CN116178565A - Bispecific polypeptide complex - Google Patents
Bispecific polypeptide complex Download PDFInfo
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- CN116178565A CN116178565A CN202211161355.XA CN202211161355A CN116178565A CN 116178565 A CN116178565 A CN 116178565A CN 202211161355 A CN202211161355 A CN 202211161355A CN 116178565 A CN116178565 A CN 116178565A
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Abstract
The invention relates to the field of biological medicine, in particular to a bispecific fusion polypeptide and application thereof, wherein the bispecific fusion polypeptide comprises a first antigen binding part, the first antigen binding part comprises a first conjugate fragment and a second conjugate fragment which can be specifically combined, and the first conjugate and the second conjugate fragment are ligand/receptor pairs.
Description
The present application claims priority from chinese patent application No. 202210072619.8, title of invention, a bispecific polypeptide complex, of 2022, 1/21 and chinese patent application No. 202111123525.0, title of invention, a bispecific polypeptide complex, of 2021/9/24, the entire contents of which 2 chinese patent applications are incorporated herein by reference.
Technical Field
The invention relates to the field of biological medicine, in particular to a bispecific polypeptide complex containing interleukin 21 and a receptor thereof.
Background
Bispecific antibodies are the most popular novel biomacromolecule drug structures in the clinic at present. Bispecific antibodies (Bispecific Antibodies, bsAb) refer to antibodies that bind two different antigens or different epitopes of one antigen simultaneously, and can exert biological functions that cannot be achieved by monoclonal antibodies by a specific mode of action.
Bispecific antibodies during assembly, two natural heavy chains and two natural light chains can randomly generate 10 possible combinations, only one of which is the target diabody product. The biochemical characteristics of the 10 different diabody products are similar, the difficulty of separating the target diabody from the products is great, the yield and purity of the target diabody are low, and the cost is increased to influence the curative effect.
With the progress of recombinant protein expression and genetic engineering techniques, bispecific antibody formats are becoming more and more diverse, and up to now, more than 20 bispecific antibody formats have been developed as a technical platform.
The core value of the double-antibody technology platform is to solve the problems of heavy chain and heavy chain mismatch and light chain and heavy chain mismatch, and the technology platform for solving the problems of heavy chain and heavy chain mismatch mainly comprises the following components: knob-into-Holes (KiH), ART-Ig, chain exchange engineering domain (SEED) technology, xmAb.
The technology for solving the mismatch of the heavy chain and the heavy chain is relatively mature, and the technology for solving the mismatch of the light chain and the heavy chain still has room for improvement, and the invention is particularly proposed in view of the problem.
Disclosure of Invention
The present invention aims to solve one of the technical problems in the related art to a certain extent.
The inventor provides a new development idea of bispecific antibody, which utilizes the specific affinity of interleukin 21 and its receptor to replace CH1 and CL in the antibody or its functional fragment, thereby avoiding or reducing the occurrence of mismatch of heavy chain and light chain; further, the substitutions may be simultaneously or independently selected from CH2, CH3, and optionally CH4, thereby promoting the formation of heavy chain heterodimers. On the other hand, the bispecific antibody provided by the invention is a multifunctional fusion protein, which can not only exert the specificity of double targets, but also exert the biological activity of interleukin 21 and the receptor conduction thereof.
In one aspect, the invention provides a bispecific fusion polypeptide comprising a first antigen binding portion comprising:
a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain VH1 of a first antibody operably linked to a first conjugate fragment, and
a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain VL1 of a first antibody operably linked to a second conjugate fragment,
the first conjugate fragment and the second conjugate fragment are capable of specific binding;
wherein the first conjugate fragment is a receptor and the second conjugate fragment is a ligand; or the first conjugate fragment is a ligand and the second conjugate fragment is a receptor.
In some embodiments, further comprising a second antigen binding portion, the second antigen binding portion being different from the first antigen binding portion;
the second antigen binding portion comprises:
a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to a third conjugate fragment, and
A fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to a fourth conjugate fragment;
wherein the third conjugate fragment and the fourth conjugate fragment are capable of specific binding; and the third conjugate segment is a receptor, the fourth conjugate segment is a ligand, or the third conjugate segment is a ligand, the fourth conjugate segment is a receptor; and
the third and/or fourth conjugate fragments are selected from different receptors and ligands than the first and/or second conjugate fragments.
In some embodiments, further comprising a second antigen binding portion, the second antigen binding portion being different from the first antigen binding portion;
the second antigen binding portion comprises:
a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to an antibody heavy chain constant region CH1, and
a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to an antibody light chain constant region CL.
In some embodiments, the receptor comprises only active sites that recognize and bind the ligand, and does not comprise functional active sites that produce a response.
In some embodiments, the receptor and the ligand comprise at least one non-natural interchain bond therebetween that is capable of enhancing the specific binding force between the receptor and the ligand; in some embodiments, the non-natural interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; in some embodiments, the VH1 and VL1 comprise at least one non-natural inter-chain bond between a first mutated residue comprised by the first heavy chain variable domain VH1 and a second mutated residue comprised by the first light chain variable domain VL 1; in some embodiments, at least one of the first and the second mutated residues is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
In some embodiments, wherein at least one native glycosylation site is absent from the receptor and/or ligand.
In some embodiments, the receptor and its ligand is IL21/IL21R. In some embodiments, the IL21 is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.44 through SEQ ID NO.53 and the IL21R is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.54 through SEQ ID NO. 60. In some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.51 and the IL21R is selected from the sequence set forth in SEQ ID No. 55; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.48 and the IL21R is selected from the sequence set forth in SEQ ID No. 57; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.50 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.53 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.52 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.49 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.45 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.46 and the IL21R is selected from the sequence set forth in SEQ ID No. 56; in some embodiments, the IL21 is selected from the group consisting of the sequences set forth in SEQ ID NO.47 and the IL21R is selected from the group consisting of the sequences set forth in SEQ ID NO. 60. In some embodiments, the IL21 is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 44-53, and the IL21R is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 54-60.
In some embodiments, the bispecific fusion polypeptide comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments, the antibody Fc constant regions are associated as heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility; in some embodiments, the antibody Fc constant region comprises CH2, CH3, and optionally CH4, the CH2, CH3, and/or optional CH4 being replaced with the receptor and its ligand.
In some embodiments, the first antigen binding portion binds to a different antigen or to a different epitope of the same antigen than the second antigen binding portion; in some embodiments, the first antigen binding portion targets immune cells and the second antigen binding portion targets tumor cells; in some embodiments, the first antigen binding portion and the second antigen binding portion both target tumor cells; in some embodiments, the first antigen binding portion and the second antigen binding portion both target immune cells. In some embodiments, the first antigen and the second antigen are capable of engaging T cells and tumor antigens upon binding; in some embodiments, the binding of the first antigen to the second antigen is capable of engaging NK cells with a tumor antigen; in some embodiments, binding to the first antigen and the second antigen is capable of synergistically inhibiting a signaling pathway; in some embodiments, the first antigen and the second antigen are capable of forming a protein complex upon binding. In some embodiments, the first antigen-binding portion targets human PD-L1 and the second antigen-binding portion targets human TIGIT; or the first antigen binding portion targets human TIGIT and the second antigen binding portion targets human PD-L1.
The invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion polypeptide as described above.
The invention also relates to vectors containing the nucleic acids as described above.
The invention also relates to a host cell containing a nucleic acid as described above or a vector as described above.
The invention also relates to a method for preparing a bispecific fusion polypeptide or a multifunctional fusion polypeptide comprising:
transforming a host cell with a vector as described above;
culturing the transformed host cell; and
collecting bispecific fusion polypeptides or multifunctional fusion polypeptides expressed in host cells.
The invention also relates to a pharmaceutical composition comprising a bispecific fusion polypeptide or a multifunctional fusion polypeptide as described above, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
The invention also relates to the use of a bispecific fusion polypeptide or a multifunctional fusion polypeptide or a pharmaceutical composition as described in any of the above for the preparation of a medicament for the treatment of a disease.
The invention also relates to a bispecific or multifunctional fusion polypeptide or a pharmaceutical composition according to any one of the preceding claims for use as a medicament, in some embodiments for the treatment of a disease or disorder.
The invention also relates to a method of treating a disease comprising administering to a subject a therapeutically effective amount of a bispecific or multifunctional fusion polypeptide or pharmaceutical composition as described above.
Drawings
Fig. 1 shows 4 classical dual antibody platforms: FIG. 1A is a KiH heterodimerization Fc engineering technique; FIG. 1B is a cross mab bispecific antibody technique; FIG. 1C shows the technology of YBODY double antibody (asymmetric scFv double antibody); FIG. 1D is a symmetrical scFv diabody;
FIG. 2 shows a novel bispecific antibody FiBody provided by the present invention with specific affinity for the ligand receptor to replace CH1, CL of one side Fab;
fig. 3 is an exemplary illustration of the possible scheme in 4 of FiBody: FIG. 3-1 is an engineered ligand having non-naturally occurring interchain bonds between the ligands; FIG. 3-2 shows that CH1 and CL of the two-sided Fab are both substituted by receptor and ligand, and two sides are selected from different ligand receptors; FIGS. 3-3 show the substitution of the CH1 and CL of the antibody except for the Fab by the ligand, and the substitution of the CH3 segment of the Fc dimer by the ligand; FIGS. 3-4 show the substitution of CH1, CL of the antibody except for one side Fab with ligand receptor, and the substitution of CH2 in the Fc dimer with ligand receptor; other feasible modification modes are numerous;
FIG. 4 is an exemplary targeted binding of the antigen binding portion of a bispecific antibody of the present invention when the bispecific antibody is used in the treatment of a tumor, comprising exemplary 3 types: FIG. 4-A first antigen binding portion targets T cells and second antigen binding portion targets tumor cells; FIG. 4-B both the first antigen binding portion and the second antigen binding portion target tumor cells; FIG. 4-C targeting T cells to both the first antigen binding portion and the second antigen binding portion; FIG. 4-D illustrates an alternative trifunctional fusion protein as a dual-characteristic antibody embodying the present invention, which, in addition to exhibiting different antigen binding, also activates the ligand pathway, stimulating ligand biological activity;
Fig. 5 is a perspective view of interleukins and their receptors, and can be divided into four classes: class A is lifting type, class B is bowknot type, class C is baseball hand type, class D is pincer type;
FIG. 6 is an example of four types of three-dimensional conformations of interleukins and their receptors, type A lifting is IL2/IL2R, type B bowtie is IL22/IL22R, type C bowtie is IL18/IL18R, type D pincer is IL21/IL21R;
FIG. 7 shows the FCM method for detecting the binding activity of the double-antibody TIGIT end and CHO-Tigit cells (R1116/R1117/R1119/R1121/R1123/R1124);
FIG. 8 shows the FCM method for detecting the binding activity of the double anti-PDL 1 end and CHO-Tigit cells (R1116/R1119/R1121/R1123/R1124/R0919);
FIG. 9 is a diagram showing FiBody two-terminal binding force detection (CHO) based on IL21/IL21R construction in accordance with an embodiment of the present invention;
FIG. 10 shows a FiBody two-terminal binding force test (Jurkat) based on IL21/IL21R construction in an embodiment of the invention;
FIG. 11 is a schematic diagram of R1155 and R1160 in example 8;
FIG. 12 example 9 disulfide bond engineered IL21/IL21RαFiBody (complexes 1-10) gel electrophoresis detection results; in the drawings, complex 1 represents R1267, complex 2 represents R1268, complex 3 represents R1269, complex 4 represents R1270, complex 5 represents R1271, complex 6 represents R1272, complex 7 represents R1273, complex 8 represents R1274, complex 9 represents R1275, complex 10 represents R1123, and the same applies;
FIG. 13 example 9 results of the detection of binding force of disulfide bond engineered to the targeting region (@ TIGIT) by unmodified IL21/IL21RαFibody;
FIG. 14 example 9 results of the detection of binding force of disulfide bond engineered to the targeting region (@ PD-L1) by unmodified IL21/IL21RαFiBody;
FIG. 15 example 9 results of a disulfide bond engineered and non-engineered IL21/IL21RαFiBody blocking assay for binding to the targeting region (@ TIGIT).
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment. All documents cited in this disclosure, including publications, patents, and patent applications, are incorporated by reference in their entirety.
Unless defined otherwise, all 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Interpretation of the terms:
The term "antigen binding portion" or "antigen binding domain" means that portion of an antigen binding molecule that confers binding specificity to an epitope. In some embodiments, the "antigen binding portion" is an antibody functional fragment.
The term "amino acid" means one of the 20 naturally occurring amino acids encoded by DNA and RNA.
The term "wild-type or WT" means an amino acid sequence or nucleotide sequence found in nature, including allelic variations. The WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
The term "antibody" encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody, or bispecific fusion polypeptide that can bind to a particular antigen. A natural whole antibody comprises two heavy chains and two light chains. Each heavy chain consists of one variable region ("HCVR" or VH) and first, second and third constant regions (CH 1, CH2, CH3, respectively), while each light chain consists of one variable region ("LCVR" or VL) and one constant region (CL). Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains can be classified as λ or κ.
The antibody is "Y" shaped, the backbone consisting of the second (CH 2), third (CH 3) and optionally fourth (CH 4) constant regions of two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure comprises a variable region (VH) and a first constant region (CH 1) of one of the heavy chains, which is associated with a variable region (VL) and a constant region (CL) of one of the light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs), the CDRs of the (light (L) chain comprise LCDR1, LCDR2, LCDR3, the CDRs of the heavy (H) chain comprise HCDR1, HCDR2, HCDR3. Wherein the three CDRs are separated by contiguous portions of the sides called Framework Regions (FRs) which are more highly conserved than the CDRs and form a scaffold supporting hypervariable loop.
The constant regions of the heavy and light chains do not participate in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes according to the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five main classes or allotypes depending on whether they contain alpha, delta, epsilon, gamma and mu heavy chains: igA, igD, igE, igG and IgM. Several major classes of antibodies can also be classified into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain), among others.
Hypervariable regions typically comprise amino acid residues from amino acid residues 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3) in the light chain variable region and 31-35B (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3) in the heavy chain variable region (Kabat et al, sequence of immunorelated proteins (SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST), 5 th edition, besseda, maryland, public health service, U.S. department of health (Public Health Service, national Institutes of Health, bethesda, md.) (1991)), or those residues forming hypervariable loops, such as residues 26-32 (LCDR 1), 50-52 (LCDR 2) and 91-96 (LCDR 3) in the light chain variable region and 26-32 (HCDR 1), 53-55 (HCDR 2) and 96-101 (HCDR 3) in the heavy chain variable region (Chothia and Lesk, 1987, J.7. Mol.901-917.
In some embodiments, the antibody is a bispecific antibody (BiAb). The term "bispecific" refers herein to two different antigens, or when both are the same antigen, each having binding specificity for a different epitope. The epitopes may be derived from different antigens or the same antigen. The terms "bispecific fusion polypeptide" and "bispecific antibody" refer herein to all produced products having full length antibodies or fragments with antigen binding sites. The antibody may be a human antibody, a non-human antibody (e.g., a mouse-derived antibody), a humanized antibody, or a chimeric antibody (e.g., a human-mouse chimeric antibody or a chimeric of different subtypes of antibodies). In some cases, the variant of the antibody is obtained by conservative modifications or conservative substitutions or substitutions in the antibody sequences provided herein. "conservative modifications" or "conservative substitutions or substitutions" refer to amino acids in other amino acid substituted proteins that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially 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, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. One of ordinary skill in the art will be able to determine suitable variants of the antigen binding molecules as set forth herein using well known techniques. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
The term "Fab" is a Fab fragment containing no or a small fraction of residual Fc fragments in the immunoglobulin, e.g., fab fragments include the variable regions of the heavy and light chains, as well as all or part of the first constant domain. For simplicity, the term "Fab" hereinafter may also refer to fragments such as F (ab) 2.
The term "Fc" or "Fc region" or "Fc domain" means a polypeptide comprising a constant region of an antibody, in some cases excluding all or a portion of a first constant region immunoglobulin domain (e.g., CH 1) or a portion thereof, and in some cases further excluding all or a portion of a hinge. Thus, fc may refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH 3) of IgA, igD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally all or a portion of the flexible hinge N-terminus of these domains. For IgA and IgM, the Fc may comprise the J chain. For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (cγ2 and cγ3) and a lower hinge region between CH1 (cγ1) and CH2 (cγ2). Although the boundaries of the Fc region may vary, a human IgG heavy chain Fc region is generally defined to include residues E216, C226 or a231 at its carboxy-terminus, with numbering according to the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, e.g., the Fc is a heterodimer, as described more fully below.
"modification" herein refers to an amino acid substitution, insertion and/or deletion in a polypeptide sequence or a change in the moiety that is chemically linked to a protein. "amino acid modification" herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, amino acid modifications are always amino acids encoded by DNA, e.g., 20 amino acids with codons in DNA and RNA, unless otherwise indicated.
An "epitope" is herein intended to mean a determinant that interacts with a specific antigen binding domain, e.g., the variable region (referred to as the paratope) of an antibody molecule. An epitope is a grouping of molecules such as amino acids or sugar side chains, and generally has specific structural features as well as specific charge characteristics. A single molecule may have more than one epitope. An epitope may comprise amino acid residues directly involved in binding (also referred to as immunodominant components of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked by a specific antigen binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen binding peptide. Epitopes may be conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may differ by loss of binding to the former but not to the latter in the presence of denaturing solvents. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antigen binding molecules recognizing the same epitope can be validated in a simple immunoassay, showing the ability of one antigen binding molecule to block the binding of another antigen binding molecule to a target antigen. As outlined below, the invention encompasses not only the antigen binding molecules and antigen binding domains listed herein, but also antigen binding molecules and antigen binding domains that compete for binding to epitopes bound by the listed antigen binding molecules or antigen binding domains.
The terms "specific binding", "selective binding", "selectively binding" and "specifically binding" refer to a directed biological binding process in which a ligand capable of being competitively blocked by the corresponding substance interacts with a specific structural site in vitro or in vivo. Such as binding between an antigen and an antibody or between a receptor and a ligand.
The strength or affinity of a specific binding may be expressed in terms of the dissociation constant (KD) of the interaction, where a smaller KD indicates a greater affinity and a larger KD indicates a lower affinity. For example, KD of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or greater antigen binding capacity. Binding properties may be determined by methods well known in the art, such as biological layer interferometry and methods based on surface plasmon resonance. One such method entails measuring the rate of association and dissociation of antigen binding site/antigen or receptor/ligand complexes, where the rate depends on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the association rate (ka) and dissociation rate (KD) can be determined, and the ratio of KD/ka is equal to the dissociation constant KD (Nature 361:186-187 (1993) and Davies et al (1990) biochemistry yearbook (Annual RevBiochem) 59:439-473).
The term "immune cell" includes cells that are involved in protecting the immune system of the body against infectious diseases or foreign substances. Immune cells may include, for example, neutrophils, eosinophils, basophils, lymphocytes, such as B cells and T cells, and monocytes. T cells may include, for example, cd4+, cd8+, T helper cells, cytotoxic T cells, γδ T cells, regulatory T cells, inhibitory T cells, and natural killer cells.
The term "multifunctional fusion polypeptide" means a non-naturally occurring binding molecule designed to target two or more antigens. The "multifunctional fusion polypeptides" described herein are typically genetically engineered fusion proteins designed to bring two different desired biological functions into a single binding molecule. For example, the multifunctional fusion polypeptide may be a multifunctional binding molecule.
The term "FiBody" is a bispecific antibody obtained by recombination by replacing CL and CH1 on one side of the bispecific antibody with a specific affinity between the ligand and its receptor, which is capable of avoiding the occurrence of mismatches between the light and heavy chains of the bispecific antibody.
The "YBody" technology mentioned in the present invention was developed by the wuhanou zhiyou company in 2012, and is based on the "Knob-into-Holes" technology, which forms heterodimers. One of them is normal heavy chain, and the other is N-terminal linked scFv of Fc functional region, so that it can form asymmetric bispecific antibody.
The term "about" or "approximately" means a quantity, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length that differs from a reference quantity, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. In particular embodiments, when the term "about" or "approximately" precedes a numerical value, the value plus or minus a range of 15%, 10%, 5%, or 1% is indicated.
Unless the context indicates otherwise, the words "comprise," "comprising," and "include" are to be construed as meaning that the recited step or element or group of steps or elements are included, but not to exclude any other step or element or group of steps or elements. What is meant by "consisting of … …" is what is encompassed by and limited to the phrase "consisting of … …". Thus, the phrase "consisting of … …" means that the listed elements are required or necessary and that no other elements may be present. "consisting essentially of … …" is intended to include any element listed after this phrase and is limited to other elements that contribute to or do not interfere with the activity or function of the listed elements as detailed in the present invention. Thus, the phrase "consisting essentially of … …" means that the listed elements are required or necessary, but that other elements are optional and may be present or absent depending on whether they affect the activity or effect of the listed elements.
Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "another embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature defined as "optional" may explicitly or implicitly include or exclude that feature.
The terms "first," "second," and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
Bispecific fusion polypeptides
The present invention provides novel bispecific fusion polypeptides comprising a ligand (or fragment thereof) and a receptor (or fragment thereof) thereof, which ligand (or fragment thereof) and receptor (or fragment thereof) independently replace CH1 and CL of an antibody-side Fab, respectively, in particular the bispecific fusion polypeptides comprise a first antigen-binding moiety comprising: a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain VH1 of a first antibody operably linked to a first conjugate fragment;
a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain VL1 of a first antibody operably linked to a second conjugate fragment,
wherein the first conjugate fragment is a receptor and the second conjugate fragment is a ligand; or the first conjugate fragment is a ligand and the second conjugate fragment is a receptor.
In some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is, in order from the N-terminal to the C-terminal: [ VH1] - [ linker 1] - [ IL21] - [ linker 2] - [ CH2] - [ CH3], a second polypeptide comprising, in order from the N-terminus to the C-terminus: [ VL1] - [ linker 3] - [ [ IL21R ]; in some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is, in order from the N-terminal to the C-terminal: [ VH1] - [ linker 1] - [ IL21R ] - [ linker 2] - [ CH2] - [ CH3], a second polypeptide comprising, in order from the N-terminus to the C-terminus: [ VL1] - [ linker 3] - [ [ IL21]. Wherein, CH2 and CH3 are heavy chain constant region subunits, and the linker 1, the linker 2 and the linker 3 are linkers for connecting polypeptides, which may be the same or different; in some embodiments, the linker 1, linker 2, and linker 3 are independently selected from (GxS) y linkers, wherein x is selected from an integer from 1 to 5 and y is selected from an integer from 0 to 6.
The bispecific fusion polypeptide further comprises a second antigen binding moiety that is different from the first antigen binding moiety. Alternative polypeptide fusion means for the second antigen binding portion include those selected from the group consisting of:
1. the CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or fragment thereof) and its receptor (or fragment thereof), i.e.
The second antigen binding portion comprises: a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to a third conjugate fragment, and
a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to a fourth conjugate fragment;
the third conjugate segment is a receptor, and the fourth conjugate segment is a ligand; or the third conjugate fragment is a ligand and the fourth conjugate fragment is a receptor; and
the third and/or fourth conjugate fragments are selected from different receptors and ligands than the first and/or second conjugate fragments, the third and fourth conjugate fragments being capable of specific binding; or alternatively
2. The Fab on the other side of the antibody retains the original CH1 and CL, i.e.,
the second antigen binding portion comprises: a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to an antibody heavy chain constant region CH1, and
a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to an antibody light chain constant region CL.
The present invention utilizes the specific binding force characteristic of the ligand and its receptor itself to creatively operably link it to an antigen binding region (antibody variable region) including one of them, the other antigen binding region still being linked to CH1 and CL; or both antigen binding regions are linked to ligand receptors, except that both antigen binding regions are linked to different classes of ligand receptors, thereby avoiding mismatches in the different antigen binding regions.
In some embodiments, the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide comprising 2 antibody fabs, wherein CH1 and CL of one Fab are independently substituted with a ligand and its receptor, and CH1 and CL of the other Fab are unsubstituted, said receptor comprising both an active site that recognizes and binds the ligand and a functional active site that produces a response; the light chain of the first antigen binding portion is not mismatched with the heavy chain of the second antigen binding portion. In some embodiments, wherein CH1 and CL of one Fab are independently substituted with a first ligand and its receptor, and CH1 and CL of the other Fab are independently substituted with a second ligand and its receptor, the first ligand and its receptor being different from the second ligand and its receptor.
In some embodiments, the multifunctional fusion protein is capable of exerting not only dual-target specificity, but also ligand-receptor transduction biological activity. For example, in a particular embodiment, the ligands and their receptors are IL21 and IL21R, and the multifunctional fusion polypeptide, in addition to having dual-target targeting, binds IL21 and IL21R, activating downstream signaling pathways, exerting corresponding biological functions.
In some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is, in order from the N-terminal to the C-terminal: [ VH1] - [ linker 1] - [ IL21] - [ linker 2] - [ Fc1], a second polypeptide, which in order from the N-terminus to the C-terminus: [ VL1] - [ linker 3] - [ [ IL21R ], a third polypeptide, which is, in order from the N-terminus to the C-terminus: [ VH2] - [ Fc2], and a fourth polypeptide, which is in order from the N-terminus to the C-terminus: [ VL2] - [ CL ]; in some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is, in order from the N-terminal to the C-terminal: [ VH1] - [ linker 1] - [ IL21R ] - [ linker 2] - [ Fc1], a second polypeptide, which in order from the N-terminus to the C-terminus: [ VL1] - [ linker 3] - [ [ IL21], a third polypeptide, which is, in order from the N-terminus to the C-terminus: [ VH2] - [ Fc2], and a fourth polypeptide, which is in order from the N-terminus to the C-terminus: [ VL2] - [ CL ]. Wherein, the Fc1 and Fc2 are 2 subunits of a heavy chain constant region Fc, which may be the same or different, preferably the Fc constant region is a heterodimer (heterodimer Fc fusion protein); in some embodiments, the Fc constant regions are associated as heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility. The linker 1, the linker 2 and the linker 3 are linkers for connecting polypeptides, and can be the same or different; in some embodiments, the linker 1, linker 2, and linker 3 are independently selected from (GxS) y linkers, wherein x is selected from an integer from 1 to 5 and y is selected from an integer from 0 to 6.
In some embodiments, VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT, and VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1. In some embodiments, VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1, and VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT. In some embodiments, the antigen-binding portion that binds TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises tigit_vh in complex 1 (i.e., a polypeptide consisting of residues 1-118 of SEQ ID No. 24) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises tigit_vl in complex 1 (i.e., a polypeptide consisting of residues 1-107 of SEQ ID No. 25) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto. In some embodiments, wherein the heavy chain variable region of the antigen binding portion that binds TIGIT comprises HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 comprising HCDR1, HCDR2, and HCDR3, respectively, in the variable region consisting of residues 1 to 118 of SEQ ID No.24, in some embodiments, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions, the LCDR1, LCDR2, and LCDR3 comprising LCDR1, LCDR2, and LCDR3, respectively, in the light chain variable region consisting of residues 1 to 107 of SEQ ID No. 25; in some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region comprising the PD-L1 heavy chain variable region in complex 1 (i.e., the polypeptide consisting of residues 1-119 of SEQ ID No. 43) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and a light chain variable region comprising the PD-L1 light chain variable region in complex 1 (i.e., the polypeptide consisting of residues 1-108 of SEQ ID No. 42) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; in some embodiments, the heavy chain variable region of the antigen binding portion that binds PD-L1 comprises HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 comprising HCDR1, HCDR2, and HCDR3, respectively, in SEQ ID No.43, in some embodiments, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions, the LCDR1, LCDR2, and LCDR3 comprising LCDR1, LCDR2, and LCDR3, respectively, in SEQ ID No. 42; in some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
In some embodiments, the IL21 is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.44 through SEQ ID NO.53 and the IL21R is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.54 through SEQ ID NO. 60. In some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.51 and the IL21R is selected from the sequence set forth in SEQ ID No. 55; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.48 and the IL21R is selected from the sequence set forth in SEQ ID No. 57; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.50 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.53 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.52 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.49 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.45 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.46 and the IL21R is selected from the sequence set forth in SEQ ID No. 56; in some embodiments, the IL21 is selected from the group consisting of the sequences set forth in SEQ ID NO.47 and the IL21R is selected from the group consisting of the sequences set forth in SEQ ID NO. 60. In some embodiments, the IL21 is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 44-53, and the IL21R is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 54-60.
In some embodiments, the bispecific fusion polypeptide is a FiBody molecule of the invention shown in table 3; in some embodiments, the bispecific fusion polypeptide is complex 1, complex 2, complex 3, complex 4, complex 5, complex 6, complex 7, complex 8, or complex 9 of example 9 of the present invention.
I.ligands and receptors
A "receptor" is a substance on or in a cell membrane that recognizes and binds to a biologically active molecule, and the biologically active substance that binds to a receptor is collectively referred to as a "ligand".
Receptors are classified into two major classes, cell surface receptors and intracellular receptors, according to their location in the cell. The receptor itself contains at least two active sites: one is an active site that recognizes and binds a ligand; the other is a functionally active site responsible for the response, which site only after binding to the ligand forms a binary complex and allosteric, will produce a response, thereby initiating a series of biochemical reactions, ultimately leading to a biological effect in the target cell.
Receptors are typically glycoproteins, and the binding between wild-type receptors and ligands is not mediated by covalent bonds, but rather is mediated by ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions. The receptor has the characteristics of saturation, high affinity, specificity and the like when being combined with the ligand.
The co-ordinated receptor and ligand have a relatively specific binding affinity and optionally a biological effect. In some embodiments, the receptor comprises only active sites that recognize and bind the ligand, and does not comprise functional active sites that produce a responsive response (e.g., functions that activate the biological effects of downstream signaling pathways). In some embodiments, the receptor and/or ligand is a natural ligand-receiving structure, the receptor comprises both an active site for recognizing the binding ligand and a functional active site responsible for generating a response reaction, and is capable of performing a corresponding biological function, and the bispecific fusion protein is a multifunctional fusion protein which has dual specificity and is capable of performing a ligand receptor function.
In some embodiments, the receptor and/or ligand is modified based on the native sequence, including, but not limited to, truncations, insertions, and/or mutations; the purposes of these modifications include, but are not limited to: increasing or decreasing the binding force of the ligand to the receptor; enhancing, reducing or eliminating the biological function of the ligand receptor; increasing, decreasing or eliminating glycosylation sites in receptor and or ligand proteins; reducing or eliminating ligand toxicity.
The binding means of the receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) may be covalent binding, non-covalent interactions or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonds, hydrophobic bonds, ionic bonds, and van der Waals bonds. In some embodiments, antibodies can be engineered to increase affinity when the affinity between the inserted or substituted conjugate fragments is lower than expected (e.g., the two variable regions in the antigen binding portion cannot be pulled together to allow them to achieve the function of specifically recognizing an antigen, or the heavy chain mismatches between 2 or more heavy chain constant regions cannot be prevented, or the mismatches between the antigen binding portions cannot be prevented to achieve a combination of specific VL-VH portions). In some embodiments, the receptor and the ligand comprise at least one non-natural interchain bond therebetween that is capable of enhancing the specific binding force between the receptor and the ligand; in some embodiments, the non-natural interchain bond is formed between a first mutated residue of a receptor and a second mutated residue of a ligand; in some embodiments, at least one of the first and the second mutated residues is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
"unnatural interchain bonds" refers to interchain bonds not found in wild-type polypeptide polymers. For example, an unnatural inter-chain bond can be formed between a mutated amino acid residue of one polypeptide and a mutated amino acid residue of another polypeptide.
In some embodiments, wherein at least one native glycosylation site is absent from the receptor and/or ligand.
In some embodiments, the receptor and ligand are selected from the group consisting of interleukins and their receptors.
The inventor performs three-dimensional conformation research on a large number of interleukins and receptors thereof, and discovers that the three-dimensional conformation of a large number of interleukins or IFN molecules can be divided into 4 types: class a-lift type, class B-bow tie type, C-baseball hand type, class D-pincer type.
In some embodiments, the ligand and its receptor are selected from the group consisting of class D interleukins and their receptors, e.g., IL21/IL21R.
The insertion or substitution positions of the mutually cooperating receptors (or fragments thereof) and ligands (or fragments thereof) may be located, for example:
the receptor or fragment thereof inserts or replaces the CL region, and the ligand or fragment thereof inserts or replaces the CH1 region; or (b)
The receptor or fragment thereof inserts or replaces the CH1 region and the ligand or fragment thereof inserts or replaces the CL region.
In some embodiments, the IL21 is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.44 through SEQ ID NO.53 and the IL21R is selected from the group consisting of the sequences set forth in any one of SEQ ID NO.54 through SEQ ID NO. 60. In some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.51 and the IL21R is selected from the sequence set forth in SEQ ID No. 55; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.48 and the IL21R is selected from the sequence set forth in SEQ ID No. 57; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.50 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.53 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.52 and the IL21R is selected from the sequence set forth in SEQ ID No. 59; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.49 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.45 and the IL21R is selected from the sequence set forth in SEQ ID No. 58; in some embodiments, the IL21 is selected from the sequence set forth in SEQ ID No.46 and the IL21R is selected from the sequence set forth in SEQ ID No. 56; in some embodiments, the IL21 is selected from the group consisting of the sequences set forth in SEQ ID NO.47 and the IL21R is selected from the group consisting of the sequences set forth in SEQ ID NO. 60. In some embodiments, the IL21 is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 44-53, and the IL21R is selected from a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the sequences set forth in SEQ ID nos. 54-60.
Ii. Antigen binding portion
The bispecific fusion polypeptide provided by the invention comprises a first antigen binding portion and a second antigen binding portion, has two antigen specificities, and can be different from the second antigen binding portion, and can be different antigens combined by the first antigen binding portion and the second antigen binding portion or different epitopes combined by the first antigen binding portion and the second antigen binding portion.
In some embodiments, the target against which the bispecific fusion protein is directed is a tumor. In some embodiments, the target point at which the first antigen binding portion binds to the second antigen binding portion is expressed in a tumor cell; in some embodiments, the target to which the first antigen binding portion binds is a tumor cell and the target to which the second antigen binding portion binds is an immune cell; in some embodiments, the target to which the first antigen binding portion binds to the second antigen binding portion is in an immune cell.
T cell redirected killing is an ideal mechanism of action in many therapeutic areas. In preclinical and clinical trials, various bispecific antibody formats are involved in T cell redirection (mayc et al (2012) Biochem Pharmacol,84 (9)): 1105 to 1112, th; franker SR, and Baeuerle PA, (2013) CURR OPIN chemical biology, volume 17 (3): 385-92, pages). All T cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen binding sites, one binding to a surface antigen on a target cell and the other binding to a T cell surface antigen. Of the T cell surface antigens, the epsilon subunit of human CD3 derived from the TCR protein complex is most often targeted as a target for redirecting T cell killing.
Tumor associated antigens that can be targeted include, but are not limited to: alpha-fetoprotein (AFP), alpha-actin-4, A3, antigen specific for the A33 antibody, ART-4, B7, ba 733, BAGE, brE 3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32B, CD33, CD37, CD38, CD40L, CD, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD L, CD, CD79a, CD80, CD83, CD95, CD132, CD138, CD147, CD80, CD133 CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR7, CXCL12, HIF-1α, colon specific antigen p (CSAp), CEA (CEACAM 5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, ep-CAM, fibroblast Growth Factor (FGF), flt-1, flt-3, folate receptor, G250 antigen, claudin18.2, GAGE, gp100, GRO- β, HLA-DR, HM1.24, human chorionic BCMA gonadotrophin (HCG) and subunits thereof, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, ia, IGF-1R, IFN- γ, IFN- α, IFN- β, IFN- λ, IL-4R, IL-6-5-13R, IL-15R, IL-17-18-IL-6, IL-12, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC 4-antigen, KS-1-antigen, KS1-4, le-Y, LDR/FUT, macrophage Migration Inhibitory Factor (MIF), MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreatic Cancer mucin, PD-1 receptor, placental growth factor p53, PLAGL2, prostatectomy phosphatase, PSA, PRAME, PSMA, plGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha, tn antigen, thomson-Friedenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factor C3, C3a, C3B, C5a, C5, angiogenesis markers, bcl-2, bcl-6, kras, oncogene markers, and oncogene products (see, such as Sensi et al, clin Cancer Res2006, 12:5023-32; parmia et al, JImmunol2007, 178:1975-79; novellino et al Cancer Immunol Immunother2005, 54:187-207).
While antibodies or other binding molecules specific for effector T cells preferably bind to CD3 antigen, other antigens expressed on effector T cells are known and can be targeted by T-cell redirecting complexes. Exemplary T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40L, CD, CD45, CD69, and CD90.
Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and modulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage. In some embodiments, the targets at which the first antigen binding portion binds to the second antigen binding portion are each an immune checkpoint or a ligand thereof, including but not limited to: TIGIT, PD-1, TIM-3, LAG3, GTLA4, BTLA, BTN1A1, VISTA, LAIR, CD96, PVRIG, LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, LLRB4, NKG-2A, CD47, CD200R1, CD300, dectin-1, ICOS, NKp30, CD28H, CRTAM, DNAM-1, 4-1-BB, BAFF, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, OX, TACI, 2B4, CD2, CD48, CD229, SLAM, SLAMF5, GRAAC, TIM1, TIM4, CD7, DPPIV.
In some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is PD-L1; in some embodiments, the first antigen binding portion binds to PD-1 at the target and the second antigen binding portion binds to TIGIT at the target; in some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is GTLA4; in some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is LAG3; in some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is TIM-3; in some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is CD47; in some embodiments, the first antigen binding portion binds to a target that is PD-1 and the second antigen binding portion binds to a target that is GTLA4; in some embodiments, the first antigen binding portion binds to a target of PD-1 and the second antigen binding portion binds to a target of 4-1-BB; in some embodiments, the first antigen binding portion binds to a target of PD-L1 and the second antigen binding portion binds to a target of 4-1-BB; in some embodiments, the first antigen-binding portion binds to a target that is PD-L1 and the second antigen-binding portion binds to a target that is TIGIT.
In some embodiments, the first antigen binding moiety targets a tumor-associated antigen and the second antigen binding moiety targets an immune checkpoint. In some embodiments, the first antigen binding portion targets HER2 and the second antigen binding portion targets PD-1; in some embodiments, the first antigen binding portion targets VEGF and the second antigen binding portion targets PD-L1; in some embodiments, the first antigen binding moiety targets claudin18.2 and the second antigen binding moiety targets PD-L1; in some embodiments, the first antigen binding moiety targets HER2 and the second antigen binding moiety targets CTLA-4; in some embodiments, the first antigen binding portion targets CD20 and the second antigen binding portion targets CD47; in some embodiments, the first antigen binding portion targets HER2 and the second antigen binding portion targets CD47.
In some embodiments, the first antigen binding portion and the second antigen binding portion simultaneously target tumor heterogeneity. Exemplary common targets for tumors include, but are not limited to, HGF and VEGF, IGF-1R and VEGF, her2 and VEGF, CD19 and CD3, CD20 and CD3, her2 and CD3, CD19 and fcγriiia, CD20 and fcγriiia, her2 and fcγriiia. The bispecific fusion polypeptide of the invention is capable of binding VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and phosphatidylserine; erbB3 and IGF1R; erbB3 and IGF1,2; c-Met and Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, and EGFR and DLL4. Other target combinations include one or more members of the EGF/erb-2/erb-3 family.
Furthermore, exemplary common targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNFα, IL-6 and IL-1, igE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and FcgammaRIIB, and CD79 and FcgammaRIIB.
Exemplary targets for treating inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and αvβ3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; igE and IL-13; IL-13 and IL23p19; igE and IL-4; igE and IL-9; igE and IL-9; igE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-1 beta; IL-1 beta and RANKL; IL-1 beta and VEGF; RANKL and CD-20; IL-1 alpha and IL-1 beta; IL-1 alpha and IL-1 beta.
Targets involved in Rheumatoid Arthritis (RA) include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1 beta; TNF and MIF; TNF and IL-17; and TNF and IL-15.
Targets for treating Systemic Lupus Erythematosus (SLE) include, but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6ST, INHA, HBA, KLF6, DPP4, FCER2, R2, IL2, ITGA2, GA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA, 5E, CTLA4, CTLA 1, GALNA-BlyS, BAFF, IFN, and TNF alpha.
Targets for the treatment of Multiple Sclerosis (MS), including but not limited to: IL-12, TWEAK, IL-23, CXCl13, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNγ, GM-CSF, FGF, C5, CD52 and CCR2.
Targets for treating sepsis include, but are not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, fasL, LPS, toll-like receptor, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NFkB 1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFkB 1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFkB 2, SERPINA1, SERPINE1, and TREM1.
To form the bispecific fusion proteins of the invention, antibodies can be made against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by the multispecific antibodies according to the invention.
In some embodiments, the first antigen binding portion and the second antigen binding portion target different epitopes of the same antigen.
In some embodiments, the at least one antigen binding fragment may further comprise a secretion signal sequence.
Secretion signal sequence refers to a sequence that induces secretion of an expressed protein or peptide by linking to the N-terminus of a coding sequence located outside of a cell membrane or outside of a cell, and may be a peptide sequence consisting of about 18-30 amino acids. All proteins capable of transporting to the outside of the cell membrane have different signal sequences that are cleaved by signal peptidases on the cell membrane. In general, for foreign proteins that are not naturally expressed by the host cell, secretion signal sequences that secrete the protein into the periplasm or medium of the cell, or modified sequences, may be employed.
In some embodiments, the first antigen binding portion and the second antigen binding portion bind TIGIT and PD-L1, respectively. In some embodiments, the antigen-binding portion that binds TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises tigit_vh in complex 1 (i.e., a polypeptide consisting of residues 1-118 of SEQ ID No. 24) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises tigit_vl in complex 1 (i.e., a polypeptide consisting of residues 1-107 of SEQ ID No. 25) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto. In some embodiments, wherein the heavy chain variable region of the antigen binding portion that binds TIGIT comprises HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 comprising HCDR1, HCDR2, and HCDR3, respectively, in the variable region consisting of residues 1 to 118 of SEQ ID No.24, in some embodiments, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions, the LCDR1, LCDR2, and LCDR3 comprising LCDR1, LCDR2, and LCDR3, respectively, in the light chain variable region consisting of residues 1 to 107 of SEQ ID No. 25; in some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region comprising the PD-L1 heavy chain variable region in complex 1 (i.e., the polypeptide consisting of residues 1-119 of SEQ ID No. 43) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and a light chain variable region comprising the PD-L1 light chain variable region in complex 1 (i.e., the polypeptide consisting of residues 1-108 of SEQ ID No. 42) or a sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; in some embodiments, the heavy chain variable region of the antigen binding portion that binds PD-L1 comprises HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 comprising HCDR1, HCDR2, and HCDR3, respectively, in SEQ ID No.43, in some embodiments, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions, the LCDR1, LCDR2, and LCDR3 comprising LCDR1, LCDR2, and LCDR3, respectively, in SEQ ID No. 42; in some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
Iii heterodimeric Fc fusion proteins
In some embodiments, it comprises a heavy chain constant region Fc that is a heterodimer (heterodimeric Fc fusion protein).
The Fc includes, but is not limited to, the following combinations:
CH2;
ch2+ch3; or (b)
CH2+CH3+CH4;
The Fc constant region introduces mutations to avoid heavy chain mismatches.
In some embodiments, the Fc constant region is mutated to be based on the KiH technique (Knob-into-Holes), i.e., an amino acid mutation is introduced into one heavy chain of the Fc constant region in a volume greater than the original amino acid residue volume to form a raised, "Knob" like structure (Knob), and another mutation is introduced into the other chain region of the Fc constant region in a volume less than the original amino acid residue volume to form a recessed, "mortar" like structure (Hole), whereby the male heavy chain is more prone to pairing with the female heavy chain, thereby avoiding heavy chain mismatches. This technique is described in patent application WO1996027011, which is incorporated in its entirety into the present invention.
In some embodiments, the Fc constant region introduction mutation is based on electrostatic interactions, such as ART-lg technology, which promotes pairing of heterologous heavy chains by specifically altering the charge of the Fc constant region domain, equivalent to the charge version of the KiH technology described in patent application WO2006106905, which is incorporated herein in its entirety.
In some embodiments, the Fc constant region introduction mutation is based on SEED technology, SEED heterodimerization being another spatial mutation-based design strategy that exploits complementarity of alternating sequences derived from IgG and IgA CH3 domains (also referred to as AG SEED CH3 and GA SEED CH 3). IgG and IgA CH3 derivatives produce complementary sequences, thus eliminating the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technique is described in patent application WO2007110205, which is incorporated in its entirety into the present invention.
In some embodiments, the Fc constant region introduction mutation is based on isoelectric point changes, facilitating engineering to increase the rate of heterodimer formation and maintain stability of the Fc region, a technique described in WO2014145806, which is incorporated herein in its entirety.
In some embodiments, the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.
In some embodiments, the Fc constant regions associate into heterodimers based on any combination of the above techniques, e.g., in some embodiments, the Fc constant regions are mutated based on a combination of KIH and electrostatic interactions. For example, the XmAb bispecific platform approach can improve the thermostability of bispecific antibodies by binding electrostatic interactions, CH3 domain conformation, and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutation of native IgG1 to S364K and K370S heterodimers to form hydrogen bonds between the two, followed by L368D/K370S substitution driven salt bridge interactions to promote heterodimer formation, patent application WO2014145907, which is incorporated herein in its entirety.
In some embodiments, the full length or part of the CH2, CH3, or CH4 region is inserted or replaced with a receptor and its ligand.
In some embodiments, the inserted or replaced regions are independently located in the CH2, CH3, or CH4 region, or any position between adjacent regions (e.g., CH1-CH2 interface, CH2-CH3 interface, CH3-CH4 interface);
in some embodiments, when any two of the constant regions described above (e.g., any of the CL-CH1, CH2-CH2, CH3-CH3, or CH4-CH4 regions) are inserted or replaced, the affinity, K, between two cooperating conjugate fragments of the replacement region D <1×10 -3 (M), e.g. as x.times.10 -4 (M)、x×10 -5 (M)、x×10 -6 (M)、x×10 -7 (M)、x×10 -8 (M)、x×10 -9 (M)、x×10 -10 (M)、x×10 -11 (M); the value of x may be selected from 1 to 9, for example 2,3,4,5, 6, 7, 8.
In some embodiments, the N-terminus and/or the C-terminus of the conjugate fragment is linked to the antigen binding fragment by a linker peptide.
The term "operably linked" refers to a linkage of components (e.g., two polypeptides) through a covalent bond, either directly or via one or more linkers (connecting peptides).
In some embodiments, the number of amino acids of the connecting peptide is 1 to 30; may be 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 or 30; preferably 5 to 20.
In some embodiments, the amino acid of the linker peptide is a nonsensical polypeptide that does not have additional functions (e.g., protein localization, cleavage site, etc.) other than ligation.
In some embodiments, the connecting peptide is a flexible connecting peptide;
in some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, ser, pro, ala and Glu.
In some embodiments, the amino acid sequence of the connecting peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, or (G) n, wherein n is selected from 1,2,3,4,5, or 6.
The linker peptide is generally flexible and can reduce steric hindrance between the fusion protein and the protein of interest, thereby further facilitating correct folding of the protein.
In further embodiments, the linker peptide is a rigid linker peptide; i.e., relatively inflexible peptide linkers. Rigid linker peptides do not require complete lack of flexibility, but are less flexible than flexible linker peptides such as glycine-rich peptide linkers. Due to its relative lack of flexibility, the rigid linker peptide reduces the movement of the two protein domains (in the present case the stabilizer protein and the thermostable reverse transcriptase) that are linked together by the rigid linker peptide. A linker peptide that provides an ordered chain (e.g., an alpha helical structure) may provide a rigid linker peptide. For example, arginine, leucine, glutamic acid, glutamine and methionine all exhibit a relatively high tendency to helix-linker structure. However, non-helical linkers comprising a number of proline residues may also exhibit significant rigidity. Examples of rigid linking peptides include polylysine and poly-DL-alanine polylysine. Further description of rigid peptide linkers is provided by Wriggers et al, biopolymers,80, pages 736-46 (2005). Furthermore, rigid linker peptides are described in the linker database described by George et al Protein Engineering,15, pages 871-79 (2003). Preferably, the rigid linker peptide is also a non-cleavable linker peptide, i.e. a non-cleavable rigid linker peptide.
Isolated nucleic acids
The invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion protein as described above.
The term "isolated nucleic acid" refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in single-or double-stranded form. The isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and unless otherwise indicated, the polypeptides also include natural polynucleotides, sugars, or base-altered analogs. According to one aspect of the invention, the polynucleotide is a light chain polynucleotide.
The isolated nucleic acid includes a nucleotide sequence encoding an amino acid sequence of a protein complex, as well as a nucleotide sequence complementary thereto. The complementary sequences include fully complementary sequences and substantially complementary sequences, which refers to sequences that hybridize under stringent conditions known in the art to nucleotide sequences encoding amino acid sequences of protein complexes.
Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such alterations include additions, deletions, or non-conservative or conservative substitutions. Polynucleotides encoding protein complex amino acid sequences may be construed to include nucleotide sequences that have substantial identity to the isolated nucleic acid. The substantial identity aligns the nucleotide sequences to additional random sequences in a manner that maximizes their correspondence, which sequences may exhibit greater than 80% homology, greater than 90% homology, or greater than 95% homology when the aligned sequences are analyzed using algorithms common in the art.
Carrier body
The invention also relates to vectors containing the nucleic acids as described above.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). The vector may contain a selectable marker (e.g., a tag that facilitates enrichment, such as his tag; or a tag that facilitates detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements such as enhancers, promoters, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.) necessary to effect expression in the specified target cell. The vector may be a cloning vector or an expression vector. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved, wherein the prokaryotic expression vectors are commonly used in PET series and pGEX series, and the eukaryotic expression vectors are commonly used in pcDNA3.1, pcDNA3.4, pcDNA4, pEGFP-N1, pSV2 and the like.
In the present invention, the vector may be a composition, for example, a mixture of plasmids, different plasmids carrying a portion of the antibody or fragment thereof.
Host cells
The invention also relates to a host cell containing a nucleic acid as described above or a vector as described above.
A variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as escherichia coli or bacillus stearothermophilus), fungal cells (such as saccharomyces cerevisiae or pichia), insect cells (such as lepidopteran insect cells including spodoptera frugiperda cells), or mammalian cells (such as Chinese Hamster Ovary (CHO) cells, NS0 cells, hamster kidney (BHK) cells, monkey kidney cells, hela cells, human hepatocellular carcinoma cells, or 293 cells, among others).
Method for preparing bispecific fusion polypeptide or multifunctional fusion protein
The bispecific fusion polypeptides or multifunctional fusion proteins of the invention can be prepared using any method known in the art.
For example: transforming a host cell with a vector as described above;
culturing the transformed host cell; and
collecting bispecific fusion polypeptides or multifunctional fusion proteins expressed in host cells.
In particular, the following method can be employed.
Early methods of constructing bispecific antibodies were chemical cross-linking or hybrid hybridomas or tetravalent body tumor methods (e.g., staerz UD et al, nature,314:628-31,1985;Milstein C et al, nature,305:537-540,1983;Karpovsky B et al, j. Exp. Med.,160:1686-1701,1984). The chemical coupling method is to connect 2 different monoclonal antibodies together in a chemical coupling mode to prepare the bispecific monoclonal antibody. For example, the chemical binding of two different monoclonal antibodies, or for example, the chemical binding of two antibody fragments, such as two Fab fragments. Hybrid-hybridoma methods produce bispecific monoclonal antibodies by means of cellular or ternary hybridomas obtained by established hybridoma fusion, or established hybridoma fusion with lymphocytes from mice. Although these techniques are used to make biabs, various problems arise that make such complexes difficult to use, such as creating mixed populations containing different combinations of antigen binding sites, difficulties in protein performance, the need to purify the target BiAb, low yields, high production costs, and the like.
Recent approaches utilize genetically engineered constructs that are capable of producing homogeneous products of a single BiAb without the need for extensive purification to remove unwanted byproducts. Such constructs include tandem scfvs, diabodies, tandem diabodies, double variable domain antibodies, and heterodimers using motifs such as Ch1/Ck domains or DNLTM (Chames & Baty, curr. Opan. Drug. Discovery. Development., 12:276-83,2009; chames & Baty, mabs, 1:539-47). Related purification techniques are well known.
Antibodies can also be produced using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for the production of a specific antibody, e.g., by Babcook J et al, proc. Natl. Acad. Sci. USA.93:7843-7848,1996; WO 92/02551; methods described in WO 2004/051268 and WO 2004/106377.
Antigenic polypeptides for producing antibodies, e.g., for immunization of a host or for panning, e.g., for phage display (or yeast or bacterial cell surface expression), may be prepared from genetically engineered host cells comprising an expression system by methods well known in the art, or they may be recovered from natural biological sources. For example, nucleic acid encoding one or both polypeptide chains of a bispecific antibody can be introduced into a cultured host cell by a variety of known methods (e.g., transformation, transfection, electroporation, microprojectile bombardment with nucleic acid coatings, and the like). In some embodiments, the nucleic acid encoding the bispecific antibody may be inserted into a vector suitable for expression in a host cell prior to introduction into the host cell. Typically the vector may comprise sequence elements that enable expression of the inserted nucleic acid at the RNA and protein levels.
Bispecific antibodies of the invention, or portions thereof, can be used to detect any or all of these antigens (e.g., in a biological sample such as serum or plasma) by conventional immunological analytical methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or histoimmunohistochemistry. The present invention provides a method of detecting an antigen in a biological sample, the method comprising: contacting the biological sample with a bispecific antibody, or antibody portion antigen, of the invention that specifically recognizes the antigen, and detecting an antibody (or antibody portion), or non-binding antibody (or antibody portion), that binds to the antigen, thereby detecting the antigen in the biological sample. The antibodies are labeled directly or indirectly with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include a variety of enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase; examples of suitable repair group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include 7-hydroxycoumarin, fluorescein isothiocyanate, basic maroon B, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of the light-emitting substance include 3-aminophthaloyl ring hydrazine; examples of suitable radioactive materials include I 125 、I 131 、 35 S or 3 H。
Pharmaceutical composition
The bispecific polypeptide complexes of the invention or nucleic acids encoding the same may be used in the preparation of pharmaceutical or sterile compositions, for example, by mixing a bispecific fusion polypeptide or multifunctional fusion protein with a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutical composition may comprise one or a combination (e.g., two or more different) of the antibodies of the invention, functional fragments thereof. For example, the pharmaceutical compositions of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) having complementary activity that bind to different epitopes on the target antigen. Formulations of therapeutic and diagnostic agents may be prepared by mixing, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions with pharmaceutically acceptable carriers, excipients or stabilizers.
Medical application and treatment method
The invention also relates to the use of a bispecific fusion polypeptide or a multifunctional fusion protein as described above for the preparation of a medicament for the treatment of a disease.
The invention also relates to a bispecific fusion polypeptide or a multifunctional fusion protein as described above for use as a medicament; the medicament is useful for treating diseases.
According to one aspect of the invention, the disease may be, for example, cancer, an immune disorder, a metabolic disease, and a microbial infection.
The term "cancer" refers to a broad class of diseases characterized by uncontrolled growth of abnormal cells in the body. "cancer" includes benign and malignant cancers, dormant tumors or micrometastases.
The invention also relates to a method of preventing and/or treating and administering a therapeutically effective amount of a pharmaceutical composition to prevent and/or treat a disease as described above.
The method of the invention may be used in human clinical medicine and veterinary applications. Thus, the host animal carrying the population of pathogenic organisms and treated with the ligand-immunogen conjugate may be a human or, in the case of veterinary applications, a laboratory animal, an agricultural animal, a domestic animal or a wild animal. The invention may be applied to host animals including, but not limited to: a human being; laboratory animals such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; domesticated animals such as dogs, cats and rabbits; farm animals such as cattle, horses, pigs, sheep, goats; and wild animals in close proximity, such as bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, and whale.
The pharmaceutical compositions may be injected into an entity, including rats, mice, domestic animals, and/or humans, by a variety of routes. All injection methods are contemplated, for example, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or topical injection is possible. The composition may be injected by other methods known in the art.
By "therapeutically effective amount" is meant herein a sufficient amount to treat a disease in view of a reasonable benefit-to-loss ratio. The therapeutically effective amount may vary for a variety of reasons caused by the patient, such as, for example, disease type, severity, onset, age, weight, rate of excretion, susceptibility to response, health, and/or complications; and/or pharmaceutical activity, route of injection, injection cycle and number of injections, and/or pharmaceutical combination; or may be appropriately selected by one of ordinary skill in the art according to the purpose of the treatment. For example, the injection amount may be divided randomly into a number of times such that the amount is about 0.001-100mg/kg of adult body weight.
The bispecific fusion polypeptides or multifunctional fusion proteins of the invention or nucleic acids or polynucleotides encoding the antibodies of the invention may also be administered in combination with, for example, standard cancer therapies (e.g., surgery, radiation, and chemotherapy). For example, anti-tumor therapies using the compositions of the invention and/or effector cells equipped with these compositions are used in combination with chemotherapy. Non-limiting examples of antibody combination therapies of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and combinations thereof.
Embodiments of the present invention will be described in detail below with reference to examples.
Example 1 FiBody design
FiBody is a bispecific antibody obtained by recombination by replacing CL and CH1 on one side of the bispecific antibody with specific affinity between the ligand and its receptor, which can avoid or reduce the occurrence of mismatch between the light chain and the heavy chain of the bispecific antibody.
In this example, fiBody was constructed using interleukin and its receptor as an example, and the interleukins and their receptors were classified into four types according to their steric conformations, see fig. 5 and table 1:
TABLE 1 FiBody Classification
Bispecific antibodies were constructed based on the above 4 classes of interleukins and their receptors, respectively.
Remarks: the classification of the steric conformation is mainly based on a summary analysis of the various IL cytokines published on PDB. Because of the complexity of the cytokine structural complexes, some cytokines can be split into multiple structural types, such as IL2, the complete IL2/IL2Rα/IL2Rβ/IL2Rγ structural complex is similar to that of one lift (A), but looking at IL2/IL2Rβ alone is that of the clamp (D). We categorize IL2 as a lift-type (a) structure based on weaker affinity for IL2/IL2rβ alone and stronger affinity for IL2/IL2rα, so more IL2rα is needed to assist in the formation of structural complexes. For another example, the complete complex of IL21, IL21/IL21R/IL2 Rgamma is the structure of lift (A), but because IL21/IL21R has a strong affinity, it can be classified as the structure of clamp (D) alone.
Example 2 construction of Interleukin-based and its receptor FiBody
Selecting VH of a targeting first antibody to be connected to a receptor protein through a Linker, and then connecting the VH with Fc of the antibody through a Hinge; the VL of the targeting primary antibody is connected to the ligand protein through a Linker to reduce or avoid the mismatch of the light chain and the heavy chain; the other end is the complete Fab structure of the targeting secondary antibody, the Fc making up the primary antibody has conventional KiH engineering with the Fc making up the secondary antibody to reduce or avoid heavy chain mismatches. Or (b)
Selecting VH of a targeting first antibody to be connected to ligand protein through Linker, and then connecting with Fc of the antibody through finger; the VL of the targeting secondary antibody is connected to the receptor protein through a Linker to reduce or avoid the mismatch of the light chain and the heavy chain; the other end is the complete Fab structure targeting the first antibody, the Fc making up the first antibody has conventional KiH engineering with the Fc making up the second antibody to reduce or avoid heavy chain mismatches. Some exemplary FiBody molecular structures constructed and the sequence listing are shown in Table 2 and Table 3, table 2.FiBody molecular structures
TABLE 3 exemplary FiBody molecular sequence Listing
Example 3 construction of bispecific antibodies based on scFv, crossMab Structure as an Experimental control
As described above, both scFv and CrossMab are common techniques for bispecific antibody construction, and are used herein as design controls in comparison to our molecules:
specifically selecting a construction method of a double antibody based on an scFv structure, wherein VH of a target secondary antibody (anti-TIGIT) is connected to VL of the secondary antibody through a Linker to form an scFv structure, and then connected with Fc of the antibody through a Hinge; the other end is a complete Fab structure of the target primary antibody, (the double-antibody platform is developed by the Zhiyou of Wuhan and named YBOdy), and the Fc of the primary antibody and the Fc of the secondary antibody have conventional KiH modification so as to avoid heavy chain mismatch. Specific examples are table 4:
table 4.Y-Body molecular sequence
Describing a construction method of the double antibody based on the scFv structure, specifically selecting a VH of a targeting second antibody (anti-TIGIT), connecting the VH to a VL of the second antibody through a Linker to form a scFv structure, and connecting the scFv structure with the C end of the Fc of the complete targeting first antibody through the Linker; a symmetrical structure is formed. Specific examples are table 5:
TABLE 5 scFv structural diabody sequences
Specifically selecting a construction method based on a cross mab structure double antibody, connecting VH of a targeting second antibody (anti-TIGIT) to a CL domain, connecting the VH of the targeting second antibody (anti-TIGIT) to a CH1 domain through a Hinge and an Fc of the antibody, and forming a light chain; the other end is the complete Fab structure targeting the first antibody, the Fc making up the first antibody has conventional KiH engineering with the Fc making up the second antibody to avoid heavy chain mismatches. Specific examples are table 6:
TABLE 6 Cross mab structural diabody sequence
The construction method based on the scFv structure double antibody comprises the steps that a specific heavy chain is selected to target the VH of a second antibody (illustratively, an anti-TIGIT antibody) and is connected to the VL of the second antibody through a Linker to form an scFv structure, and then the scFv structure is connected with the VH-CH1 of a complete targeting first antibody (illustratively, an anti-PD-L1 antibody) through the Linker, and then the scFv structure is connected with Fc; the light chain is VL-CL of the primary antibody (illustratively, an anti-PD-L1 antibody) and forms a symmetrical structure. See in particular fig. 11, sequences such as table 7:
TABLE 7 scFv structural diabody sequences
Example 4, antibody heavy-light chain mismatch test
Light chain mismatch is a difficult problem faced by the dual antibody platform. In order to verify the anti-mismatch performance of the platform, we specially designed Fab with receptor and ligand distributed on both sides of the antibody, deliberately designed mismatched heavy and light chain structures, and perform expression verification.
Description of the construction method of Fab-IL21/IL21R_Fc mismatch:
VH of a targeting secondary antibody (illustratively an anti-PD-L1 antibody) was selected to be linked to the receptor protein (IL 21R) by Linker, and then to Fc of the antibody by finger; VL targeting the primary antibody (illustratively selected as an anti-TIGIT antibody) is linked to ligand protein (IL 21) by Linker; the other end is a VL of a targeting secondary antibody (an anti-PD-L1 antibody is selected as an example) connected to CL, the VH of a targeting structure primary antibody (an anti-TIGIT antibody is selected as an example) is connected to CH1, then the VL is connected with Fc of the antibody through finger, and the Fc at the two ends is modified by a conventional KiH. Specific examples are table 8:
TABLE 8 mismatched FiBody antibody sequences
Example 5 preparation of FiBody sample
Transient protein expression:
the plasmid containing the target gene is introduced into host cell Expi293 after forming a cationic complex with transfection reagent PEI, and the exogenous gene on the plasmid is transcribed and translated in the cell during the period of the plasmid in the cell, thereby obtaining the target protein.
The Expi293 was incubated at 37℃with 8% carbon dioxide at 130rpm and 2E6 cells were inoculated into 1L shake flasks by cell counting prior to transfection, the culture system being approximately 300ml. Preparing transfection complex for transfection: firstly, 750 mug of target plasmid is added into a 50ml centrifuge tube containing 15ml of Opti-MEM reagent, and the mixture is gently mixed and marked as a tube A; 1.5mg of the transfection reagent PEI was added to a 50ml centrifuge tube containing 15ml of Opti-MEM reagent, gently mixed, incubated at room temperature for 5min, labeled as tube B; and (3) dropwise adding the PEI diluent of the B tube into the DNA diluent of the A tube, slightly mixing, incubating for 15min at room temperature, adding the PEI-target plasmid complex into the Expi293 cells after incubation, and placing the mixture in a shaking table at 37 ℃ for continuous culture. Until D7-D10, the sample is collected.
Protein purification:
the transient cell expression liquid is centrifuged at 9000rpm/20min, and the supernatant is collected and sterilized and filtered by a 0.22 mu m filter membrane. ProA affinity chromatography is adopted for purification. The procedure is as follows, using an AKTA avant 150 chromatography apparatus, a chromatography column (e.g., mabselectsurex LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and a sample is loaded onto the column to allow the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.
Example 6 FiBody physicochemical detection
The samples were purified in one step and then tested for purity by HPLC-SEC (analytical column TOSOH, TSKgel G2000), and the expression levels and purity results of the respective samples are shown in Table 9.
TABLE 9 physical and chemical detection results of bispecific antibodies
The results show that the fibody class a molecules and class D molecular platforms produce bispecific antibodies (including various engineered optimized antibodies) with higher expression levels and/or higher purity than bispecific antibodies of asymmetric scFv (Y-Body, R0809), symmetric scFv (R0810), crossMab (R0959) structures.
It was unexpected that bispecific antibodies with a wrong paired form (sample R1124) could also be expressed and have an expression level similar to normal molecules, but with significantly lower purity.
Example 7 FiBody antigen affinity detection
TIGIT end binding Activity assay
The binding activity of the diabody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay. Preparing 3% BSA buffer: weighing 4.5g BSA into 150mL 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly adding 50 mu L of antibodies with different concentrations into cells, incubating for 0.5h at 2-8 ℃, then adding 50 mu L of ligand, and incubating for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE gold anti-human IgG Fc and PE gold anti-mouse IgG Fc (diluted 1:500x) were formulated with 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well, and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; and (5) performing on-machine detection according to the standard operation procedure of a CytoFLEX flow cytometer.
The results are shown in FIG. 7: interleukin 21 and its receptor replaced CH1 and CL with best results, the binding force of the targeting region was not affected, and the binding force was comparable to that of the positive control (R0774, the same anti-TIGIT hIgG1 monoclonal antibody for the heavy and light chain variable regions as for TIGIT_VH/TIGIT_VL of R1116); the replacement of CH1 and CL post-targeting regions of class C molecule R1119 is severely affected.
R1124 is a mismatch test molecule, the TIGIT binding activity of which is significantly reduced.
PD-L1 end binding Activity assay
The binding activity of the diabody molecule (PD-L1 end) to CHO-PD-L1 cells was examined by FCM assay. Preparing 3% BSA buffer: weighing 4.5g BSA into 150mL 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly adding 50 mu L of antibodies with different concentrations into cells, incubating for 0.5h at 2-8 ℃, then adding 50 mu L of ligand, and incubating for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE gold anti-human IgG Fc and PE gold anti-mouse IgG Fc (diluted 1:500x) were formulated with 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well, and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; and (5) performing on-machine detection according to the standard operation procedure of a CytoFLEX flow cytometer.
As a result, as shown in FIG. 8, each FiBody type showed an affinity comparable to that of a positive antibody (the hIgG1 monoclonal antibody against PD-L1 in which the variable region of the heavy chain and the variable region of the light chain were identical to that of PD-L1_VH/PD-L1_VL of R1116), and the targeting affinity of the mismatched molecule R1124 to the PDL1 terminus was significantly reduced.
Example 8 FiBody two-terminal binding force detection based on IL21/IL21R construction
Crosslinking of PDL1+cells and tigit+cells (PDL 1/Tigit cross-linked with cells):
1. CHO-S-hPDL1 cells (CHO-S recombinant cells recombinantly expressing human PDL1 on the cell membrane by lentiviral transfection) were first labeled with CFSE (BD biosices, cat: 565082), jurkat-Tigit cells (Jurkat recombinant cells recombinantly expressing Tigit on the cell membrane by lentiviral transfection) or CHO-Tigit cells were labeled with CellTraceTM Violet (Thermo Fisher, cat: C34557);
2. mixing the labeled cells according to a ratio of 1:1 (1E 5 respectively) and adding an antibody (20 nM) to incubate in a 96-well U-shaped plate, and incubating for 1h at room temperature;
3. and (3) after the incubated cells are gently mixed, detecting the cells by a flow-through machine, wherein the double positive Cells of FITC+ (CFSE)/PB450+ (Violet) are crosslinked cells.
Compared to scFv diabodies (R1155-3.12%, R1160-3.72%), the bispecific sample R1123 (30.84%/59.69) can bind CHO-PDL1 (CFSE labeled) with CHO-TIGIT [ fig. 9 ] or CHO-PD-L1 (CFSE labeled) with Jurkat-TIGIT (Violet labeled) [ fig. 10] significantly better simultaneously, demonstrating that the bispecific antibody fibdy of the invention can exert a very strong crosslmking effect.
Example 9 bispecific antibodies with disulfide engineering
To further enhance bispecific antibody stability and extend the half-life of bispecific antibodies, disulfide engineering of bispecific antibodies is exemplified by IL21/IL21 ra, disulfide engineering of IL21/IL21 ra, as shown in tables 10 and 11.
TABLE 10 IL21/IL21Rα disulfide engineering
Combination of two or more kinds of | IL21 | IL21Rα | |
1 | | E38C | |
| Q17C | A71C | |
3 | | D72C | |
4 | S5C | S92C | |
5 | S4C | S92C | |
6 | R10C | D72C | |
7 | I13C | D72C | |
8 | I71C | A127C | |
9 | K82C | Y36C |
Remarks: in the table, the sites of the IL21 mutation are numbered according to the natural sequence of human IL21 (SEQ ID NO. 44), and the sites of the IL21Rα mutation are numbered according to the natural sequence of human IL21Rα (SEQ ID NO. 54), for example, the R81C mutation of IL21 means that the 81 st site of SEQ ID NO.44 is mutated from R to C.
TABLE 11 IL21/IL21R alpha amino acid sequence
Ligand disulfide bond modification: VH selected to target a second antibody (illustratively an anti-TIGIT antibody) is linked to the receptor protein (il21rα) by Linker, and then to Fc of the antibody by finger; the VL targeting the second antibody is linked to ligand protein (IL 21) by Linker; the other end is the complete Fab structure targeting the primary antibody (illustratively selected anti-PD-L1 antibody), the Fc making up the primary antibody having conventional KiH engineering with the Fc making up the secondary antibody to avoid heavy chain mismatches. Meanwhile, mutation is carried out on the receptor and ligand proteins, so as to form intermolecular disulfide bonds, further improve the stability of molecules, and the sequence of the constructed FiBody bispecific antibody is shown in table 12:
TABLE 12 IL21/IL21Rα side sequence in FiBody bispecific antibodies
The other side of the compound 1-9 is polypeptide targeting PD-L1, and the sequence is as follows:
polypeptide light chain of PD-L1:
EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKLLIYGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.42)
heavy chain:
EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.43)
(1) Transient protein expression:
the plasmid containing the target gene is introduced into host cell Expi293 after forming a cationic complex with transfection reagent PEI, and the exogenous gene on the plasmid is transcribed and translated in the cell during the period of the plasmid in the cell, thereby obtaining the target protein.
The Expi293 was incubated at 37℃with 8% carbon dioxide at 130rpm and 2E6 cells were inoculated into 1L shake flasks by cell counting prior to transfection, the culture system being approximately 300mL. Preparing transfection complex for transfection: firstly, 750 mug of target plasmid is added into a 50mL centrifuge tube containing 15mL of Opti-MEM reagent, and the mixture is gently mixed and marked as a tube A; 1.5mg of the transfection reagent PEI was added to a 50ml centrifuge tube containing 15ml of Opti-MEM reagent, gently mixed, incubated at room temperature for 5min, labeled as tube B; and (3) dropwise adding the PEI diluent of the B tube into the DNA diluent of the A tube, slightly mixing, incubating for 15min at room temperature, adding the PEI-target plasmid complex into the Expi293 cells after incubation, and placing the mixture in a shaking table at 37 ℃ for continuous culture. Until D7-D10, the sample is collected.
(2) Purification of disulfide bond engineered samples:
the transient cell expression liquid is centrifuged at 9000rpm/20min, and the supernatant is collected and sterilized and filtered by a 0.22 mu m filter membrane. ProA affinity chromatography is adopted for purification. The procedure is as follows, using an AKTA avant 150 chromatography apparatus, a chromatography column (e.g., mabselectsurex LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and a sample is loaded onto the column to allow the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.
(3) Disulfide bond engineered IL21/IL21rαfibody gel electrophoresis detection:
SDS-PAGE electrophoresis detection is carried out on the disulfide bond modified IL21/IL21Rα complex, the detection result is shown in FIG. 12, and the disulfide bond modified complex (complex 10, same as R1123) has a band between the molecular weight of 25KD and 35KD, which indicates that a free light chain exists; the compounds 1 to 9 have no band between the molecular weights of 25KD to 35KD, which indicates that the disulfide bond modification is successful.
(4) Targeting moiety affinity detection:
TIGIT end binding Activity assay
The binding activity of the diabody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay. Preparing 3% BSA buffer: weighing 4.5g BSA into 150mL 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly, 50 mu L of antibodies with different concentrations are added into cells, and the cells are incubated for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE gold anti-human IgG Fc and PE gold anti-mouse IgG Fc (diluted 1:500x) were formulated with 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well, and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; the results of the on-press detection according to the standard protocols of the CytoFLEX flow cytometer are shown in fig. 13, which shows that the disulfide bond modification does not attenuate the affinity of the targeting region, compared to the molecule without disulfide bond modification.
PD-L1 end binding Activity assay
The binding activity of the diabody molecule (PD-L1 end) to CHO-PD-L1 cells was examined by FCM assay. Preparing 3% BSA buffer: weighing 4.5g BSA into 150mL 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly, 50 mu L of antibodies with different concentrations are added into cells, and the cells are incubated for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE gold anti-human IgG Fc and PE gold anti-mouse IgG Fc (diluted 1:500x) were formulated with 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well, and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; and (5) performing on-machine detection according to the standard operation procedure of a CytoFLEX flow cytometer. The results of the assay are shown in FIG. 14 to be comparable in affinity to the molecule without disulfide engineering, indicating that disulfide engineering does not affect the affinity of the targeting region.
(5) TIGIT end blocking Activity assay
The binding activity of the blocking ligand to CHO-TIGIT cells was detected by FCM assay methods for diabody molecules (TIGIT ends). Preparing 3% BSA buffer: weighing 4.5g BSA into 150mL 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly adding 50 mu L of antibodies with different concentrations into cells, incubating for 0.5h at 2-8 ℃, then adding 50 mu L of ligand, and incubating for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE gold anti-human IgG Fc and PE gold anti-mouse IgG Fc (diluted 1:500x) were formulated with 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well, and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; the results of the on-press detection according to the standard protocols of CytoFLEX flow cytometry are shown in figure 15, which shows that the affinity of the disulfide-engineered molecules is comparable to that of the molecules not subjected to disulfide-engineering, indicating that disulfide-engineering does not attenuate the affinity of the targeting region. The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (15)
1. A bispecific fusion polypeptide comprising a first antigen-binding portion comprising:
a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain VH1 of a first antibody operably linked to a first conjugate fragment, and
a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain VL1 of a first antibody operably linked to a second conjugate fragment,
the first conjugate fragment and the second conjugate fragment are capable of specific binding;
wherein the first conjugate fragment is a receptor and the second conjugate fragment is a ligand; or the first conjugate fragment is a ligand and the second conjugate fragment is a receptor;
Alternatively, the ligand and receptor are IL21/IL21R; optionally, the receptor and ligand comprise at least one non-native interchain bond therebetween.
2. The bispecific fusion polypeptide of claim 1, further comprising a second antigen-binding portion that is different from the first antigen-binding portion, the second antigen-binding portion comprising:
a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to a third conjugate fragment, and
a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to a fourth conjugate fragment;
wherein,,
a) The third conjugate fragment and the fourth conjugate fragment are capable of specific binding;
b) The third conjugate segment is a receptor, and the fourth conjugate segment is a ligand; or the third conjugate fragment is a ligand and the fourth conjugate fragment is a receptor; and
c) The third and/or fourth conjugate fragments are selected from different receptors and ligands than the first and/or second conjugate fragments.
3. The bispecific fusion polypeptide of claim 1, further comprising a second antigen-binding portion that is different from the first antigen-binding portion, the second antigen-binding portion comprising:
a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain variable domain VH2 of a second antibody operably linked to an antibody heavy chain constant region CH1, and
a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain variable domain VL2 of a second antibody operably linked to an antibody light chain constant region CL.
4. A bispecific fusion polypeptide according to any one of claims 1 to 3, comprising at least one non-natural inter-chain bond between the receptor and the ligand, said non-natural inter-chain bond being capable of enhancing the specific binding force between the receptor and the ligand; optionally, the non-natural interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; optionally, at least one of the first mutant residue and the second mutant residue is a cysteine residue; optionally, the non-natural interchain bond is a disulfide bond.
5. The bispecific fusion polypeptide of any one of claims 1 to 4, comprising at least one non-native inter-chain bond between the first heavy chain variable domain VH1 and the first light chain variable domain VL 1; the non-natural interchain bond is formed between a first mutated residue comprised by the first heavy chain variable domain VH1 and a second mutated residue comprised by the first light chain variable domain VL 1; optionally, at least one of the first mutant residue and the second mutant residue is a cysteine residue; optionally, the non-natural interchain bond is a disulfide bond.
6. The bispecific fusion polypeptide of any one of claims 1 to 5, wherein at least one native glycosylation site is absent in the receptor and/or ligand.
7. The bispecific fusion polypeptide of any one of claims 1 to 6, wherein IL21 is selected from the sequence set forth in any one of SEQ ID No.44 to SEQ ID No.53, and IL21R is selected from the sequence set forth in any one of SEQ ID No.54 to SEQ ID No. 60.
8. The bispecific fusion polypeptide of any one of claims 1 to 7, comprising an antibody Fc constant region; optionally, the antibody Fc constant region is a heterodimer; alternatively, the antibody Fc constant regions are associated as heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility.
9. The bispecific fusion polypeptide of claim 8, the antibody Fc constant region comprises CH2 and CH3 or the antibody Fc constant region comprises CH2, CH3, and CH4, and two CH2, two CH3, and/or two CH4 on the Fc constant region are replaced by the receptor and its ligand.
10. The bispecific fusion polypeptide of any one of claims 1 to 9, wherein the first antigen-binding portion binds to a different antigen or to a different epitope of the same antigen than the second antigen-binding portion;
optionally, the first antigen binding portion targets immune cells and the second antigen binding portion targets tumor cells;
optionally, both the first antigen binding portion and the second antigen binding portion target a tumor cell;
optionally, both the first antigen binding portion and the second antigen binding portion target immune cells;
alternatively, the first antigen-binding portion targets human PD-L1 and the second antigen-binding portion targets human TIGIT; or the first antigen binding portion targets human TIGIT and the second antigen binding portion targets human PD-L1.
11. An isolated nucleic acid encoding the bispecific fusion polypeptide of any one of claims 1 to 10.
12. A vector comprising the nucleic acid of claim 11.
13. A host cell comprising the nucleic acid of claim 11 or the vector of claim 12.
14. A pharmaceutical composition comprising the bispecific fusion polypeptide of any one of claims 1 to 10, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
15. Use of a bispecific fusion polypeptide according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of a disease.
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