NO: 1. In more preferred embodiments of the first aspect, the linking moiety binds to at least one of the DETCEQR amino acids (SEQ ID NO: 4). SEQ ID NO: 4 corresponds to human C5a amino acids 31-37.
In more preferred embodiments of the first aspect, the linking moiety binds to at least one of the HKDMQ amino acids (SEQ ID NO: 5), more preferably to at least one of the KDM amino acids.
SEQ ID NO: 5 corresponds to human C5a amino acids 67-71; the KDM sequence corresponds to human C5a amino acids 68-70.
In particularly preferred embodiments, the linking moiety binds at least one amino acid in the DETCEQR amino acid sequence (SEQ ID NO: 4), and at least one amino acid in the KDM amino acid sequence at the same time.
In preferred embodiments of the first aspect, the two sequences that form the conformational epitope (for example, sequence pairs according to SEQ ID NO: 18 and 19; SEQ ID NO: 2 and 3; SEQID NO: 22 and 23; SEQ ID NO: 24 and 25; SEQ ID NO: 26 and 27, SEQID NO: 28 and 29; SEQ ID NO: 30 and 31; SEQ ID NO: 32 and 33; SEQ ID NO: 34 and 35; SEQ ID NO: 36 and 37) are separated by contiguous amino acids 1-50 that do not participate in the linking portion of the invention.
In the following, such amino acids that do not participate in the binding in the linking portion of the invention will be referred to as "unbound amino acids". The two sequences that form the conformational epitope are preferably separated by 6-45 contiguous unbound amino acids, more preferably by 12-40 contiguous unbound amino acids, more preferably by 18-35 contiguous unbound amino acids, more preferably by 24-30 contiguous amino acids contiguous linkages, more preferably by 25-29 contiguous unrelated amino acids, even more preferably by 26-28 contiguous unrelated amino acids, and above all preferably by 27 contiguous unrelated amino acids.
In preferred embodiments of the first aspect, the binding portion has a binding constant in C5a, preferably human C5a, with a value of K; g of 10 nM or less, preferably 9 nM or less, more preferably 8 nM or less, more preferably 7 nM or less, more preferably 6 nM or less, more preferably 5 nM or less, more preferably 4 nM or less, more preferably 3 nM or less, more preferably 2 nM or less, and even more preferably 1 nM or less .
In preferred embodiments of the first aspect, a binding portion exhibits at least 75% blocking activity, preferably at least 80% blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% activity blocking, more preferably at least 95% blocking activity for biological effects induced by a C5a molecule, preferably human C5a. These preferred blocking activities refer to those embodiments in which the linking portion comprises a single parope that binds to C5a, preferably human C5a. In embodiments where the binding portion comprises two or more specific C5a paropes, said blocking activities of at least 75%, preferably at least 80%, more preferably at least 85%, etc. they are reached when a molecule of the binding portion is brought into contact with innumerable molecules of C5a, equal to the number of specific C5a particles in the binding portion. In other words, - when the paratopes of a binding portion of the first aspect and C5a are present in equimolar concentrations, the binding portion according to the first aspect exhibits at least 75% of blocking activity, preferably at least 80% of blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, and most preferably at least 95% blocking activity for C5a-induced biological effects. A preferred biological effect to be blocked is the release of C5a-induced lysozyme from whole human blood cells. The assays to determine this C5a-induced lysozyme release and blockade are described in the examples section.
In preferred embodiments of the first aspect, the binding portion does not inhibit CH50 activity in human plasma. Assays to determine CH50 activity are known to those skilled in the art and - are described below in the examples section.
In preferred embodiments of the first aspect, the binding portion does not exhibit a blocking activity on at least one biological effect induced by C5b, preferably the binding portion does not exhibit a blocking activity on any biological effect induced by C5b.
In preferred embodiments of the first aspect, the binding portion is able to reduce E. coli-induced IL-8 production in whole human blood. Assays to measure IL-8 production in whole blood are known to those skilled in the art and will be described below in the examples section.
In preferred embodiments of the first aspect, the binding portion is selected from: (a) antibodies or antigen-binding fragments thereof; (b) oligonucleotides; (c) antibody-like proteins or (d) peptidomimetics.
In preferred embodiments of the first aspect, the binding portion is an Antibody or antigen-binding fragment thereof, said antibody being selected from the group consisting of polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, antibodies - heteroconjugates, antibodies “Multispecific, de-immunized antibodies, chimeric antibodies, humanized antibodies (in particular grafted with CDR) and human antibodies.
In preferred embodiments of the first aspect, the binding portion is an antigen-binding fragment of an antibody, said fragment being selected from the group consisting of Fab fragments, Fab 'fragments, F (ab') fragments, Fd fragments, Fv fragments, disulfide-bound Fvs (Fvds), single domain antibodies (also known as nanobodies), and single chain Fv antibodies (Fvsc). In a particularly preferred embodiment of the first aspect, the binding moiety is an antibody or antigen binding fragment thereof comprising: (i) a heavy chain CDR3 sequence as shown in SEQ ID NO: 6; or (ii) a heavy chain CDR3 sequence as shown in SEQ ID NO: 7; wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the antibody or fragment thereof further comprises: (1) a light chain CDR3 sequence as shown in SEQ ID NO: 8; or (11) a light chain CDR3 sequence - as shown in SEQ ID NO: 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the antibody or fragment further comprises at least one of the following sequences: (1) a heavy chain CDR) 2 sequence according to SEQ ID NO: 10; (11) a heavy chain CDR2 sequence according to SEQ ID NO: 11; (iii) a light chain CDR2 sequence according to SEQ ID NO: 12; (iv) a light chain CDR2 sequence according to SEQ ID NO: 13; (v) a heavy chain CDR1 sequence according to SEQ ID NO: 14; (vi) a heavy chain CDRI1 sequence according to SEQ ID NO: 15; (vii) a light chain CDR1 sequence according to SEQ ID NO: 16; or (viii) a light chain CDR1 sequence according to SEQ ID NO: 17; wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDRI sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the total number of these optional exchanges in each of the amino acid sequences according to SEQ ID NO: 6, SEQ ID NO: 7, SEQIDNO: 38, SEQID NO: 59, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, that is, the total number of changes, deletions and additions in each sequence,
is lou2. In some embodiments of the first aspect, the linker is an oligonucleotide.
In these embodiments, it is further preferable that the oligonucleotide is a nucleic acid aptamer, such as a DNA aptamer, or RNA aptamer, or a mixed aptamer comprising DNA and RNA nucleotides.
In some embodiments, one or more nucleotides can be replaced by modified nucleotides,
such as 2º-fluorine-substituted pyrimidines. The nucleic acid aptamers can also be conjugated to fluorescent reporter molecules, affinity tags and / or macromolecules. For example, conjugating the aptamer with polyethylene glycol (PEG) or a comparable macromolecule will increase the biological half-life of the aptamer.
In some embodiments of the first aspect, the binding moiety is an antibody-like protein, for example, an antibody-like protein as exemplified earlier in the "Definitions" section.
In some embodiments of the first aspect, the linker is a peptidomimetic. Peptidomimetics suitable for practicing the present invention are preferably based on antibody-like proteins in the manner described above.
A preferred embodiment of the first aspect refers to the binding portion for the prevention and or treatment of various diseases involving acute inflammation, such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / related injuries reperfusion such as ischemic heart disease, acute lung injury, pneumonia, acute or chronic graft rejection in transplant patients, host graft reactions, but also diseases involving chronic types of inflammation such as renal glomerular diseases, such as glomerulonephritis and others renal failure entities, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, tumor growth or solid organ cancer.
In a second aspect, the present invention relates to an Antibody or Antigen-binding fragment thereof comprising: (i) a heavy chain CDR3 sequence as set forth in SEQ ID NO: 6; or (ii) a heavy chain CDR3 sequence as shown in
SEQ ID NO: 7; wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NC: 6, that is, the total number of changes, deletions and additions, is 1 or 2. Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NO: 7,
that is, the total number of changes, deletions and additions, is 1 or 2. In preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof further comprises: (1) a light chain CDR3 sequence as shown SEQ ID NO: 8; or (il) a light chain CDR3 sequence as shown in SEQ ID NO: 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NO: 8, that is, the total number of changes, deletions and additions, is 1 or 2. Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NO: 9,
that is, the total number of changes, deletions and additions, is 1 or 2. The second aspect of the present invention also relates to an antibody or antigen-binding fragment thereof comprising: (1) a CDR3 sequence of light chain as shown in SEQ ID NO: 8; or (11) a light chain CDR3 sequence as shown in SEQ ID NO 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NO: 8, that is, the total number of changes, deletions and additions, is | or 2.
Preferably, the total number of these optional exchanges in the amino acid sequence of SEQ ID NO: 9, that is, the total number of changes, deletions and additions, is 1 or 2. In preferred embodiments of the second aspect, the antibody or fragment thereof antigen-binding additionally comprises at least one of the following sequences: (1) a heavy chain CDR2 sequence according to SEQ ID NO: 10; (1) a heavy chain CDR2 sequence according to SEQ ID NO: 11; (111) a light chain CDR2 sequence according to SEQ ID NO: 12; (iv) a CDR2 - light chain sequence according to SEQ ID NO: 13; (v) a heavy chain CDRI1 sequence according to SEQ ID NO: 14; (vi) a heavy chain CDRI1 sequence according to SEQ ID NO: 15; (vile) a light chain CDRI1 sequence according to SEQ ID NO: 16; or (vil) a light chain CDR1 sequence according to SEQ ID NO: 17; wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
Preferably, the total number of these optional exchanges in each of the amino acid sequences according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ
ID NO: 17, that is, the total number of changes, deletions and additions in each sequence, is 1 or 2. In preferred embodiments of the second aspect, the antibody is selected from the group consisting of polyclonal antibodies, —monoclonal antibodies, antibodies monovalent, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, de-immunized antibodies, chimeric antibodies, humanized antibodies (in particular grafted with CDR) and human antibodies. In preferred embodiments of the second aspect, the fragment — antigen binding of an antibody is selected from the group consisting of Fab fragments, Fab fragments, F (ab) fragments, Fd fragments, Fv fragments, disulfide-bound Fvs (Fvds) , single domain antibodies (also known as nanobodies) and single chain Fv antibodies (Fvsc).
In preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof binds to a conformational epitope formed by amino acid sequences XXETCEX; RX, (SEQ ID NO: 18) and XsXsKX; XsXoL (SEQ ID NO: 19) from C5a, where X, is selected from the group consisting of N, H, D, EF, K, Y, and T; X; is selected from the group consisting of D, L, Y, and H; X; 3 is selected from the group consisting of Q, E, and K; X, is selected from the group consisting of A, V, and L; Xs is selected from the group consisting of S, H, P, and N; X; is selected from the group consisting of H and N; X; is selected from the group consisting of D, N, H, P, and G; X; is selected from the group consisting of M, L, LL and V. and Xo is - selected from the group consisting of Q, L, and I. In other words, an antibody or antigen-binding fragment according to the second aspect it binds at least one amino acid in the amino acid sequence according to SEQ ID NO: 18, and at least one amino acid in the amino acid sequence according to SEQ ID NO: 19.
Preferably, the antibody or antigen-binding fragment thereof binds to at least one amino acid of the amino acid sequence XçETCEX; R (SEQ ID NO: 20), where X; and X; are defined in the previous way.
Preferably, the antibody or antigen-binding fragment thereof binds only to an amino acid of the amino acid sequence XçKX; XgX, (SEQ ID NO: 21), more preferably KX; X; g, where Xç, X7, Xg, € X9 are defined in the previous way.
In particularly preferred embodiments, the antibody or antigen-binding fragment binds to at least one amino acid in the 10 ° XETCEX amino acid sequence; R (SEQ ID NO: 20), and to at least one amino acid in the same sequence. amino acids KX; X ;, where X>, X3, X7, and X; are defined in the previous way.
In preferred embodiments of the second aspect, the conformational epitope is formed by (a) amino acid sequences NDETCEQRA (SEQIDNO: 2) and SHKDMQL (SEQ ID NO: 3) of C5a (sequences of Homo sapiens and Pan troglodytes); (b) amino acid sequences HDETCEQRA (SEQ ID NO: 22) and SHKDLQL (SEQ ID NO: 23) of C5a (Macaca mulatta sequences); (c) DDETCEERA (SEQ ID NO: 24) and SHKNIQL (SEQ ID NO: 25) amino acid sequences from C5a (Sus scrofa sequences); - (d) DLETCEQRA (SEQ ID NO: 26) and SHKHIQL (SEQ ID NO: 27) amino acid sequences from C5a (Equus caballus sequences) ;, (e) DDETCEQRA (SEQ ID NO: 28) and HHKNMQL amino acid sequences (SEQ ID NO: 29) from C5a (Bos taurus sequences); (f) amino acid sequences FYETCEERV (SEQ ID NO: 30) and PHKPVQL (SEQ ID NO: 31) of C5a (Mus musculus sequences); (g) KYETCEQRV (SEQ ID NO: 32) and HHKGMLL (SEQ ID NO: 33) amino acid sequences (Rattus norvegicus sequences); (h) amino acid sequences YDETCEQRA (SEQ ID NO: 34) and SNKPLQL (SEQ ID NO: 35) of C5a (Canis lupus sequences); or (1) THETCEKRL amino acid sequences (SEQ ID NO: 36) and
NHKPVIL (SEQ ID NO: 37) from C5a (Monodelphis domestica sequences). In more preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof binds to at least one amino acid in an amino acid sequence selected from the group consisting of: (a) DETCEQR (SEQ ID NO: 4); (b) DETCEER (SEQ ID NO: 38); (0) LETCEQR (SEQ ID NO: 39); (e) YETCEER (SEQ ID NO: 40); () YETCEQR (SEQ ID NO: 41) and (g) HETCEKR (SEQ ID NO: 42). In even more preferred embodiments of the first aspect, the antibody or antigen-binding fragment thereof binds to at least one amino acid in an amino acid sequence selected from the group consisting of (a) HKDMQ (SEQ ID NO: 5), preferably KDM; (b) HKDLQ (SEQ ID NO: 43), preferably KDL; (c) HKNIQ (SEQ ID NO: 44), preferably KNI; (d) HKHIQ (SEQ ID NO: 45), preferably KHI; (e) HKNMQ (SEQ ID NO: 46), preferably KNM; (1) HKPVYQ (SEQID NO: 47), preferably KPV; (8) HKGML (SEQ ID NO: 48), preferably KGM; (h) NKPLQ (SEQ ID NO: 49), preferably KPL;
and (1) HKPVI (SEQ ID NO: 50), preferably KPV.
In particularly preferred embodiments of the second aspect, C5a is human C5a.
Thus, it is preferable that the antibody or antigen-binding fragment thereof binds to a conformational epitope formed by amino acids NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3) from human C5a.
In other words, an antibody or antigen-binding fragment thereof in accordance with this preferred embodiment of the second aspect, binds at the same time in at least - one amino acid in the amino acid sequence according to SEQ ID NO: 2, and in at least at least one amino acid in the amino acid sequence according to SEQ ID NO: 3. In more preferred embodiments of the second aspect, the antibody or antigen binding fragment thereof binds to at least one amino acid in the DETCEQR amino acid sequence (SEQ
ID NO: 4). In more preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof binds to at least one amino acid in the HKDMQ amino acid sequence (SEQ ID NO: 5), more preferably to at least one amino acid in the amino acid sequence - In particularly preferred embodiments, the antibody or antigen-binding fragment thereof binds to at least one amino acid in the DETCEQR amino acid sequence (SEQ ID NO: 4) and to at least one amino acid in the KDM amino acid sequence at the same time .
In preferred embodiments of the second aspect, the antibody or antigen binding fragment thereof has a binding constant for C5a, preferably human C5a, with a K value; 10 nM or less, preferably 9 nM or less, more preferably 8 nM or less, more preferably 7 nM or less, more preferably 6 nM or less, more preferably 5 nM or less, more preferably 4 nM or less, more preferably 3 nM or less, more preferably 2 nM or less, and even more preferably 1 nM or less.
In preferred embodiments of the second aspect, an antibody or antigen binding fragment thereof exhibits at least 75% blocking activity, preferably at least 80% deblocking activity, more preferably at least 85% blocking activity, plus preferably at least 90% blocking activity, more preferably at least 95% blocking activity for biological effects induced by a C5a molecule, preferably human C5a. These preferred blocking activities refer to those modalities, in which the antibody (or the antigen-binding fragment thereof) comprises a single parope that binds to C5a, preferably human C5a. In embodiments in which the antibody (or the antigen binding fragment thereof) comprises two or more specific C5a paropes, said blocking activities of at least 75%, preferably at least
80%, more preferably at least 85%, etc., are achieved when a binding portion molecule is brought into contact with numerous C5a molecules, equal to the number of specific C5a paropes in the antibody (or the fragment thereof) antigen binding). For example, a typical anti-C5a antibody of type I8G comprises two paratopes capable of binding to C5a, whereas a typical anti-C5a antibody of type IM comprises ten parapsides capable of binding to C5a. Thus, the blocking activity of an IgG-type antibody can be determined by placing said antibody in contact with C5a in a molar ratio of 1: 2. The blocking activity of an antibody of type IZM can be determined by putting said antibody in contact with C5a in a molar ratio of 1:10. When choosing these molar ratios, the paratopes in the antibody and C5a are present in equimolar concentrations. In other words, when the antibodies of an antibody (or antigen-binding fragment thereof) according to the second aspect and C5a are present in equimolar concentrations, the antibody or antigen-binding fragment exhibits at least 75% of blocking activity, preferably at least 80% blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, and most preferably at least 95% blocking activity for induced biological effects by C5a. A preferred biological effect to be blocked is the C5a-induced lysozyme release from whole human blood cells. Assays to determine this C5a-induced lysozyme release and its blockages are described in the examples section.
In preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof does not inhibit CH50 activity in human plasma. Assays to determine CH50 activity are known to those skilled in the art and are described below in the examples section.
In preferred embodiments of the second aspect, the antibody or antigen binding fragment thereof does not exhibit a blocking activity in at least one 'biological effect induced by C5b, preferably the antibody or antigen binding fragment thereof does not exhibit an activity of blockade in any biological effect induced by C5b.
In preferred embodiments of the second aspect, the antibody or antigen-binding fragment thereof is capable of reducing E. coli-induced IL-8 production in whole human blood.
Assays to measure the production of IL-8 in whole blood are known to those skilled in the art - they will be described below in the examples section.
A preferred embodiment of the second aspect concerns the antibody, or antigen-binding fragment thereof, for the prevention and / or treatment of various diseases involving acute inflammation such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, shock septic, ischemia / reperfusion-related injuries such as ischemic heart disease, acute lung injury, pneumonia, acute or chronic graft rejection in transplant patients, graft-versus-host reactions, but also diseases involving chronic types of inflammation such as renal glomerular diseases , such as glomerulonephritis and other renal failure entities, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, tumor growth or solid organ cancer.
In preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises one of the sets of sequences of heavy chain CDR3, heavy chain CDR2 and heavy chain CDR1, as listed below in table 1 , wherein each heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; and wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions: Table 1: Suitable heavy chain CDR sequence sets - for use in the antibodies, or fragments thereof, of the present invention CDR3 Sequence Symbol CDR2 Sequence | CDR1 heavy chain assembly sequence SEQID NO: 6 SEQ ID NO: 10 | —B | SEQIDNOS SEQ ID NO: 10 SEQ ID NO: 15 SEQ ID NO: 6 SEQ ID NO: 11 SEQ ID NO: 14 | —D | SEQIDNOS6 SEQ1ID NO: 11 SEQ ID NO: 15 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 10 SEQID NO: 15 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 11 SEQ ID NO: 14 SEQ ID NO: 7 SEQ1ID NO: 11 SEQ ID NO: 15 In preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises one of the following sets of light chain CDR3, light chain CDR2 sequences and light chain CDRI1 listed in table 2, wherein each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2.0 or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; and wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions: Table 2: Light chain CDR sequence sets suitable for use in the antibodies or fragments thereof of the present invention Since the light chain CDR2 sequence of the INab308 antibody (SEQ ID NO: 12) is identical to the light chain CDR2 sequence of the INab708 antibody (SEQ ID NO: 13), the sets that include SEQ ID NO: 13 would be redundant with the sets that include SEQ ID NO: 12. Therefore, the table lists only four sets of light chain CDR sequences. set of In preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises one of the AH heavy CDR sets listed above in Table 1, and one of the light chain I-IV CDR sets listed above in table 2, that is, one of the following combinations of sets: AI, A-II, A-III, A-IV, B-1, B-II, B-III, B-IV, CI, C-II , C-III, C-IV, DI, D-II, D-III, D-IV, EI, E-II, E-II, E-IV, FI, F-II, F-III, F-IV , GI, G-II, G-III, G-IV, HI, H-II, H-III, or H-IV, wherein each heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, - preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; and wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
In preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises a VH domain that comprises, consists essentially of or consists of (1) the INab308 VH domain or (11) the INab708 VH domain.
The FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequences that define the INH308 and INab708 VH domains are shown below in table 4.
In preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises a VL domain that comprises, essentially consists of or consists of (1) the INab308 VL domain or (11) the INab708 VL domain .
The sequences of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 that define the VL domains of INab308 and INab708 are shown below - natabelad4.
In additional preferred embodiments of the first and second aspects, the antibody or antigen binding fragment thereof comprises a VH domain and a VL domain, in which: (1) said VH domain comprises, essentially consists of or consists of the VH domain of INab308, and said VL domain comprises, essentially consists of or consists of the INL308 VL domain; or (1) said VH domain comprises, essentially consists of or consists of the INab708 VH domain, and said VL domain. comprises, consists essentially of, or consists of the VL domain of INab708.
In preferred embodiments of the first and second aspects, the antibody or antigen-binding fragment thereof comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs, as described herein, comprises said CDRs in one human antibody structure.
The reference here to an antibody comprising, with respect to its heavy chain, a particular chain, or a particular region or sequence, preferably refers to the situation in which all the heavy chains of said antibody comprise said particular chain, region or sequence . This applies correspondingly to the light chain of an antibody.
The precept provided here with respect to specific nucleic acid and amino acid sequences, for example, those shown in the sequence listing, must be interpreted in a way that is also related to the modifications of said specific sequences, resulting in sequences that are functionally equivalent to those said specific sequences, for example, amino acid sequences that exhibit properties identical or similar to those of specific amino acid sequences, and nucleic acid sequences that encode the sequences - of amino acids that exhibit properties identical or similar to those of the amino acid sequences encoded by the sequences nucleic acids. An important property is to maintain the binding of an antibody to its target or to sustain the effector functions of an antibody. Preferably, a sequence modified with respect to a specific sequence, when it replaces the specific sequence in an antibody, maintains the binding of said antibody in C5a, in particular in the conformational epitope of C5a identified herein, and preferably maintains the functions of said antibody in the manner described herein, for example, by blocking the release of C5a-induced lysozyme from whole human blood cells, and / or by reducing E. coli-induced IL-8 production in whole human blood.
It will be understood by those skilled in the art that, in particular, CDR sequences, hypervariable and variable regions can be modified without losing the ability to bind to C5a. For example, the CDR regions will be both identical and highly homologous to the regions specified here. It is contemplated as "highly homologous" that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions, deletions, or additions can be performed on CDRs. In addition, the hypervariable and variable regions can be modified in such a way that they show homology - substantial with the regions specifically disclosed herein.
Furthermore, it may be desired in accordance with the present invention to modify the amino acid sequences described herein, in particular those of human heavy chain constant regions to adapt the sequence to a desired allotype, for example, an allotype found in the - population Caucasian.
The present invention further comprises antibodies in which changes have been made in the Fc region in order to change the functional or pharmacokinetic properties of the antibodies. Such changes may result in a decrease or increase in the binding of Clq and CDC or in the binding of FeyR and ADCC. Substitutions, for example, can be prepared on one or more of the amino acid residues of the heavy chain constant region, thereby causing a change in an effector function, while maintaining the ability to bind to the antigen, compared to the antibody. modified, cf. U.S. patent 5,624,821 and U.S. patent
5,648,260.
The in vivo antibody half-life can be improved by modifying the recovery of the receptor epitope from the Ig constant domain or an Ig constant domain, in such a way that the molecule does not comprise an intact CH2 domain or an intact Ig Fc region, cf . U.S. patent 6,121,022 and U.S. patent 6,194,551. In vivo half-life, moreover, can be increased by mutating in the Fc region, for example, replacing threonine with leucine at position 252, replacing threonine with serine at position 254, or replacing threonine with phenylalanine at position 256, cf. U.S. patent 6,277,375.
Furthermore, the pattern of antibody glycosylation can be modified in order to change the effector function of antibodies. For example, antibodies can be expressed in a transfectome that does not add the fucose unit normally attached to Asn at position 297 of the Fc region, in order to improve the affinity of the Fc region for Fc receptors which, in turn, will result in greater ADCC of antibodies in the presence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733. Furthermore, galactosylation modification can be carried out in order to modify CDC.
Alternatively, in another embodiment, mutations can be introduced randomly over all or part of a sequence encoding anti-C5a antibody, such as by saturation mutagenesis, and the resulting modified anti-C5a antibodies can be selected with respect to bonding activity.
In a third aspect, the present invention relates to a pharmaceutical composition comprising (a) the binding portion according to the first aspect, or (b) the antibody or antigen binding fragment thereof according to the second aspect, and which additionally comprises one or more carriers, diluents, excipients, fillers, binders, lubricants, nonstick, disintegrants, adsorbents and / or pharmaceutically acceptable preservatives.
In a fourth aspect, the present invention relates to the use of (a) a binding portion according to the first aspect, or (b) an antibody or antigen binding fragment thereof according to the second aspect, for the preparation of a pharmaceutical composition in the prevention and or treatment of various diseases involving acute inflammation such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injuries such as ischemic heart disease, lung injury acute, pneumonia, acute or chronic graft rejection in transplant patients, graft-versus-host reactions, but also diseases involving chronic types of inflammation - such as renal glomerular diseases, such as glomerulonephritis and other renal failure entities, rheumatoid arthritis and diseases similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, t growth sweat or solid organ cancer.
In a fifth aspect, the present invention relates to a method of preventing and or treating various diseases involving acute inflammation such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injuries such as ischemic heart disease, acute lung injury, pneumonia, acute or chronic graft rejection in transplant patients, graft-versus-host reactions, but also diseases involving chronic types of inflammation - such as renal glomerular diseases, such as glomerulonephritis and other entities renal failure, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, tumor growth or solid organ cancer in a patient who needs it, the method comprising administering to the patient an effective amount of (a) a binding portion according to the first aspect, or (b) an ant antibody or antigen-binding fragment thereof according to the second aspect. In the practice of any aspect of the present invention, a pharmaceutical composition, in the manner described above, or a "binding" portion (for example, an antibody or antigen-binding fragment thereof), can be administered to a patient by any route established in the technique that provides a sufficient level of the connecting portion to the patient. It can be administered systemically or locally. Such administration can be parenterally, transmucosally, for example, orally, nasally, rectally, intravaginally, sublingually, submucosally, transdermally or by inhalation. Preferably, the administration is parenteral, for example, by intravenous or intraperitoneal injection, and also includes, but is not limited to, intra-arterial, intramuscular, intradermal and subcutaneous administration. If the pharmaceutical composition of the present invention is administered locally, it can be injected directly into the organ or tissue to be treated, for example, the organ affected by a tumor. Pharmaceutical compositions adapted for oral administration can be supplied as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or creams; or as emulsions. Hard gelatin tablets or capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. The solutions and syrups can comprise water, polyols and sugars.
An active agent intended for oral administration can be coated or mixed with a material that slows down the disintegration and / or absorption of the active agent in the gastrointestinal tract (for example,
glyceryl monostearate or glyceryl distearate can be used). Thus, the continuous release of an active agent can be achieved for many hours and, if necessary, the active agent can be protected from being degraded in the stomach. Pharmaceutical compositions for oral administration can be formulated to facilitate the release of an active agent at a particular gastrointestinal site, by virtue of specific pH or enzyme conditions. Pharmaceutical compositions adapted for transdermal administration can be supplied as discrete adhesives intended to - remain in close contact with the recipient's epidermis for an extended period of time. Pharmaceutical compositions adapted for topical administration can be supplied as plasters, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye, or other external tissues, a topical patch or cream is preferably used. When formulated in a plaster, the active ingredient can be used with either a paraffinic plaster base or a water miscible. Alternatively, the active ingredient can be formulated into a cream with an oil-in-water or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the active ingredient can be dissolved or suspended in a suitable carrier, for example, in an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include tablets, lozenges and chewable tablets.
Pharmaceutical compositions adapted for nasal administration may comprise solid carriers such as powders (preferably with a particle size in the range of 20 to 500 microns). Powders can be administered in a manner in which sneezing is obtained, that is, by rapid inhalation through the nose, from a powder container held close to the nose. Alternatively, the compositions adopted for nasal administration may comprise liquid carriers, for example, nasal sprays or nasal drops. These compositions can comprise aqueous or oily solutions of the active ingredient. Compositions for administration by inhalation can be supplied in specially - adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed to provide predetermined dosages of the active ingredient. In a preferred embodiment, the pharmaceutical compositions of the invention are - administered via the nasal cavity to the lungs.
Pharmaceutical compositions adapted for rectal administration can be supplied as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration can be supplied as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
Pharmaceutical compositions adapted for parenteral administration include sterile aqueous or non-aqueous injectable solutions or suspensions that may contain antioxidants, buffers, bacteriostats and solutes that make the compositions substantially isotonic with the blood of a desired container. Other components that may be present in such compositions include water, alcohols, polyols, glycerin and vegetable oils, for example. Compositions adapted for parenteral administration can be presented in single or multiple dose containers, for example, sealed ampoules and vials, and can be stored in a condition — frozen-dry (lyophilized), which requires only the addition of a liquid carrier sterile, for example, sterile saline solution for injections, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration in humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to decrease pain at the injection site. In general, the ingredients are supplied either separately or mixed together in a single dosage form, for example, as a dry or concentrated lyophilized powder without water in an airtight container, such as an ampoule or sachet, indicating the amount of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, a sterile saline ampoule can be provided, so that the ingredients can be mixed before administration. In another embodiment, for example, a chemoattraction inhibitor can be delivered in a controlled release system. For example, the inhibitor can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or - other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al. (1980) Surgery 88: 507; Saudek et al. (1989) N. Eng J. Med. 321: 574). In another embodiment, the compound can be distributed in a vesicle, in particular a liposome (see Langer (1990) Science 249: 1527-1533; Treat etal (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, NY, 353-365; WO 91/04014; US
4,704,355). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (1974) Langer and Wise (eds.), CRC Press: Boca Raton, Fla .; Controlled Drug Bioavailability,
Drug Product Design and Performance, (1984) Smolen and Ball (eds.), Wiley: N.Y .; Ranger and Peppas (1953) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al. (1985) Science 228: 190; During et al. (1989) Ann. Neurol. 25: 351; Howard et al. (1989) J. Neurosurg. 71: 105).
In yet another modality, a controlled release system can be placed in close proximity to the therapeutic target, that is, the target cells, tissue or organ, thus requiring only a portion of the systemic dose (see, for example, Goodson (1984) 115 -138 in Medical Applications of Controlled Release, vol. 2). Other controlled-release systems are - discussed in Langer's review (1990, Science 249: 1527-1533).
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, for example, together with a dressing after surgery, by injection, by means of a catheter, by means of a suppository , or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including membranes, such as silastic membranes or fibers.
The selection of the preferred effective dose will be determined by those skilled in the art, who are based on considering various factors that will be known by such experts. Such factors include the particular form of the pharmaceutical composition, for example, polypeptide or vector, and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be established during the common developmental procedures typically employed in obtaining regulatory approval. for a pharmaceutical compound. Additional factors that consider the dose include the condition or disease to be prevented and or treated, or the benefit of being achieved in a normal individual, the patient's body mass, the route of administration, whether the administration is acute or chronic,
concomitant medications, and other well-known factors that affect the efficiency of administered pharmaceutical agents. Thus, the exact dosage can be decided according to the judgment of the medical professional and the circumstances of each patient, for example, depending on the condition and immune status of the individual patient according to standard clinical techniques. In the practice of any aspect of the present invention with respect to preventing and or treating tumor growth or solid organ cancer, the subject who is administered with the binding or antibody portion of the invention is treated additionally with an agent - chemotherapeutic, radiation or an agent that modulates, for example, improves or inhibits the expression or activity of an Fc receptor, for example, an Fc-gamma receptor, such as a cytokine. Typical cytokines for administration during treatment include granulocyte colony stimulating factor (G-CSF), macrophage and granulocyte colony stimulating factor (GM-CSF), interferon-y (IFN-y), and tumor necrosis factor (TNF). Typical therapeutic agents include, but are not limited to, anti-neoplastic agents such as doxorubicin, cisplatin, taxotere, 5-fluorouracil, methotrexate, genzitabine and cyclophosphamide. The following figures and examples are merely illustrative of the present invention and cannot be construed as limiting the scope of the invention in any way indicated by the appended claims.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the effect of C5a mutants on the release of enzyme from blood cells. The potential amino acids in C5a molecules for the constitution of antibody epitopes have been mutated to alanine, and these C5a mutants have been tested with regarding its bioactivity to induce the release of lysozyme from whole human blood cells. The mutation at the C5a site, which results in more than 50% loss of bioactivity compared to human C5a, was considered to be an important site for the biological function of C5a.
These sites are 24, 29, 31, 37.68 and 69. Figure 2 shows the binding capacity of INab308 in C5a C5a mutants and C5a mutants were coated on a 96-well plate.
The binding capabilities of INab308 to these proteins were assessed by an ELISA approach.
Loss of binding capacity greater than 50% is considered to be significant.
The data indicate that INab308 is linked to the two regions, 31-37 and 68-69. Figure 3 shows the binding capacity of INab708 to C5a C5a mutants and C5a mutants were coated on a 96-well plate.
The binding capabilities of INab708 to these proteins were assessed by ELISA approach.
Loss of binding capacity greater than 50% is considered to be significant.
The data indicate that INab708 is linked to the two regions, 31-37 and 68-70. Figure 4 shows that INab308 and INab708 do not affect CHS50 activity in human plasma The hemolytic activity of human plasma was determined by a classic CH50 assay.
Human C5a Mabs, including INab708, INab308 and F20, were preincubated with human plasma, and then the CHS50 assay was performed subsequently.
Among these antibodies, F20 strongly inhibits CH50 activity, while INab708 and INab308 have no influence when used at a concentration of approximately 5 µM, which is significantly higher than the C5 concentration that occurs in whole human blood (approximately 0.4 one). Figure 5 shows a comparison of the blocking effects of INab308, INab708 and L2B23 on C5a bioactivity The blocking activity of INab308, INab708 and L2B23 was assessed by lysozyme induced by the C5a release assay. The antibody to C5a molar ratio was adjusted to 1: 2 to assess the blocking activity of an antibody on two C5a molecules elicited by the biological effect. The data show that INab308 and INab708 have very high - blocking activity (290%) in C5a bioactivity, while L2B23 shows only a minimal effect.
Figure 6 shows the inhibitory effect of INab308 and INab708 on E.coli-induced IL-8 production in whole human blood E. coli was incubated with whole blood for 4 hours, and IL-8 levels were assessed by ELISA. In the presence of INab308 and INab708 during the incubation, IL-8 levels were significantly attenuated (P <0.01), although there was no significant reduction in the presence of L2B23. EXAMPLES
1. Methods
1.1 Preparation of recombinant and mutant C5a C5a: The DNA sequences encoding human C5a were obtained by polymerase chain reaction using reverse transcriptase (RT-PCR) using RNA isolated from peripheral blood leukocytes. C5a - mutants were generated using PCR methods by introducing GCT (alanine) at the mutation site. The C5a DNA was then ligated with pET-32a (Novagen, Gibbstown, NJ), and the ligation mixture was used to transform competent JM109 cells. The expression plasmids were transformed into BL21 using a standard calcium chloride method. A single colony of a plate with Luria-Bertani broth (LB) was chosen, inoculated in the LB medium with ampicillin, and incubated at 37ºC for the entire night. The culture was transferred to 2 L of LB medium and incubated at 37ºC until the medium-exponential phase (A600 = 06), and then isopropyl-b-D-thiogalactopyranoside (IPTG) was added in a final concentration of 0.1 mM. The cells continued to grow naturally at 30 ° C overnight, and were collected by centrifuging the culture at 7,000 rpm, 4 ° C, for 15 minutes. After washing with phosphate buffered saline (PBS; 10 mM PB, 150 mM NaCl [H 7.4]) once, the bacterial precipitates were resuspended in embPBS and sonicated on ice. After centrifugation at 12,000 rpm, 4ºC, for 15 minutes, the soluble portion was separated from the insoluble precipitate. To purify recombinant human C5a, the cell lysate supernatant was loaded onto a nickel-chelated affinity column pre-equilibrated with PBS. Then, the column was washed with 50 mM imidazole and 200 mM imidazole in PBS, - respectively. Finally, the bound proteins were eluted in 500 mM imidazole, dialyzed against PBS overnight, and analyzed by polyacrylamide dodecyl sulfate gel electrophoresis (SDS-PAGE).
1.2 Immunization and hybridoma selection by ELISA: Monoclonal antibodies were prepared using hybridoma methods. The immunization and production of MAbs were performed using standard protocols. Five 4-week-old female BALB / c mice were immunized subcutaneously with 100 µg of purified recombinant C5a in complete Freund's adjuvant per animal. The animals were re-immunized twice at 4-week intervals using 100 µg of antigen in incomplete Freund's adjuvant. Three days after the final re-immunization, the mice were sacrificed, and their splenocytes were fused with NS-1 in a 5: 1 ratio, and 200 µL of cells were plated in each well in five 96-well plates. The hybrids were selected in a Dulbecco-modified Eagle's medium (DMEM), supplemented with 20% fetal bovine serum and hypoxanthine 5x10 M, 2x10 ”M aminopterin, and 8x10 * M thymidine (HAT). After 8 days, cell clones that secrete antibodies against human C5a were selected by enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well plate was coated with
2 µg / ml of recombinant human C5a at 4 ° C overnight. After being blocked with 5% skimmed milk in PBS at 37 ° C for 1 hour, 50 μl of the culture medium of the growing clones were added to each well and incubated at 37 ° C for 1 hour, followed by 100 μL of anti-mouse antibody. goat marked with horseradish peroxidase (HRP) for 1 hour. The peroxidase reaction was developed with a solution that shows color containing 5.5 mM o-phenylene diamine hydrochloride (OPD); 8.5 mM. The absorbance of the light was measured at 492 nm with an ELISA reader (Anthos, Wals / Salzburg, Austria).
1.3 Production and purification of monoclonal antibodies: To produce Mab in greater quantity, 5x10º hybridoma cells were injected into the peritoneal cavity of mice. After 14 days, ascites were removed and centrifuged at 1,500 rpm, 4ºC, for 5 minutes. The supernatant was collected and applied to a protein A-Sepharose 4B column, which was pre-equilibrated in PBS. The bound Mab was eluted with citric acid (pH 4.0) and dialyzed against PBS overnight. The purified proteins were analyzed by SDS-PAGE.
1.4 Enzyme release assay with respect to C5a bioactivity The induction of enzyme release by degranulation is an important biological characteristic of C5a. In this study, fresh whole human blood from healthy volunteers was used to assess the effect of C5a on lysozyme release. The levels of lysozyme released from whole blood cells were analyzed by the EnzChek & lysozyme assay kit (Invitrogen, CA, USA). To study the blocking activity of anti-C5a antibodies, rhC5Sa (100 nM) was mixed with different concentrations of antibody. Next, whole blood cells were added immediately to avoid pre-incubating antibodies with C5a. After incubation, 50 µl of the supernatant samples were added to 50 µl of the diluted substrate solution. The plate was incubated at
37ºC for 30 minutes in the dark and then deals with Perkin Elmer 1420 multi-brand counter (Massachusetts, USA). The fluorescence intensity was measured with an excitation at 490 nm and an emission at 525 nm, and the standard zero value (white) was subtracted from all samples. Antibody blocking activity was calculated after subtracting the fluorescence intensity of rhC5a - independent lysozyme release (control buffer) from the fluorescence intensity of rhC5a - induced lysozyme release. Blocking activity was calculated with the following formula, blocking activity = C5a Fluorescence - (CSa + Ab) Fluorescence / C5a - Fluorescence - Rgluorescence Control Buffer-
1.5 ELISA analysis of the binding capacity of INab308 and INab708 to C5a or human C5a mutants A binding ELISA was performed to determine the binding activities of INab308 and INab708 to C5a and human C5a mutants. C5a mutants are produced by replacing the corresponding amino acid with alanine by introducing GCT into the mutation site from the cDNA level. These C5a mutants include mutation at 24, 29, 30, 31, 32, 35, 36, 37, 30/37 double mutation, 40, 53, 64, 65, 66, 68, 64/68, 66/68, 69, 70, and C-del (12 amino acids were eliminated from the C-terminus of C5a. Human C5a (Sigma C5788), recombinant C5a and C5a mutants (2 µg / mL) were coated with 96-well EIA plates (Costar 9018) at 4ºC for the whole night, after being blocked with 5% skimmed milk in PBS, at 37ºC, for 1 hour, 0.08, 0.4, 2 ug / mL of anti-C5a antibodies (INab308 and lINab708) prepared with dilution buffer was added to each well and incubated at 37ºC per | hour, followed by 100 μl of horseradish peroxidase-labeled goat anti-mouse antibody (HRP) for 1 hour. The peroxidase reaction developed color with the color development containing o-phenylene diamine hydrochloride (OPD) 5.5 mM and H75O; 8.5 mM The absorbance of light was measured at 492 nm with an ELISA reader (Anthos, Wals / Salzburg, Austria). The OD value for recombinant C5a has been adjusted to 100% of connection activity. The binding capacity of C5a mutants is calculated by c5a / ODc5a mutant OD
1.6 Plasma hemolytic activity (CH50): In summary, red sheep blood cells (sRBC) were prepared from fresh whole sheep blood by centrifugation, and were then sensitized with anti-sRBC. Plasma samples from healthy volunteers were serially diluted and incubated with sensitized -sRBC. After half an hour of incubation, the non-lysed cells were centrifuged, and the supernatants were read at 542 nm on a plate reader. To determine the effect of anti-C5a antibodies on C5 activation, equal volumes of anti-C5a antibody and plasma were preincubated for 1 hour before adding sensitized sRBC.
1.7 Production of IL-8 in whole blood from the EF infection model. coli: To assess the efficiency of anti-C5a antibodies in the condition close to clinical sepsis, 250 µL of whole blood from healthy volunteers were boosted with anti-C5a antibodies, and 250 µL of E. coli diluted in saline buffer with a concentration of 1 x 10 '/ ml was then added. After 4 hours of incubation at 37ºC, the supernatants were centrifuged and collected for analysis by ELISA for IL-8. The levels of IL-8 in the supernatants were analyzed using the IL-8 ELISA kit (BioLegend, USA).
1.8 Assay to select antibodies that bind to the new conformational epitope: In the 3-D structure of C5a obtained from the computer modeling method, the space epitopes containing peptide C5a, 28-40 (VYNNDETCEQRAAR, SEQ ID NO: 67), and C5a 65-70 peptide
(ISHKDM, SEQ ID NO: 68), can be viewed as random springs. When the two peptides are linked by a flexible linker peptide, GGGGS (SEQ ID NO: 69), the spatial epitopes are reconstructed resembling the conformation of the parental antigen, since the weak hydrophobic interaction of the two peptides guarantees a conformation in the form of pocket. The computer modeling analysis of the NH peptide; - 28-40- Linker (GGGGS) -65-70-COOH maintains the same conformation as the parental antigen. This new 24-AA peptide can be synthesized and conjugated to Californian keyhole limpet hemocyanin (KLH) to form an immunogen that — immunizes mice, and traditional hybridoma technology can be applied subsequently to obtain INab308 and INab708 using the new 24-peptide -AA, based on ELISA as a selection tool. A 96-well ELISA plate is coated with 1-2 µg / mL synthetic peptides with the conformational epitope at 4 ° C overnight. After being blocked with 5% skimmed milk in PBS, at 37ºC for 1 hour, 50 µL of hybridoma culture medium that grows in the clones is added to each well and incubated at 37ºC for | hour, followed by 100 μl of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) for 1 hour. The peroxidase reaction developed color with the development solution containing 5.5 mM o-phenylene diamine hydrochloride (OPD) and H5O; 8.5 mM. The absorbance of the light is measured at 492 nm with an ELISA reader.
2. Results
2.1 Identification of amino acids relevant to C5a activity Several amino acids in the C5a molecule, which constitute possible antibody epitopes, have been mutated to alanine. In particular, C5a mutants include site mutations at 24, 29, 30, 31, 32, 35, 36, 37, 30/37 double mutation, 40, 53, 64, 65, 66, 68, 64/68, 66/68, 69, 70, and C-del (12 amino acids were deleted from the C terminus
T2 of C5a). C5a mutants were tested for their bioactivity to induce lysozyme release from whole human blood cells (Figure 1).
The mutation at the C5a site that results in more than 50% of - loss of bioactivity, compared to human C5a, was considered an important site for the biological function of C5a. Thus, amino acid residues 24, 29, 31, 37, 68 and 69 were identified as important sites for function (Figure 1).
2.2 Characterization of epitopes in C5a linked by INab308 and INab708 antibodies
2,000 cell clones that secrete antibodies against human C5a were selected with the functional assay (enzyme release). Only two antibodies exhibited superior blocking activities.
These two antibodies, INab308 and INab708, were further characterized with respect to the particular amino acids in C5a recognized by the two antibodies.
In particular, several C5a mutants have been generated, in which one or more amino acids have been replaced by alanine. The INab308 and INab708 antibodies were placed in contact with these mutants and the degree of binding was determined by ELISA (see section 1.5 above). A loss of binding capacity greater than 50% (compared to wild type C5a) was considered significant.
The data indicate that INab308 is linked to the two regions, 31-37 and 68-69 (Figure 2). In the same way, INab708 binds in the two regions, 31-37 and68-70 (Figure 3).
Notably, the regions identified by both antibodies span four amino acid residues (31, 37, 68, and 69) that have been identified as important sites for C5a function in the section
2.1 previous.
2.3 Effect of INab308, INab708, and F20 antibodies on hemolytic activity of human plasma Total hemolytic complement titration (CH50) is a conventional method for determining activation of the classical complement pathway (see section 1.6). Monoclonal antibodies to C5a (INab708, INab308, and F20) were preincubated with human plasma at a concentration of approximately 5 µM, and then the CH50 assay was performed. Among these antibodies, F20 strongly inhibits CH50 activity, while INab708 and —INab308 have no influence (Figure 4). These results demonstrate that INab708 and INab308 do not interfere with C5b-mediated complement activation.
2.4 Effect of INab308, INab708, and L2B23 antibodies on C5a bioactivity The blocking activity of INab308, INab708, and L2B23 antibodies on C5a was assessed by the C5a-induced lysozyme release assay (see section 1.4). The antibody to C5a molar ratio was adjusted to 1: 2 to assess the blocking activity of an antibody on two C5a molecules elicited by the biological effect. These antibodies are divalent antibodies. In this way, choosing the anterior molar ratio of 1: 2, the antibodies on one side and C5a on the other side are present in equimolar concentrations. The data in figure 5 show that INab308 and INab708 have very high blocking activity on C5a bioactivity (94% and 100%, respectively), while L2B23 shows only a minimal effect (about 12%).
2.5 Effect of INab308 and INab708 antibodies on the production of IL-8 induced by E. coli in whole human blood To assess the efficiency of anti-C5Sa antibodies in a condition close to clinical sepsis, the production of IL-8 induced by E. coli in whole blood was determined. This test can be related to a model of E. coli infection (see also section 1.7). In the presence of INab308 and INab708 during incubation, IL-8 levels were significantly attenuated (P <0.01), although there is no significant reduction in the presence of L2B23 (Figure 6).
3. Summary Important properties of the preferred antibodies of the invention, INab308 and INab708, are summarized in Table 3 below. Also included are the comparative antibodies 888, MAb 137-26, Ab11876, G57, F20, L2B23 and G13 that do not simultaneously bind to both the amine sequences of the conformational epitope identified in the present invention, that is, the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Comparative antibodies 888, MAb 137-26, F20, G57, L2B23 and G13 bind only to one of these two amino acid sequences (888, MAb 137-26, F20, G57, and G13), or to a different sequence of amino acids (L2B23). The target epitope of Ab11876 is not known. Table 3: Neutralizing antibodies to C5a and the epitopes Monoclonal antibodies to human C5Sa were generated using classical hybridoma technology. The binding sites of Mabs in human C5a were determined by the alanine selection method. Mabs blocking activities were quantified by inhibiting C5a-induced lysozyme release from whole human blood cells.
(Kd: nM) for C5a blocking | | | | | ne [Oo o a a | | | | ss Sea O and ND: Not determined; * ATCC clone no.
PTA-3650 related to an antibody disclosed in European patent publication 1 878 441 A2; ** AbI 1876 is a mouse monoclonal anti-C5 / C5Sa antibody obtained from ABCAM (Cambridge, United Kingdom). As shown in Table 3, some comparative antibodies (8g8, MAb 137-26) exhibit better binding affinities with C5a than the preferred antibodies of the invention, INab308 and INab708. Despite this, INab308 and INab708 exhibit better blocking activities than any comparative antibody studied.
More specifically, each of INab308 and INab708 exhibits a very high blocking activity (> 90%), even when used in stoichiometric quantities, that is, in a ratio of 1 parotope to | epitope; that is, 0.5 antibody molecules for | target molecule.
Antibodies of the prior art (MAb 137-26, Ab11876) achieved reasonable blocking activities only when used in super-stoichiometric amounts.
These findings demonstrate that high binding affinity cannot always be equated with high blocking activity.
In summary, the present invention provides antibodies that exhibit extremely high blocking activity for the first time - even when used only in stoichiometric amounts.
The amino acid sequences of the complementarity determining regions of the heavy and light chains of the INab308 and INab708 antibodies are listed below in Table 4. Table 4: CDR and FR sequences of the INab308 and INab708 antibodies (Chothia classification mode) INab308 : INab708: Heavy chain: Heavy chain: FR1: QVQLOQSGPQLVRPGTSVKIS FR1: VOLLESGAELMKPGAS VKIS AND SEQ ID NO: 51) (SEQ ID NO: 59) CDRI1: CKASGYSFTTFWMD CDRI1: CKATGNTFSGY NO (14): SEQ: NO ) FR2: WVKQRPGQGLEWIGR FR2: WVKQRPGHGLEWIGE (SEQ ID NO: 52) (SEQ ID NO: 60) CDR2: IDPSDSESRLDQ CDR2: ILPGSGSTNYNE (= SEQ ID NO: 10) (= SEQ ID NO: 11) FR3: FR3: RFKDRATLTVDKSSSTVYMQLSSPTS - KFKGKATLTADTSSNTAYMQLSSLTSE
EDSAVYY DSAVYY (SEQ ID NO: 53) (SEQ ID NO: 61) CDR3: CARGNDGYYGFAY CDR3: CTRRGLYDGSSYFAY (= SEQ ID NO: 6) (= SEQID NO: 7) FR4: WGQGTLVTVSSA FR4: WGQGTLVTV (SEG IDL: 54) (SEQ ID NO: 62) Light chain: Light chain: FR1: DIVLTOSPASLAVSLGQRATIS FR1: DIVLTOSPASLAVSLGQRATIS (SEQ ID NO: 55) (SEQ ID NO: 63) CDRI1: CKASQSVYDYDGDSYMK CDRI1: SEQDDY NO: 17) FR2: WYQQKPGQPPKLL FR2: WYQQKPGQPPKLL (SEQ ID NO: 56) (SEQ ID NO: 64) CDR2: IYAASNL CDR2: IVAASNL (AND SEQ ID NO: 12) (= SEQID NO: 13) FR3: FR3: QSGIPS - GSGIPARFSGSGSGTDFTLNIHPVEEEV
DAATYY AATYY (SEQ ID NO: 57) (SEQ ID NO: 65) CDR3: COQSNEDPYT CDR3: COQNNEDPLT (= SEQID NO: 8) (= SEQID NO: 9) FR4: FGGGTKLEIK FR4: FGAGTLLELK (SEQ ID NO: 58) ( SEQ ID NO: 66) YOU
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SEQUENCE LISTING WITHOUT INFORMATION IN THE TEXT SEQ ID NO: 6 INab308 heavy chain CDR3 155 SEQIDNO: 7 INab708 heavy chain CDR3 SEQ ID NO: 8 CDR; 3 INab308 light chain SEQ ID NO: 9 CDR; 3 of INab708 light chain SEQIDNO: 10 "INab308 heavy chain CDR2 SEQ ID NO: 11 CDR) 2 INab708 heavy chain SEQIDNO: 12 INab308 light chain CDR32 SEQ ID NO: 13 INab708 light chain CDR32 SEQIDNO: 14. - INab308 SEQIDNO heavy chain CDRI: 155 INab708 SEQIDNO heavy chain CDRI SEQ ID NO: 16 INab308 SEQIDNO light chain CDRI: I17 INab708 SEQIDNO decadeiel light CDRI: 18! SEQIDNO: 19º "C5a consensus sequence in the region of amino acids 66-72
SEQIDNO: 20 C5a consensus sequence in the amino acid region 31-37 SEQ ID NO: 21 C5 consensus sequence in the 67-71 amino acid region SEQIDNO: 51I INab308 heavy chain FR1
SEQIDNO: 52 - INab308 heavy chain FR2 SEQIDNO: 53 —INab308 heavy chain FR3 SEQIDNO: 54 —INab308 heavy chain FRR SEQ ID NO: 55 INab308 light chain FR1
/ SEQIDNO: 56 INab308 light chain FR2 SEQ ID NO: 57 INab308 light chain FR3 SEQ ID NO: 58 INab308 light chain FR4 SEQIDNO: 59 —INab708 heavy decade FRI SEQIDNO: 60 INab708 heavy chain FR2
155 SEQIDNO: 61 INab708 heavy chain FR3 SEQIDNO: 62 INab708 heavy chain FR4 SEQ ID NO: 63 INab708 light chain FR1 SEQIDNO: 64 INab708 light chain FR2 SEQ ID NO: 65 INab708 light chain FR3
—SEQIDNO: 66 FR4 of INab708 light chain SEQIDNO: 69 Binding peptide

权利要求:
Claims (17)
[1]
1. Linking portion that binds to a conformational epitope formed by amino acid sequences X, X &ETCEX; RX, (SEQ ID NO: 18) and XsXsKX; XgXoL (SEQ ID NO: 19) of C5a, characterized by the fact that: X is selected from the group consisting of N,, D,) FE, K, Y, and T; X, is selected from the group consisting of D, L, Yy, and H; X; is selected from the group consisting of Q, E, and K; X, is selected from the group consisting of A, V, and L; Xs is selected from the group consisting of S, H, P, and N; Xçs It is selected from the group consisting of H and N; X, is selected from the group consisting of D, N, H, P, and G; X is selected from the group consisting of M, L, Le V; and X, is selected from the group consisting of Q, L, and L, wherein said binding portion is selected from the group consisting of: (a) antibodies or antigen binding fragments thereof; (b) oligonucleotides; (c) antibody-like proteins; and (d) peptidomimetics.
[2]
2. Linking moiety according to claim 1, characterized in that the linking moiety binds to at least one amino acid of the amino acid sequence XçETCEX; R (SEQ ID NO: 20), where X, and X; are as defined in claim 1.
[3]
3. Connection portion according to claim | or 2, characterized by the fact that the linking moiety binds to at least one amino acid of the amino acid sequence X 'KX; X:; Xs (SEQ ID NO: 21), preferably KX; X; wherein Xç, X7, Xg, and Xo are defined according to claim 1.
[4]
Binding portion according to claim 1, 2, or 3, characterized by the fact that the conformational epitope is formed by: (a) amino acid sequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3) from C5a; (b) amino acid sequences HDETCEQRA (SEQ ID NO: 22) and SHKDLQL (SEQ ID NO: 23) from C5a; (c) amino acid sequences DDETCEERA (SEQ ID NO: 24) and SHKNIQL (SEQ ID NO: 25) of C5a; (d) DLETCEQRA (SEQ ID NO: 26) and SHKHIQL (SEQ ID NO: 27) amino acid sequences from C5a; (e) amino acid sequences DDETCEQRA (SEQ ID NO: 28) and HHKNMQL (SEQ ID NO: 29) from C5a; (f) amino acid sequences FYETCEERV (SEQ ID NO: 30) and PHKPVQL (SEQ ID NO: 31) of C5a; (g) KYETCEQRV (SEQ ID NO: 32) and HHKGMLL (SEQ ID NO: 33) amino acid sequences of C5a; (h) amino acid sequences YDETCEQRA (SEQ ID NO: 34) and SNKPLQL (SEQ ID NO: 35) of C5a; or (1) THETCEKRL (SEQ ID NO: 36) and NHKPVIL (SEQID NO: 37) amino acid sequences from C5a.
[5]
Connection portion according to any one of the claims | to 4, characterized by the fact that the linking moiety binds to at least one amino acid of an amino acid sequence selected from the group consisting of: (a) DETCEQR (SEQ ID NO: 4); (b) DETCEER (SEQ ID NO: 38); (0) LETCEQR (SEQ ID NO: 39); (e) YETCEER (SEQ ID NO: 40); (O) YETCEQR (SEQ ID NO: 41); and
(8) HETCEKR (SEQ ID NO: 42).
[6]
6. Linking moiety according to any one of claims 1 to 5, characterized in that the linking moiety binds to at least one amino acid of a selected amino acid sequence - group consisting of: (a) HKDMQ (SEQ NC ID: 5), preferably KDM; (b) HKDLQ (SEQ ID NO: 43), preferably KDL; (c) HKNIQ (SEQ ID NO: 44), preferably KNI; (d) HKHIQ (SEQ ID NO: 45), preferably KHI; (e) HKNMQ (SEQ ID NC: 46), preferably KNM; (D HKPVQ (SEQ ID NO: 47), preferably KPV; (8) HKGML (SEQ ID NO: 48), preferably KGM; (h) NKPLQ (SEQ ID NO: 49), preferably KPL; and (1) HKPVI ( SEQ ID NO: 50), preferably KPV.
[7]
Connection portion according to any one of the claims | to 6, characterized by the fact that said binding portion exhibits one or more of the following properties: - said binding portion has a binding constant in C5a with a Kg value of 10 nM or less; a binding portion exhibits at least 80% blocking activity for biological effects induced by a C5a molecule; - said binding portion does not inhibit CH50 activity in human plasma; - said binding portion is capable of reducing the production of IL-8 — induced by choline whole human blood.
[8]
Binding portion according to any one of claims 1 to 7, characterized in that the binding portion is an antibody or antigen-binding fragment thereof, said antibody being selected from the group consisting of polyclonal antibodies,
monoclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies.
[9]
Binding portion according to any one of claims 1 to 7, characterized in that the binding portion is an antigen-binding fragment of an antibody, said fragment being selected from the group consisting of Fab fragments, fragments Fab ”, F (ab ') fragments, Fd fragments, Fv fragments, disulfide-linked Fvs (Fvds), single domain antibodies and single chain Fv antibodies (Fvsc).
[10]
10. Antigen or antigen-binding fragment thereof, - characterized by the fact that it comprises: (1) a heavy chain CDR3 sequence as shown in SEQ ID NO: 6; or (11) a heavy chain CDR3 sequence as shown in SEQ ID NO: 7; wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions, which further comprises at least one of the sequences next: (1) a heavy chain CDR2 sequence according to SEQIDNO: IO, (11) a heavy chain CDR2 sequence according to SEQ ID NO: 11; (111) a light chain CDR2 sequence according to SEQ ID NO: 12; (iv) a light chain CDR2 sequence according to SEQ ID NO: 13; (v) a heavy chain CDR1 sequence according to SEQ ID NO: 14; (vi) a heavy chain CDR1 sequence according to
SEQ ID NO: 15; (vii) a light chain CDR1 sequence according to SEQ ID NO: 16; or (viii) a light chain CDRI1 sequence according to 5 - SEQIDNO: 17; wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the heavy chain CDRI1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDRI1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[11]
Antibody or fragment thereof according to claim 10, characterized in that it further comprises: (1) a light chain CDR3 sequence as shown in SEQ ID NO: 8; or (11) a light chain CDR3 sequence as shown in SEQID NO: 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[12]
12. Antibody or antigen-binding fragment thereof, characterized by the fact that it comprises: (1) a light chain CDR3 sequence as presented in SEQ ID NO: 8; or
(11) a light chain CDR3 sequence as shown in SEQID NO: 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[13]
Antibody or fragment thereof according to claim 12, characterized in that it additionally comprises at least one of the following sequences: (1) a heavy chain CDR2 sequence according to SEQIDNO: 10; (11) a heavy chain CDR2 sequence according to SEQ ID NO: 11; (111) a light chain CDR2 sequence according to SEQ ID NO: 12; (iv) a light chain CDR2 sequence according to SEQ ID NO: 13; (v) a heavy chain CDR1 sequence according to SEQ ID NO: 14; (vi) a heavy chain CDR1 sequence according to SEQIDNO: IS, (vii) a light chain CDR1 sequence according to SEQ ID NO: 16; or (viii) a light chain CDRI1 sequence according to SEQ ID NO: 17; wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; where the chain CDR2 sequence light optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the heavy chain CDRI1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein the light chain CDRI1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[14]
14. Antigen or antigen-binding fragment thereof, characterized by the fact that it comprises a set of sequences of heavy chain CDR3, heavy chain CDR2 and heavy chain CDRI1 - selected from one of the following sets: (1) one heavy chain CDR3 sequence according to SEQ ID NO: 6, a heavy chain CDR2 sequence according to SEQ ID NO: 10, and a heavy chain CDR1 sequence according to SEQ ID NO: 14; (11) a heavy chain CDR3 sequence according to SEQ ID NO: 6, a heavy chain CDR2 sequence according to SEQ ID NO: 10, and a heavy chain CDR1 sequence according to SEQ ID NO: 15; (111) a heavy chain CDR3 sequence according to SEQID NO: 6, a heavy chain CDR2 sequence according to SEQ ID NO: 11, and a heavy chain CDR1 sequence according to SEQ ID NO: 14 ; (iv) a heavy chain CDR3 sequence according to SEQ ID NO: 6, a heavy chain CDR2 sequence according to —SEQIDNO: I11, and a heavy chain CDRI1 sequence according to SEQ ID NO: 15 ; (v) a heavy chain CDR3 sequence according to SEQ ID NO: 7, a heavy chain CDR2 sequence according to SEQ ID NO: 10, and a heavy chain CDR1 sequence according to
SEQ ID NO: 14; (vi) a heavy chain CDR3 sequence according to SEQ ID NO: 7, a heavy chain CDR2 sequence according to SEQ ID NO: 10, and a heavy chain CDR1 sequence according to - SEQIDNO: IS5 ; (vii) a heavy chain CDR3 sequence according to SEQ ID NO: 7, a heavy chain CDR2 sequence according to SEQ ID NO: 11, and a heavy chain CDR1 sequence according to SEQ ID NO: 14; or (viii) a heavy chain CDR3 sequence according to SEQ ID NO: 7, a heavy chain CDR2 sequence according to SEQ ID NO: 11, and a heavy chain CDR1 sequence according to SEQ ID NO : 15; wherein each heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; and wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[15]
15. Antigen or antigen-binding fragment thereof, characterized by the fact that it comprises a set of sequences of light chain CDR3, light chain CDR2 and light chain CDRI1 - selected from one of the following sets: (1) one light chain CDR3 sequence according to SEQ ID NO: 8, a light chain CDR2 sequence according to SEQ ID NO: 12, and a light chain CDR1 sequence according to SEQ ID NO: 16;
(11) a light chain CDR3 sequence according to SEQ ID NO: 8, a light chain CDR2 sequence according to SEQ ID NO: 12, and a light chain CDRI1 sequence according to SEQ ID NO: 17; (111) a light chain CDR3 sequence according to SEQ ID NO: 9, a light chain CDR2 sequence according to SEQ ID NO: 12, and a light chain CDR1 sequence according to SEQ ID NO: 16; or (iv) a light chain CDR3 sequence according to / SEQID NO: 9, a light chain CDR2 sequence according to SEQ ID NO: 12, and a light chain CDR1 sequence according to SEQ ID NO : 17; wherein each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions; and wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 amino acid additions.
[16]
16. Pharmaceutical composition, characterized by the fact that it comprises: (a) the binding portion as defined in any of the claims | to 9, or (b) the antibody or antigen-binding fragment thereof as defined in any one of claims 10 to 15, and which further comprises one or more carriers, diluents, excipients, fillers, binders, lubricants, non-stick, disintegrants , pharmaceutically acceptable adsorbents and / or preservatives.
[17]
17. Use of: (a) a binding moiety as defined in any one of claims 1 to 9, or (b) an antibody or antigen binding fragment thereof as defined in any one of claims 10 to 15, characterized by the fact that it is for the preparation of a pharmaceutical composition for the prevention and or treatment of various diseases involving acute inflammation such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injuries such as illness ischemic heart disease, acute lung injury, pneumonia, acute or chronic graft rejection in transplant patients, graft-versus-host reactions, as well as diseases involving chronic types of inflammation such as renal glomerular diseases, such as glomerulonephritis and other renal failure entities, rheumatoid arthritis and similar autoimmune diseases such as Bechterew's disease, lupus-like disease, inflammatory bowel disease, d disease and Crohn's, tumor growth or solid organ cancer.

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法律状态:
2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-08-31| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-09-14| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US26469609P| true| 2009-11-26|2009-11-26|

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EP09014745.5|2009-11-26|

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EP09014745A|EP2327725A1|2009-11-26|2009-11-26|Anti-C5a binding moieties with high blocking activity|

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US61/264,696|2009-11-26|

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PCT/EP2010/007197|WO2011063980A1|2009-11-26|2010-11-26|Anti-c5a binding moieties with high blocking activity|

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