anti-BTLA antibodies
阅读说明：本技术 抗btla抗体 (anti-BTLA antibodies ) 是由 武海 姚剑 姚盛 冯辉 张静 周岳华 于 2018-08-02 设计创作，主要内容包括：本发明涉及抗BTLA抗体或其抗原结合片段,所述抗体或其抗原结合片段包含至少一个选自SEQ ID NO：7、8、9、10、11、12、16、17、18、22、23、24、31、32和33的轻链CDR结构域和/或至少一个选自SEQ ID NO：1、2、3、4、5、6、13、14、15、19、20、21、25、26、27、28、29和30的重链CDR结构域。本发明还涉及编码所述抗体或其抗原结合片段的核酸分子、相应的表达载体和宿主细胞,以及所述抗体或其抗原结合片段、核酸分子、表达载体和宿主细胞的治疗用途。(The present invention relates to an anti-BTLA antibody or antigen-binding fragment thereof comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11, 12, 16, 17, 18, 22, 23, 24, 31, 32, and 33 and/or at least one light chain CDR domain selected from SEQ ID NOs: 1.2, 3, 4, 5, 6, 13, 14, 15, 19, 20, 21, 25, 26, 27, 28, 29 and 30. The invention also relates to nucleic acid molecules encoding said antibodies or antigen-binding fragments thereof, corresponding expression vectors and host cells, and therapeutic uses of said antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors and host cells.)
1. An isolated antibody or antigen-binding fragment thereof, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11, 12, 16, 17, 18, 22, 23, 24, 31, 32, and 33 and/or at least one light chain CDR domain selected from SEQ ID NOs: 1.2, 3, 4, 5, 6, 13, 14, 15, 19, 20, 21, 25, 26, 27, 28, 29 and 30.
2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 in the light chain CDRs of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of any of the CDR sequences of SEQ ID NOs 7, 10, 16, 22 and 31; and/or LCDR2 thereof is selected from any one of the CDR sequences of SEQ ID NOs 8, 11, 17, 23 and 32; and/or LCDR3 thereof is selected from any one of the CDR sequences of SEQ id nos 9, 12, 18, 24 and 33; and/or
The HCDR1 in the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof is selected from any one of the CDR sequences of SEQ ID NOs 1, 4, 13, 19, 25 and 28; and/or its HCDR2 is selected from any CDR sequence of SEQ ID NO. 2, 5, 14, 20, 26 and 29; and/or its HCDR3 is selected from any CDR sequence of SEQ ID NO. 3, 6, 15, 21, 27 and 30.
3. The isolated antibody or antigen-binding fragment thereof of claim 2,
in the light chain CDRs of the isolated antibody or antigen-binding fragment thereof, LCDR1 is selected from the group consisting of any of the CDR sequences of SEQ ID NOs 7, 10, 16, 22, and 31; LCDR2 is selected from any one of the CDR sequences of SEQ ID NOs 8, 11, 17, 23 and 32; and LCDR3 is selected from any one of the CDR sequences of SEQ ID NOs 9, 12, 18, 24 and 33; and/or
In the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof, HCDR1 is selected from any one of the CDR sequences of SEQ ID NOs 1, 4, 13, 19, 25 and 28; HCDR2 is selected from any one of the CDR sequences of SEQ ID NOs 2, 5, 14, 20, 26 and 29; and HCDR3 is selected from any one of the CDR sequences of SEQ ID NOs 3, 6, 15, 21, 27 and 30.
4. The isolated antibody or antigen-binding fragment thereof of claim 2,
the LCDR1, LCDR2, and LCDR3 sequences of the light chain of the isolated antibody or antigen-binding fragment thereof are as set forth in any one of the following groups a-E:
and/or
The amino acid sequences of the HCDR1, HCDR2 and HCDR3 of the heavy chain CDRs of the isolated antibody or antigen-binding fragment thereof are as set forth in any one of the following F-K groups:
5. the isolated antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 of the heavy chain CDRs and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 of the light chain CDRs are as set forth in any one of the following groups I-IX:
6. the isolated antibody or antigen-binding fragment thereof of claim 1,
the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 36. 37, 39, 41, 44, 46, 47 and 48; and/or
The amino acid sequence of the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 34. 35, 38, 40, 42, 43 and 45.
7. The isolated antibody or antigen-binding fragment thereof of claim 6,
the amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 36, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 34 or 35; or
The amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 37, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 34 or 35; or
The amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 39, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 38; or
The amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 41, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 40 or 42; or
The amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 44, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 43 is shown; or
The amino acid sequence of the light chain variable region of the isolated antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 46. 47 or 48, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 45.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody is a chimeric, humanized, or fully human antibody.
9. An isolated nucleic acid selected from the group consisting of:
(1) a polynucleotide sequence encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7; and
(2) (1) the complement of the polynucleotide sequence.
10. An expression vector or a host cell comprising the expression vector, wherein the expression vector comprises the isolated nucleic acid of claim 9.
11. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7, the nucleic acid of claim 9, the expression vector or host cell of claim 10, or any combination thereof.
12. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7, the nucleic acid of claim 9, the expression vector or host cell of claim 10 in the manufacture of a medicament for treating or preventing a BTLA-mediated disease.
13. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 coupled to a therapeutic agent, preferably the therapeutic agent is a toxin, radioisotope, drug, or cytotoxic agent.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antibody or an antigen binding fragment thereof for binding BTLA and application thereof. More particularly, the present invention relates to active antibodies that recognize human BTLA and can be used for the treatment or prevention of tumors, infectious diseases, inflammatory, autoimmune diseases, and the like.
Background
Positive and negative co-stimulatory signals play a crucial role in the regulation of B-cell and T-cell activity, and molecules that mediate these signals have been shown to be effective targets for immune modulators. In addition to T Cell Receptor (TCR) involvement, positive co-stimulation is also required for optimal activation of naive T cells, whereas negative co-stimulation is thought to be required for acquisition of autoimmune tolerance and termination of effector T cell function. Upon interaction with B7.1 or B7.2 on the surface of Antigen Presenting Cells (APCs), the prototype T cell costimulatory molecule CD28 signals promotion of T cell proliferation and differentiation in response to TCR engagement, but the CD28 homolog cytotoxic T lymphocyte antigen-4 (CTLA-4) mediates inhibition of T cell proliferation and effector function (Chambers et al, Ann. Rev. Immunol., 19: 565-. Several new molecules homologous to the B7 family have been found (Abbas et al, nat. Med., 5: 1345-6, 1999; Coyle et al, nat. Immunol., 2: 203-9, 2001; Carreno et al, Annu. Rev. Immunol., 20: 29-53, 2002; Liang et al, curr. Opin. Immunol., 14: 384-90, 2002) and their role in T cell activation has just begun to be elucidated.
B and T Lymphocyte Attenuator (BTLA) are members of the CD28 family, which also includes CD28, ICOS, CTLA-4 and PD-1. According to the increase of T cell proliferation after adding monoclonal antibodyReproductive function, the first members of this family, CD28 and ICOS, were found to have immune-activating effects (Hutloff et al, 1999). While BTLA, CTLA-4, PD-1, etc. are described as negative regulatory proteins. Several in vivo studies have demonstrated the inhibitory effect of BTLA in lymphocyte responses. BTLA-deficient mice prepared by Murphy and colleagues (washington university st. louis) showed a 3-fold increase in IgG production in response to T-dependent antigen. In addition, from BTLA-Mouse isolated T and B cells showed greater proliferative responses to antigen-receptor stimulation with CD 3-and anti-IgM, respectively (Watanabe, 2003). In overexpression studies, BTLA was found to associate with B cell receptor complexes and with T cell receptors. Consistent with the results of this study, antigen-receptor independent stimulation using ConA (T cells) or LPS (B cells) was not affected in BTLA deficient lymphocytes, and could not be modulated using anti-BTLA antibodies. BTLA knockout mice have been shown to develop spontaneous autoimmune disease over time and to have a shortened lifespan (Oya, 2008). BTLA knockout mice show increased disease severity in autoimmune encephalomyelitis (EAE) and allergic airway inflammation models, both of which rely on T cell activity (Watanabe, 2005; decappng, 2006).
Herpes Virus Entry Mediators (HVEM) have been shown to be ligands of BTLA (Scully et al, 2005). HVEM is a type I transmembrane glycoprotein belonging to a member of the TNF receptor superfamily, with 4 extracellular cysteine-rich regions (CDRs) containing 6 pseudorepetitive cysteines. BTLA and HVEM regulate T cell and APC functions primarily through dynamic expression on the cell surface. BTLA binding to ligand not only inhibits T cell proliferation and down-regulates the T cell activation marker CD25, but also inhibits the production of IFN-gamma, IL-2, IL-4, IL-10, etc., but does not induce apoptosis. Binding of HVEM to BTLA results in down-regulation of T cell activation and proliferation (Sedy, 2005). These findings indicate that BTLA expression or BTLA-HVEM binding is strongly associated with T cell activation and proliferation.
The antibodies are useful as therapeutic agents. Certain antibodies may cause unwanted immunogenicity of the antibody when used as a therapeutic in vivo. Because most monoclonal antibodies are of rodent origin, repeated use in humans results in the generation of an immune response against the therapeutic antibody (e.g., human anti-mouse antibody or HAMA). Such immune responses result in at least a loss of therapeutic efficacy and, at best, a potentially lethal allergic reaction. One method of reducing the immunogenicity of rodent antibodies involves the generation of chimeric antibodies in which the mouse variable region (Fv) is fused to human constant regions (Liu et al (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, mice injected with hybrids of human variable regions and mouse constant regions develop strong antibody responses against the human variable regions, suggesting that the retention of the complete rodent Fv region in such chimeric antibodies may still cause deleterious immunogenicity in patients.
In addition, grafting of Complementarity Determining Region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences. Jones et al (1986) Nature 321: 522; verhoeyen et al (1988) Science 239: 1534. however, CDR loop exchange still does not uniformly produce antibodies with the same binding properties as the starting antibody. In humanized antibodies, Framework Residue (FR) (residues involved in CDR loop support) changes are often also required to maintain antigen binding affinity. Kabat et al (1991) J.Immunol.147: 1709. although the use of CDR grafting and framework residue retention in many humanized antibody constructs has been reported, it is difficult to predict whether a particular sequence will produce an antibody with the desired binding properties and, occasionally, biological properties. See, e.g., Queen et al, (1989) Proc.Natl.Acad.Sci.USA86: 10029; gorman et al, (1991) proc.natl.acad.sci.usa 88: 4181; and Hodgson, (1991) biotechnology (ny), 9: 421-5. Furthermore, most of the existing studies use different human sequences for animal light and heavy chain variable sequences, rendering the predictability of such studies problematic. The sequences of known antibodies, or more generally antibodies having a known X-ray crystal structure, such as the antibodies NEW and KOL, have been used. See, e.g., Jones et al, supra; verhoeyen et al, supra; and Gorman et al, supra. Exact sequence information has been reported for a few humanized constructs.
There is a need for anti-BTLA antibodies, particularly anti-BTLA monoclonal antibodies, for use in treating human disorders (e.g., inflammatory disorders, autoimmune disorders, and proliferative disorders). Such antibodies may preferably have low immunogenicity in human subjects, allowing repeated administration without adverse immune responses.
Disclosure of Invention
The present invention relates to one or more anti-human BTLA antibodies or antigen-binding fragments thereof, and the use of the antibodies or antigen-binding fragments thereof in the treatment of disease.
In one or more embodiments, the invention relates to the interaction with human BTLA (B)-And T-Lymphocyte attenuator) that specifically binds to an isolated antibody or antigen-binding fragment thereof that comprises one or more properties selected from the group consisting of:
A) block BTLA binding to HVEM (herpes virus entry mediator);
B) cross-reacting with cynomolgus monkey BTLA; and
C) k binding to human BTLAD≤0.28nM。
In one or more embodiments, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11, 12, 16, 17, 18, 22, 23, 24, 31, 32 and 33 and at least one light chain CDR domain selected from SEQ ID NOs: 1.2, 3, 4, 5, 6, 13, 14, 15, 19, 20, 21, 25, 26, 27, 28, 29 and 30.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, whose light chain CDR amino acid sequences of CDR1, CDR2, and CDR3 are as set forth in any one of the following groups a-E:
group of
LCDR1
LCDR2
LCDR3
A
SEQ ID NO：7
SEQ ID NO：8
SEQ ID NO：9
B
SEQ ID NO：10
SEQ ID NO：11
SEQ ID NO：12
C
SEQ ID NO：16
SEQ ID NO：17
SEQ ID NO：18
D
SEQ ID NO：22
SEQ ID NO：23
SEQ ID NO：24
E
SEQ ID NO：31
SEQ ID NO：32
SEQ ID NO：33
And/or the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain CDR thereof are as set forth in any one of the following groups F-K:
group of
HCDR1
HCDR2
HCDR3
F
SEQ ID NO：1
SEQ ID NO：2
SEQ ID NO：3
G
SEQ ID NO：4
SEQ ID NO：5
SEQ ID NO：6
H
SEQ ID NO：13
SEQ ID NO：14
SEQ ID NO：15
I
SEQ ID NO：19
SEQ ID NO：20
SEQ ID NO：21
G
SEQ ID NO：25
SEQ ID NO：26
SEQ ID NO：27
K
SEQ ID NO：28
SEQ ID NO：29
SEQ ID NO：30
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, whose amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain CDR and CDR1, CDR2 and CDR3 of the light chain CDR are as set forth in any one of the following groups I-IX:
group of
LCDR1
LCDR2
LCDR3
HCDR1
HCDR2
HCDR3
I
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
II
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
III
SEQ ID NO:16
SEQ ID NO:17
SEQ ID NO:18
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
IV
SEQ ID NO:22
SEQ ID NO:23
SEQ ID NO:24
SEQ ID NO:19
SEQ ID NO:20
SEQ ID NO:21
V
SEQ ID NO:22
SEQ ID NO:23
SEQ ID NO:24
SEQ ID NO:25
SEQ ID NO:26
SEQ ID NO:27
VI
SEQ ID NO:31
SEQ ID NO:32
SEQ ID NO:33
SEQ ID NO:28
SEQ ID NO:29
SEQ ID NO:30
VII
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
VIII
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
IX
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
In one or more embodiments, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 36. 37, 39, 41, 44, 46, 47 or 48, and the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34. 35, 38, 40, 42, 43 or 45.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 36 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 34.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 36 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 35.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 37 is shown in the figure; the amino acid sequence of the heavy chain variable region is SEQID NO: shown at 34.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 37 is shown in the figure; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 35.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 39; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 38.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 41 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 40.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 41 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 42.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 43.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 45.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:47 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 45.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:48 is shown; the amino acid sequence of the heavy chain variable region is shown as SEQID NO: shown at 45.
In one or more embodiments, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA is a single chain Fv antibody; in certain embodiments, the antibody or antigen binding fragment thereof is a Fab antibody; in certain embodiments, the antibody or antigen binding fragment thereof is a Fab' antibody; in certain embodiments, the antibody or antigen-binding fragment thereof is (Fab')2An antibody.
In additional embodiments, the invention relates to an isolated polypeptide comprising the VL domain or VH domain of any of the antibodies or antigen-binding fragments thereof described herein.
In additional embodiments, the invention relates to an isolated nucleic acid encoding the VL domain and the VH domain of any of the antibodies or antigen-binding fragments described herein.
In additional embodiments, the invention relates to compositions comprising one or more antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier or diluent.
In additional embodiments, the invention relates to methods of preventing or treating disease by abrogating, inhibiting, or reducing BTLA activity using one or more antibodies or antigen binding fragments thereof described herein, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof disclosed herein, a nucleic acid, an expression vector, a host cell, an immunoconjugate, or a pharmaceutical composition. Preferably, the prevention or treatment of the disease or disorder is benefited by the elimination, inhibition, or reduction of BTLA activity; preferably, the disease or condition is selected from cancer, infectious disease or inflammatory disease.
In a further embodiment, the invention also relates to the use of the antibody or antigen binding fragment, nucleic acid, expression vector, host cell, immunoconjugate or pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of a disease or disorder, preferably a BTLA-mediated disease, which may be selected from cancer, infectious disease or inflammatory disease.
Drawings
FIG. 1: SDS-PAGE electrophoresis of human BTLA extracellular domain protein.
FIG. 2: flow cytometry examined the binding capacity of BTLA to HVEM.
FIG. 3: ELISA assay of chimeric antibody binding to human BTLA.
FIG. 4: the ability of the chimeric antibody to block BTLA-HVEM binding.
FIG. 5: effect of chimeric antibodies on T cell activity experiments.
FIG. 6: humanized antibodies were tested for specific binding to BTLA.
FIG. 7: binding of humanized antibodies to hBTLA on 293F cells.
FIG. 8: experiments in which the humanized antibody blocked BTLA binding to HVEM on the cell surface.
FIG. 9: humanized antibodies facilitate T cell activation experiments.
FIG. 10: comparative experiments with humanized
Detailed Description
The mammalian immune system has developed several pathways that control the potentially harmful activity of T and B lymphocytes. They include various cytokine-receptor pathways as well as costimulatory pathways involving receptors like CD28, CTLA-4, PD-1, and BTLA. Although the CD28-B7 interaction is an example of a positive costimulatory pathway (i.e., CD28 trigger enhances T cell responses to antigen-specific triggers), the other 3 receptors appear to be inhibitory costimulatory pathways. CTLA-4, PD-1 and BTLA show overlapping but unique expression profiles and limit the activity of T and B lymphocytes and other immune cells (see Deppong et al, JImmunol 2006; Tao et al, J Immunol 2005). Although CTLA-4 competes with CD28 for binding to B7.1 and B7.2(CD80 and CD86) and sets the original threshold for naive T cell activation in lymph nodes and spleen, PD-1 and BTLA each have their own unique ligands (PD-L1/-L2 and HVEM, respectively) and appear to control peripheral T cell homeostasis and reactivation (see Krieg et al, Nat Immunol 2007).
BTLA down-regulates B-cell and T-cell activation. As its name suggests, B and T Lymphocyte Attenuators (BTLA) are expressed on both resting and activated B and T lymphocytes. BTLA is a type I transmembrane glycoprotein with a cytoplasmic tail containing several tyrosine-inhibiting motifs (Watanabe, 2003). BTLA shares some structural similarity with members of the CD28/CTLA-4 family, but it has unique properties. BTLA is associated with a variety of immunological, inflammatory and proliferative diseases. Therefore, the development of monoclonal antibody drugs capable of blocking human BTLA-HVEM binding is a continuing need for disease treatment.
Definition of
In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined as follows. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Herein, "activation", "stimulation" and "treatment" for a cell or receptor may have the same meaning, e.g., the cell or receptor is activated, stimulated or treated with a ligand, unless the context otherwise or specifically dictates otherwise. "ligands" include natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "ligands" also include small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies. "activation" may refer to the activation of a cell by internal mechanisms as well as regulated by external or environmental factors. A "response/response", e.g., a response of a cell, tissue, organ or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration, gene expression rate or differentiation status within a biological compartment), where the change is associated with activation, stimulation or treatment, or with an internal mechanism such as genetic programming.
The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; an antigenic activity; modulating the activity of other molecules, etc. "activity" of a molecule can also refer to activity in modulating or maintaining cell-cell interactions (e.g., adhesion), or in maintaining cellular structure (e.g., cell membrane or cytoskeleton). "activity" may also refer to specific activities, such as [ catalytic activity ]/[ mg protein ] or [ immunological activity ]/[ mg protein ], concentration in a biological compartment, and the like. "activity" may refer to the modulation of a component of the innate immune system or the adaptive immune system. "proliferative activity" includes activity that is promoting, essential for, or specifically related to: such as normal cell division, as well as cancer, tumors, dysplasia, cellular transformation, metastasis and angiogenesis.
"administration" and "treatment" as applied to an animal, human, subject, cell, tissue, organ, or biological fluid means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treatment" also mean in vitro and ex vivo treatment, e.g., in vitro and ex vivo treatment of a cell with an agent, a diagnostic, a binding compound, or with another cell. "treating" also includes administering a therapeutic agent, e.g., a composition comprising any of the antibodies or antigen-binding fragments thereof of the invention, internally or externally to a patient in need thereof. In general, an antibody or antigen-binding fragment thereof or corresponding pharmaceutical composition described herein is administered in an amount effective to reduce one or more symptoms of a disease in a patient or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the agent to elicit a desired response in the patient. Whether a symptom is reduced can be assessed by any clinical measure commonly used by physicians or other healthcare providers to assess the severity or progression status of a disease symptom.
The term "subject" or "patient" includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), most preferably a human.
Herein, "administering" or "treatment" can be accomplished by an invasive route, e.g., by injection administration of any of the anti-BTLA antibodies or antigen-binding fragments thereof described herein or corresponding pharmaceutical compositions thereof. Administration by non-invasive routes (e.g., oral; e.g., oral in pills, capsules, or tablets) is also within the scope of the invention. In one embodiment of the invention, the anti-BTLA antibody or antigen-binding fragment thereof or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intra-arterially, intra-articularly (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumorally.
Any of the anti-BTLA antibodies or antigen-binding fragments thereof or corresponding compositions described herein can be administered using medical devices known in the art. For example, the pharmaceutical compositions of the present invention may be administered by injection using a hypodermic needle; or the pharmaceutical composition of the present invention may be administered by injection using an intravenous injection needle.
In certain embodiments, any of the anti-BTLA antibodies or antigen-binding fragments thereof described herein or corresponding pharmaceutical compositions thereof can be used alone or in combination to treat or prevent any disease or condition in a subject in need of such treatment or prevention.
Herein, the terms "B and T lymphocyte attenuator" and "BTLA" gene/protein are used interchangeably and include variants, isoforms, homologues, orthologues (orthologues) and paralogues (paralogs). For example, in certain embodiments, a human BTLA-specific antibody can cross-react with BTLA from a non-human species. In other embodiments, a human BTLA-specific antibody can be fully specific for human BTLA and not have species cross-reactivity or other types of cross-reactivity. Unless otherwise indicated, the term "human BTLA" or "hBTLA" refers to the human BTLA sequence. Unless otherwise indicated, the human BTLA sequence includes all human isoforms and BTLA variants, e.g., the complete amino acid sequence of human BTLA with Genbank accession number AAP 44003. There are also at least two human BTLA transcript variants,
BTLA is a negative regulator of immune response with a C-terminal inhibitory motif involved in the inhibition of IL-2 production and T cell expansion (Watanabe et al, nat. Immunol., 4, 670-. In addition, human BTLA can be an epitope in the extracellular domain of BTLA that specifically binds to an antibody of the invention.
Specific BTLA sequences the extracellular nucleotide sequence of which can be generally compared to SEQ ID NO:49 has at least 90% identity to the nucleotide sequence of the extracellular region or other isoform of human BTLA and contains amino acid residues of the amino acid sequence identified as human when compared to BTLA amino acid sequences of other species (e.g., murine). In certain instances, the human BTLA extracellular region can hybridize to SEQ ID NO:49 or other isoforms or variants thereof, have at least 95% or even at least 96%, 97%, 98% or 99% identity.
The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement), which results in the selective damage, destruction, or elimination from the human body of cells or tissues invading, infected with, cancerous cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
The term "antibody" as used herein refers to any form of antibody having a desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized (camelized) single domain antibodies. As used herein, the term "anti-BTLA antibody" or "antigen-binding fragment" of an antibody refers to an antibody that binds to BTLA and blocks BTLA binding to HVEM, including fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of an antibody, which retains at least some of the binding specificity of the antibody. Examples of antibody binding fragments include, but are not limited to, Fab ', F (ab')2And Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When BTLA binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its BTLA binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the BTLA binding affinity of the antibody. It is also contemplated that anti-BTLA antigen-binding fragments may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of antibodies) that do not significantly alter their biological activity.
By "isolated antibody" is meant the purified state of the antibody or antigen-binding fragment thereof, and in this case means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the antibodies or antigen binding fragments thereof described herein.
The term "functional fragment" or "antigen-binding fragment" as used herein refers in particular to an antibody fragment such as an Fv, scFv, Fab, F (ab ')2, Fab', scFv-Fc fragment or diabody, or any fragment capable of increasing half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (pegylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ')2-PEG or Fab' -PEG) ("PEG" being polyethylene glycol), or by incorporation into liposomes, said fragment having EGFR binding activity. Preferably, the functional fragment consists of or comprises a partial sequence of the heavy or light variable chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably 1/100 being at least equal to the affinity of the antibody from which it is derived, and in a more
A "Fab fragment" consists of one light chain with the variable regions of CH1 and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
The "Fc" region contains 2 heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The 2 heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
A "Fab ' fragment" comprises a light chain and a portion or fragment of a heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains such that an interchain disulfide bond can form between 2 heavy chains of 2 Fab ' fragments to form F (ab ')2A molecule.
An "F (ab')2 fragment" contains 2 light chains and 2 heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the 2 heavy chains. Thus, F (ab')2The fragment consists of 2 Fab' fragments held together by 2 inter-heavy chain disulfide bonds.
The "Fv region" comprises variable regions derived from both the heavy and light chains, but lacks a constant region.
The term "single chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. scFv polypeptides also typically comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The 2 VH regions of the bivalent domain antibody may target the same or different antigens.
A "bivalent antibody" comprises 2 antigen binding sites. In some cases, 2 binding sites have the same antigen specificity. However, bivalent antibodies may be bispecific.
As used herein, unless otherwise specified, "anti-BTLA antibody" refers to an antibody raised against human BTLA or a variant thereof or any antigenic fragment thereof.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibody used in the present invention can be prepared by a hybridoma method, or can be prepared by a recombinant DNA method. "monoclonal antibodies" can also be isolated using phage antibody libraries.
In certain embodiments, monoclonal antibodies include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they possess the desired biological activity.
As used herein, a "chimeric antibody" is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies are from different species. Typically, the variable domains are obtained from an antibody of an experimental animal such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject as compared to the parent rodent antibody.
In certain embodiments, the monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muydermans et al (2001) Trends biochem. Sci.26: 230; reichmann et al (1999) J.Immunol.methods 231: 25.
the term "diabodies" as used herein refers to small antibody fragments having two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is short enough not to allow pairing between two domains of the same strand, this domain is forced to pair with the complementary domain of the other strand and two antigen binding sites are created.
The term "humanized antibody" as used herein refers to antibody forms containing sequences from both human and non-human (e.g., murine, rat) antibodies. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the Framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
In general, it is known that the basic structural unit of an antibody comprises tetramers, each tetramer comprising two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" (about 50-70kDa) chain, the amino-terminal portion or fragment of each chain may comprise a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition, the carboxy-terminal portion or fragment of each chain may define a constant region primarily responsible for effector function.
The variable region pairs of each light/heavy chain pair form antibody binding sites. Thus, an intact IgG antibody typically has 2 binding sites. The 2 binding sites are generally identical except for bifunctional or bispecific antibodies.
Typically, each chain has the same general structure of relatively conserved Framework Regions (FRs) connected by 3 hypervariable regions (also known as complementarity determining regions or CDRs). The 2-chain CDRs of each pair are typically aligned by a framework region to enable binding to a particular epitope. In general, both the light and heavy chains comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and
The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable regions comprise the amino acid residues of the "complementarity determining regions" or "CDRs". The term "framework" or "FR" residues, as used herein, refers to variable domain residues other than the hypervariable region residues which are defined herein as CDR residues.
An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject can vary depending upon factors such as the condition to be treated, the general health of the patient, the method of administration and the dosage, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The results may result in an improvement in the diagnostic measure or parameter of at least 5%, typically at least 10%, more typically at least 20%, most typically at least 30%, preferably at least 40%, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%, most ideally at least 90%, where 100% is defined as the diagnostic parameter exhibited by normal subjects (see, e.g., Maynard et al (1996) A Handbook of SOPs for Good clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good laboratory and Good clinical Practice, Urch publication, London, UK).
"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in two sequences being compared is occupied by the same base or amino acid monomer subunit, for example if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions common to both sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies in 10 positions of the two sequences when optimally aligned. Comparisons are typically made when aligning two sequences to obtain the maximum percent homology.
As used herein, "immune disorder" includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "immune disorder" also refers to infections, persistent infections, and proliferative conditions such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist eradication by the immune system. "cancerous conditions" include, for example, cancers, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia. The "immune foci" and "cancerous conditions" described herein are preferably both BTLA mediated.
By "inflammatory disorder" is meant a disorder or pathological condition in which the pathology is caused, in whole or in part, by, for example, a change in the number, rate of migration, or activation of cells of the immune system. Immune system cells include, for example, T cells, B cells, monocytes or macrophages, Antigen Presenting Cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or any other cell of particular relevance to immunology, such as cytokine-producing endothelial or epithelial cells. The "inflammatory disorder" as described herein is preferably mediated by BTLA.
An "isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is not associated with all or part of a polynucleotide with which the isolated polynucleotide is naturally occurring, or is linked to a polynucleotide with which it is not naturally associated. For the purposes of this disclosure, it is understood that "a nucleic acid molecule" comprising "a particular nucleotide sequence does not include an entire chromosome. In addition to the specified sequences, an isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may comprise a coding sequence for up to 10 or even up to 20 or more other proteins or parts or fragments thereof, or may comprise operably linked regulatory sequences controlling the expression of the coding regions of the recited nucleic acid sequences, and/or may comprise vector sequences.
As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom regardless of the number of deliveries. It is also understood that the DNA content of all progeny may not be identical due to deliberate or inadvertent mutation. Mutant progeny selected for the same function or biological activity in the originally transformed cell are included. Although different names are specified, it will be clear from the context.
The host cell of the present invention may be a prokaryotic host cell, a eukaryotic host cell or a phage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the host cell of the invention is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell or a COS cell.
As used herein, "polymerase chain reaction" or "PCR" generally requires that sequence information at the end of or beyond the target region be available so that oligonucleotide primers can be designed; these primers may be identical or similar to the sequences of opposite strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the amplification material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from whole genomic DNA, and cDNA transcribed from cellular total RNA, phage or plasmid sequences, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Quant. biol. 51: 263; erlich eds (1989) PCRTECHNOLOGY (Stockton Press, N.Y.). PCR, as used herein, is considered to be an example, but not the only, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method involves the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific nucleic acid fragments.
Human BTLA-specific antibodies
The present invention relates generally to isolated antibodies or antigen-binding fragments thereof that bind BTLA and the use of such antibodies or antigen-binding fragments thereof. More specifically, the invention provides isolated anti-BTLA antibodies and the use of these antibodies or antigen-binding fragments thereof in the treatment and prevention of disease. Examples of anti-BTLA antibodies include, but are not limited to, ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19 as described herein.
The present invention provides BTLA (B) related to human-And T-Lymphocyte attenuator) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises one or more of the following properties: A) block BTLA binding to HVEM (herpes virus entry mediator); B) cross-reacting with cynomolgus monkey BTLA; C) k binding to human BTLAD≤0.28nM。
In one or more embodiments, the light chain CDRs of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention comprise at least one CDR selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11, 12, 16, 17, 18, 22, 23, 24, 31, 32, and 33. For example, in certain embodiments, LCDR1 in the light chain CDRs of an isolated antibody or antigen-binding fragment thereof provided herein that binds to human BTLA can be selected from the group consisting of the CDR sequences of any one of
In certain embodiments, the LCDR1 is selected from the group consisting of the CDR sequences of any one of
In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds human BTLA, wherein the LCDR1, LCDR2 and LCDR3 sequences of its light chain are as set forth in any one of the following groups a-E:
group of
LCDR1
LCDR2
LCDR3
A
SEQ ID NO：7
SEQ ID NO：8
SEQ ID NO：9
B
SEQ ID NO：10
SEQ ID NO：11
SEQ ID NO：12
C
SEQ ID NO：16
SEQ ID NO：17
SEQ ID NO：18
D
SEQ ID NO：22
SEQ ID NO：23
SEQ ID NO：24
E
SEQ ID NO：31
SEQ ID NO：32
SEQ ID NO：33
In one or more embodiments, the heavy chain CDRs of an isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention comprise at least one CDR selected from the group consisting of SEQ ID NOs: 1.2, 3, 4, 5, 6, 13, 14, 15, 19, 20, 21, 25, 26, 27, 28, 29 and 30. For example, in certain embodiments, the HCDR1 in the heavy chain CDR of an isolated antibody or antigen-binding fragment thereof provided herein that binds to human BTLA can be selected from the CDR sequences of any one of
In certain embodiments, the invention provides an isolated antibody that binds to human BTLA, or an antigen-binding fragment thereof, wherein the HCDR1 is selected from the group consisting of the CDR sequences of any one of
In certain embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof that binds human BTLA, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain CDRs thereof are as set forth in any one of the following groups F-K:
group of
HCDR1
HCDR2
HCDR3
F
SEQ ID NO：1
SEQ ID NO：2
SEQ ID NO：3
G
SEQ ID NO：4
SEQ ID NO：5
SEQ ID NO：6
H
SEQ ID NO：13
SEQ ID NO：14
SEQ ID NO：15
I
SEQ ID NO：19
SEQ ID NO：20
SEQ ID NO：21
G
SEQ ID NO：25
SEQ ID NO：26
SEQ ID NO：27
K
SEQ ID NO：28
SEQ ID NO：29
SEQ ID NO：30
In one or more embodiments, the invention provides an isolated antibody that binds to human BTLA or an antigen-binding fragment thereof, wherein the LCDR1 is selected from the group consisting of the CDR sequences of any one of
In one or more embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds human BTLA, wherein the LCDR1, LCDR2, and LCDR3 sequences of its light chain are as set forth in any one of the following groups a-E:
group of
LCDR1
LCDR2
LCDR3
A
SEQ ID NO：7
SEQ ID NO：8
SEQ ID NO：9
B
SEQ ID NO：10
SEQ ID NO：11
SEQ ID NO：12
C
SEQ ID NO：16
SEQ ID NO：17
SEQ ID NO：18
D
SEQ ID NO：22
SEQ ID NO：23
SEQ ID NO：24
E
SEQ ID NO：31
SEQ ID NO：32
SEQ ID NO：33
And/or the amino acid sequences of the HCDR1, HCDR2 and HCDR3 of the heavy chain CDRs thereof are as set forth in any one of the following F-K groups:
group of
HCDR1
HCDR2
HCDR3
F
SEQ ID NO：1
SEQ ID NO：2
SEQ ID NO：3
G
SEQ ID NO：4
SEQ ID NO：5
SEQ ID NO：6
H
SEQ ID NO：13
SEQ ID NO：14
SEQ ID NO：15
I
SEQ ID NO：19
SEQ ID NO：20
SEQ ID NO：21
G
SEQ ID NO：25
SEQ ID NO：26
SEQ ID NO：27
K
SEQ ID NO：28
SEQ ID NO：29
SEQ ID NO：30
It is to be understood that each CDR sequence of the light chain, in particular each LCDR1, LCDR2 and LCDR3 sequence, disclosed herein may be combined with each CDR sequence of the heavy chain, in particular each HCDR1, HCDR2 and HCDR3 sequence, as desired. For example, the entire 6 CDR domains comprised by the isolated antibody or antigen-binding fragment thereof that binds human BTLA described herein can be formed from any one of the sequences defined herein as LCDR1 in combination with any one of the sequences defined herein as LCDR2, any one of the sequences of LCDR3, any one of the sequences of HCDR1, any one of the sequences of HCDR2, and any one of the sequences of
Thus, for example, in one or more embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof that binds human BTLA, whose amino acid sequences of the HCDR1, HCDR2 and HCDR3 of the heavy chain CDRs and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 of the light chain CDRs are as set forth in any one of the following groups I-IX:
group of
LCDR1
LCDR2
LCDR3
HCDR1
HCDR2
HCDR3
I
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
II
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
III
SEQ ID NO:16
SEQ ID NO:17
SEQ ID NO:18
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
IV
SEQ ID NO:22
SEQ ID NO:23
SEQ ID NO:24
SEQ ID NO:19
SEQ ID NO:20
SEQ ID NO:21
V
SEQ ID NO:22
SEQ ID NO:23
SEQ ID NO:24
SEQ ID NO:25
SEQ ID NO:26
SEQ ID NO:27
VI
SEQ ID NO:31
SEQ ID NO:32
SEQ ID NO:33
SEQ ID NO:28
SEQ ID NO:29
SEQ ID NO:30
VII
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
VIII
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
IX
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
In one or more embodiments, the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 36. 37, 39, 41, 44, 46, 47, and 48; and/or is selected from SEQ ID NO: 34. 35, 38, 40, 42, 43 and 45, or a pharmaceutically acceptable salt thereof.
In one or more embodiments, the isolated antibody of the invention comprises a heavy chain constant region, preferably a human constant region, e.g., a
Herein, the term "conservatively modified variant" or "conservative substitution" or "conservative mutation" refers to the substitution of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes may be made frequently without altering the biological activity of the protein. Those skilled in The art recognize that single amino acid substitutions in non-essential regions of a polypeptide will generally not significantly alter biological activity (see, e.g., Watson et al, (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., page 224 (4 th edition)). In addition, substitutions of structurally or functionally similar amino acids are unlikely to destroy biological activity. The antibodies or antigen-binding fragments thereof of various embodiments of the invention comprise polypeptide chains having the following sequences: when compared to a particular amino acid sequence disclosed herein (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48), it includes up to 0 (NO change), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions; for example, from 0 to 20 amino acid substitutions may be included, or from 1 to 15, 1 to 10, 1 to 8, 1 to 5 substitutions may be included, or the number of substitutions may be within the range consisting of any two of the above values.
Thus, the invention also includes function-conservative variants of an antibody or antigen-binding fragment thereof of the invention, i.e., variants in which one or more amino acid residues in the antibody or antigen-binding fragment thereof of the invention are altered without altering the overall conformation and function of the antibody, including, but not limited to, the replacement of an amino acid with an amino acid having similar properties. Typically, the number of substitutions may be in the ranges described above, such as 0-20 conservative substitutions.
In one or more embodiments, an isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is a single chain Fv antibody; in certain embodiments, the antibody or antigen binding fragment thereof is a Fab antibody; in certain embodiments, the antibody or antigen binding fragment thereof is a Fab' antibody; in certain embodiments, the antibody or antigen-binding fragment thereof is (Fab')2An antibody.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 36, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 34 or 35.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 37, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 34 or 35.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 39, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 38.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 41, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 40 or 42.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 44, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 43.
In one or more embodiments, the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA of the invention is set forth in SEQ ID NO: 46. 47 or 48, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 45.
The invention also provides an isolated nucleic acid, e.g., a DNA, that encodes an isolated antibody or antigen-binding fragment thereof of the invention. In certain embodiments, an isolated nucleic acid of the invention encodes an antibody or antigen-binding fragment thereof comprising at least one mature antibody light chain Variable (VL) domain and at least one mature antibody heavy chain Variable (VH) domain, wherein the VL domain comprises at least 3 cdrs having a sequence selected from SEQ ID NOs: 7-8, 10-12, 16-18, 22-24 and 31-33, the VH domain comprising at least 3 CDRs having a sequence selected from SEQ ID NOs: 1-3, 4-6, 13-15, 19-21, 25-27, and 28-30. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 36 and SEQ ID NO: 34, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 37 and SEQ ID NO: 35, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 39 and SEQ ID NO: 38, and mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 41 and SEQ ID NO: 40, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 41 and SEQ ID NO: 42, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode seq id NOs: 44 and SEQ ID NO: 43, and mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 46 and SEQ ID NO:45, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 47 and SEQ ID NO:45, mature light and heavy chain variable region sequences. In one embodiment, the isolated nucleic acids encode SEQ ID NOs: 48 and SEQ ID NO:45, mature light and heavy chain variable region sequences. In one or more embodiments, the isolated nucleic acid encodes both a light chain and a heavy chain on a single nucleic acid molecule, while in other embodiments, the light chain and the heavy chain are encoded on two or more separate nucleic acid molecules.
The invention also provides expression vectors comprising the isolated nucleic acids of the invention. Host cells comprising the expression vectors of the invention are also provided. The invention also relates to methods of producing the antibodies of the invention or antigen binding fragments thereof.
The invention also relates to antibodies or antigen-binding fragments thereof that bind to the same epitope on human BTLA as the antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19 described herein, e.g., antibodies that are capable of cross-blocking the binding of any of the antibodies of the invention.
Diseases and their treatment or prevention
The invention also provides methods of treating or preventing a subject, including a human subject, in need of treatment with an isolated antibody or antigen-binding fragment thereof, with an antibody or antigen-binding fragment thereof, preferably a humanized antibody, of the invention. The methods generally comprise administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody, or antigen-binding fragment thereof, according to any embodiment of the invention, or a pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof. Suitable modes of administration may be selected as appropriate, including but not limited to oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration, and the like.
The subject typically suffers from a BTLA-mediated disease, i.e., a disease that benefits from elimination, inhibition, or reduction of BTLA activity. Generally, BTLA-mediated diseases are all diseases associated with immunosuppression, including autoimmune diseases, transplant rejection, tumors, and the like. The tumor comprises melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, cancer of the endocrine system, thyroid cancer, adrenal gland, soft tissue cancer, cancer of the urethra, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, cancer of the kidney, prostate cancer, prostate, Epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and combinations of said cancers. The autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases; the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.; the systemic autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis and the like.
Such treatment may also include administration of one or more other therapeutic agents, such as tumor vaccines, standard tumor chemotherapy therapeutic agents, other immune inducers.
Medicine box
The invention also provides a kit comprising the components of the combination according to the invention in kit form. Kits of the invention include one or more components, including but not limited to antibodies or antigen-binding fragments that specifically bind BTLA as described herein (e.g., antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19, but not limited to the antibodies described above) and one or more other components, including but not limited to pharmaceutically acceptable carriers and/or chemotherapeutic drugs as described herein. The antibody or antigen binding fragment and/or chemotherapeutic agent may be formulated as a pure composition or combined in a pharmaceutical composition with a pharmaceutically acceptable carrier.
In one embodiment, a kit comprises an antibody or antigen-binding fragment thereof of the invention (e.g., antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19, but not limited to the above antibodies) or a pharmaceutical composition thereof in one container (e.g., a sterile glass or plastic vial), and an antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof and/or a chemotherapeutic drug in another container (e.g., a sterile glass or plastic vial).
In another embodiment of the invention, a kit comprises a combination of the invention comprising an antibody or antigen-binding fragment thereof (such as antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18 and hu19, but not limited to the above antibodies) in a single common container, optionally in combination with one or more chemotherapeutic drug components formulated together, optionally in a pharmaceutical composition.
The kit may include a package insert including information about the pharmaceutical composition and dosage form in the kit. Such information generally helps patients and physicians to use the attached pharmaceutical compositions and dosage forms effectively and safely. For example, the following information may be provided in the pharmaceutical specification regarding the combination of the invention: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overuse, proper dosage and administration, supply specifications, proper storage conditions, references, manufacturer/wholesaler information, and patent information.
Pharmaceutical compositions and administration
The invention also includes pharmaceutical compositions comprising any of the anti-human BTLA antibodies or antigen-binding fragments thereof described herein. The term "pharmaceutical composition" as used herein denotes a combination of at least one drug, optionally together with a pharmaceutically acceptable carrier or adjuvant, combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition (e.g. the antibody, nucleic acid molecule combination and/or conjugate according to the invention) may be administered to the subject in bulk or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives. The immunoconjugates of the invention can comprise an antibody or antigen-binding fragment thereof according to any one of the embodiments herein coupled to a therapeutic agent, preferably an immunotoxin, radioisotope, drug or cytotoxic agent well known and commonly used in the art for preparing immunoconjugates.
To prepare a pharmaceutical composition or a sterile composition of an anti-human BTLA antibody or antigen-binding fragment thereof of the present invention, the antibody or antigen-binding fragment thereof can be mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and u.s.pharmacopeia: national Formulary, Mackpublishing Company, Easton, Pa (1984).
The pharmaceutical compositions of the present invention may be prepared in a variety of suitable dosage forms known in the art, including but not limited to lyophilized powders, ointments, aqueous solutions, or suspensions. Dosage forms of the following forms of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients or stabilizers: such as a lyophilized powder, a paste, an aqueous solution or a suspension (see, e.g., Hardman et al (2001) Goodman and Gilman's The Pharmacological Basis of therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al (eds.) (1993) Pharmaceutical Dosaffrons: fractional medicine, Marcel Dekker, NY; Linyerman et al (1990) Pharmaceutical Dodge Forms: tables, Decell Dekker, Marny; Lidmeman et al (1990) Pharmaceutical Dokker: disks, Mark Aker, Inc.; company et al (1990) Pharmaceutical Dokker, Inc.; company, Inc. 2000).
The dosage regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the dosing regimen delivers sufficient therapeutic antibody to achieve an improvement in the target disease state while minimizing adverse side effects. Thus, the amount of biological agent (biologic) delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic Antibodies is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios scientific Pub. Ltd, Oxfordshire, UK; Kresina (eds.) (1991) Monoclonal Antibodies, Cytokines and Althrtis, Marcel Dekker, New York, N.Y.; Bach (eds.) (1993) Monoclonal Antibodies and peptide Therapy in Autoimmu Diseases, Marcel Dekker, N.Y.; Baert et al (2003) New Engl. J.J.Med.348: 601 Buck.; Milgrom et al (1999) New Engl. J.341: 1966; Slam. J.2001. Med.348: 601. 619; Milgrom. et al (1999) New Engl. J.32; Begln. 2000: 10 J.3; New Engln. 103: 2000: 10. J.103: 76).
Applications of
The invention provides the use of antibodies against BTLA and antigen-binding fragments thereof according to any of the embodiments herein for the treatment, prevention and diagnosis of BTLA-mediated diseases. In certain embodiments, the invention provides BTLA antibodies and antigen binding fragments thereof according to any of the embodiments herein for use in the treatment, prevention, and diagnosis of BTLA-mediated diseases.
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