Patent Application
20250215092
The invention relates to the biomedical or biopharmaceutical technology field, more specifically to a human epidermal growth factor receptor (EGFR) binding molecule and its application.
Human epidermal growth factor receptor (EGFR, ErbB-1 or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (ErbB1, EGFR), HER2 (ErbB2, neu), HER3 (ErbB3) and HER4 (ErbB4). EGFR is expressed on the surface of normal epithelial cells. There is high or abnormal expression of EGFR in many solid tumor cells, including head and neck cancer, breast cancer, bladder cancer, ovarian cancer, kidney cancer, colon cancer and non-small cell lung cancer, especially lung cancer. The EGFR mutation rate in Asian lung cancer population can reach 50% (Seshacharyulu P et al., Expert Opin Ther Targets, 2012; 16:15-31). EGFR is a popular target for tumor targeted therapy.
EGFR is located on the cell membrane surface and is activated by binding to ligands, including EGF and TGF α. After activation, EGFR is converted from monomers to dimers, although there is also evidence that dimers also exist before activation. EGFR may also be activated by aggregation with other members of the ErbB receptor family. EGFR dimerization can activate its kinase pathways located in cells, including Y992, Y1045, Y1068, Y1148 and Y1173 activation sites, and autophosphorylation can guide downstream phosphorylation, including MAPK, Akt and JNK pathways. EGFR is related to the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. There are mainly two signal transduction pathways downstream of EGFR: one is Ras/Raf/MEK/ERK-MAPK pathway, and the other is PI3K/Akt/mTOR pathway (R Roskoski Jr. Pharmacological research, 2014:79: 34-74.).
At present, there are four EGFR mAbs approved for marketing worldwide, namely cetuximab, panitumumab, anti cetuximab and nimotuzumab. They are mainly used to treat rectal cancer, and head and neck cancer. Studies have shown that EGFR extracellular segment is very prone to mutation and drug resistance. The binding epitope of nanobody is much smaller than that of scFv antibody, and it is less affected by mutations, which can improve the effective anti-tumor activity of EGFR to a certain extent. Moreover, nanobodies have the natural advantages of high stability, strong penetration and wide binding epitopes (Muyldermans S. Annu Rev Biochem. 2013; 82:775-97.). There is little research on nanobodies targeting the membrane proximal end of EGFR. It has become an urgent problem to develop a new type of anti human epidermal growth factor receptor nanobody, which has good specificity, blocking activity, clinical efficacy, simple production, low cost, and reduced drug burden.
The invention aims to provide a novel anti-EGFR binding molecule and use thereof.
The first aspect of the invention provides a human epidermal growth factor receptor binding molecule, which comprises an anti-EGFR single domain antibody, wherein the complementarity determining region (CDR) of the single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3.
In one or more embodiments, SEQ ID NO: 1 is GX1X2X3X4X5X6X7X8, wherein X1 is G, X2 is S, F, G, L or R, X3 is G, T, V, P, A, S or I, X4 is F, L or D, X5 is T, S, E, D, L or I, X6 is I, D, S or T, X7 is Q, Y, H or F, and X8 is A or T.
In one or more embodiments, SEQ ID NO: 1 is GX1X2X3X4X5X6X7X8, wherein X1 is G, X2 is F or L, X3 is T, P, or A, X4 is F or L, X5 is T, S, E, D, L or I, X6 is D or T, X7 is Y, and X8 is A or T.
In one or more embodiments, CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-12, preferably, CDR1 comprises the sequence shown in any one of SEQ ID NOs:5, 7, 8, 9 or 11.
In one or more embodiments, SEQ ID NO: 2 is X1X2X3X4X5X6X7X8, wherein X1 is I, L or V, X2 is H, T, F, A or S, X3 is Q, G, S, P, T, I or N, X4 is G, T, D, A, S or Y, X5 is G, H, E or N, X6 is S, E, K, D, G or A, X7 is T, H, I, K, S or null, and X8 is S, T, I, P or null.
In one or more embodiments, SEQ ID NO: 2 is X1X2X3X4X5X6X7X8, wherein X1 is I or L, X2 is T, F, A or S, X3 is G, S, P, T or I, X4 is T, D, A, or S, X5 is G or E, X6 is E, K, D or G, X7 is T, H, I, K, S or null, and X8 is S, T, I or P.
In one or more embodiments, CDR2 comprises the sequence shown in any one of SEQ ID NOs: 13-21, preferably, CDR2 comprises the sequence shown in any one of SEQ ID NOs: 14, 16, 17, 18, or 20.
In one or more embodiments, SEQ ID NO:3 is X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21 X22X23X24X25, wherein X1 is N, S, A, K or H, X2 is V, I, R, K, A or L, X3 is V, Y, S, D, L, T or N, X4 is P, H, L, V, S, K or Y, X5 is P, Y, S, R, T or A, X6 is L, P, T, F, A, S or D, X7 is R, P, F, S, A, I or D, X8 is V, D, G, A or Y, X9 is Y, N, T, R, F or null, X10 is P, A, W, E or null, X11 is S, H, L, Y or null, X12 is F, D, N, L or null, X13 is Y, V or null, X14 is I, G or null, X15 is G or null, X16 is Y, A or null, X17 is G or null, X18 is G or null, X19 is G or null, X20 is E or null, X21 is V or null, X22 is R or null, X23 is Y or null, X24 is E or null, X25 is Y or null.
In one or more embodiments, SEQ ID NO:3 is X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21 X22X23X24X25, wherein X1 is N, S, A or K, X2 is I, K or A, X3 is Y, D, L or T, X4 is H, L, V, S or K, X5 is P, S, R or T, X6 is P, F, A, S or D, X7 is R, P, F, S or I, X8 is V, D, G or A, X9 is Y, N, T, R or F, X10 is P, A, W or null, X11 is S, H, L or null, X12 is F, D, N, L or null, X13 is Y, V or null, X14 is I, G or null, X15 is G or null, X16 is Y, A or null, X17 is G or null, X18 is G or null, X19 is G or null, X20 is E or null, X21 is V or null, X22 is R or null, X23 is Y or null, X24 is E or null, X25 is Y or null.
In one or more embodiments, CDR3 comprises the sequence shown in any one of SEQ ID NOs: 22-30, preferably, CDR3 comprises the sequence shown in any one of SEQ ID NOs: 23, 25, 26, 27, or 29.
In one or more embodiments, the anti human epidermal growth factor receptor single domain antibody is a specific single domain antibody to the membrane proximal region III or IV of the human epidermal growth factor receptor.
In one or more embodiments, CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-12, CDR2 comprises the sequence shown in any one of SEQ ID NOs: 13-21, and CDR3 comprises the sequence shown in any one of SEQ ID NOs:22-30.
In one or more embodiments, the single domain antibody comprises CDR1, CDR2, and CDR3 shown in any one of the following groups a1 to a9:
In one or more embodiments, FR1 region of the single domain antibody VHH is the FR1 region of any VHH selected from SEQ ID NOs: 31-82, FR2 region of VHH is the FR2 region of any VHH selected from SEQ ID NOs: 31-82, FR3 region of VHH is the FR3 region of any VHH selected from SEQ ID NOs: 31-82, and FR4 region of VHH is the FR4 region of any VHH selected from SEQ ID NOs: 31-82.
In one or more embodiments, the FR region of the single domain antibody is the FR region of any VHH selected from SEQ ID NOs: 31-82.
In one or more embodiments, the single domain antibody VHH is as shown in any one of SEQ ID NOs: 31-82, preferably, the single domain antibody VHH is as shown in any one of SEQ ID NOs:40-82.
In one or more embodiments, the human epidermal growth factor receptor binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen binding fragment thereof, an antibody or an antigen binding fragment thereof comprising one, two or more of the anti-EGFR single domain antibodies described herein.
In one or more embodiments, the multivalent single domain antibody or multispecific single domain antibody connects a plurality of single domain antibodies through a linker. The linker consists of 1-15 amino acids selected from G and S.
In one or more embodiments, the antigen binding fragment of the heavy chain antibody is a single chain heavy chain antibody.
In one or more embodiments, the heavy chain antibody is a camelid heavy chain antibody or a shark heavy chain antibody.
In one or more embodiments, the heavy chain antibody further comprises a heavy chain constant region.
In one or more embodiments, the heavy chain constant region is a constant region of camelid heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG1 or IgG4. Preferably, the heavy chain constant region is CH2 and CH3 of IgG4, and its amino acid sequence is shown in SEQ ID NO: 83.
In one or more embodiments, the heavy chain constant region is a constant region of the shark heavy chain antibody, comprising CH1, CH2, CH3, CH4, and CH5.
In one or more embodiments, the antibody is an antibody comprising the anti-EGFR single domain antibody as the heavy chain variable domain.
In one or more embodiments, the antibody further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.
In one or more embodiments, the antigen binding fragment of the antibody is selected from Fab, F(ab′)2, Fv, scFv.
In one or more embodiments, the binding molecule described in any embodiment of the present description is a chimeric antibody or a fully human antibody; preferably, a fully human antibody.
The description also provides a polynucleotide, comprising a sequence selected from:
In one or more embodiments, the fragment is primer.
The description also provides a nucleic acid construct comprising the polynucleotide described herein.
In one or more embodiments, the nucleic acid construct is a recombinant vector or expression vector.
The description also provides a phage comprising the human epidermal growth factor receptor binding molecule according to any embodiment herein.
In one or more embodiments, the human epidermal growth factor receptor binding molecule is displayed on the surface of the phage.
The description also provides a host cell selected from:
The description also provides a method for producing a human epidermal growth factor receptor binding molecule, comprising culturing the host cells described herein under conditions suitable for producing human epidermal growth factor receptor binding molecule (such as monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies, antibodies or antigen binding fragments thereof), and optionally purifying the human epidermal growth factor receptor binding molecule from culture.
The description also provides a pharmaceutical composition, comprising the human epidermal growth factor receptor binding molecule, polynucleotide, nucleic acid construct, phage or host cell according to any embodiment herein, and a pharmaceutically acceptable excipient.
In one or more embodiments, the pharmaceutical composition is used for treating cancer.
In one or more embodiments, the cancer is the human epidermal growth factor receptor related cancer. Preferably, the cancer is selected from the group consisting of: head and neck cancer, breast cancer, bladder cancer, ovarian cancer, renal carcinoma, colon cancer, non-small cell lung cancer, and the like.
The description also provides use of the human epidermal growth factor receptor binding molecule according to any embodiment herein in the preparation of a medicament for the prevention or treatment of a cancer.
In one or more embodiments, the cancer is a human epidermal growth factor receptor related cancer. Preferably, the cancer is selected from the group consisting of: head and neck cancer, breast cancer, bladder cancer, ovarian cancer, renal carcinoma, colon cancer, non-small cell lung cancer, and the like.
The description also provides a method for treating or preventing a cancer, comprising administrating a patient in need thereof an effective amount of a human epidermal growth factor receptor binding molecule according to any embodiment of the description, or a pharmaceutical composition comprising a human epidermal growth factor receptor binding molecule according to any embodiment of the description.
In one or more embodiments, the cancer is a human epidermal growth factor receptor related cancer. Preferably, the cancer is selected from the group consisting of: head and neck cancer, breast cancer, bladder cancer, ovarian cancer, renal carcinoma, colon cancer, non-small cell lung cancer, and the like.
The description also provides a kit for detecting human epidermal growth factor receptor, for use in evaluating the therapeutic effect of a medicament or diagnosing cancer.
The kit comprises a human epidermal growth factor receptor binding molecule, polynucleotide, nucleic acid construct, phage or host cell according to any embodiment of the description.
In one or more embodiments, the kit further comprises a reagent for detecting the binding of human epidermal growth factor receptor to a single domain antibody, an antibody, or an antigen binding fragment thereof. For example, the bound reagent is detected by the enzyme-linked immunosorbent assay.
In one or more embodiments, the detection reagent for binding is a detectable marker, such as biotin, that can be linked to a human epidermal growth factor receptor binding molecule. The detectable marker is connected to the human epidermal growth factor receptor binding molecule or present in the kit separately.
The description also provides a non diagnostic method for detecting the presence of human epidermal growth factor receptor in a sample. The method comprises: incubating a human epidermal growth factor receptor binding molecule according to any embodiment herein with the sample, and detecting the binding of human epidermal growth factor receptor to a single domain antibody, antibody, or antigen binding fragment thereof, thereby determining the presence of human epidermal growth factor receptor in the sample. The detection is an enzyme-linked immunosorbent assay.
The description also provides use of a human epidermal growth factor receptor binding molecule according to any embodiment herein in the preparation of a kit for detecting human epidermal growth factor receptor in a sample, evaluating the therapeutic effect of a medicament or diagnosing a cancer.
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After extensive and in-depth research and extensive screening, the inventor found a class of human epidermal growth factor receptor binding molecules containing anti human epidermal growth factor receptor single domain antibodies. The human epidermal growth factor receptor binding molecule of the invention can bind to the membrane proximal region III or IV of EGFR antigen with high specificity, and high affinity, resulting in high biological activity, and has low immunogenicity, has stable structure, and has good druggability. The single domain antibody of the invention is simple to generate.
“Human epidermal growth factor receptor binding molecule” or “EGFR binding molecule” as used herein is a protein that specifically binds Human epidermal growth factor receptor, including but not limited to antibodies, antigen binding fragments of antibodies, heavy chain antibodies, nano antibodies, micro antibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.
“Human epidermal growth factor receptor membrane proximal region III” (EGFR Region III) refers to the region of amino acids 311-480 of human epidermal growth factor receptor (uniprot, P00533-1, C311-L480).
“Human epidermal growth factor receptor proximal membrane region IV” (EGFR Region IV) refers to the region of amino acids 481-645 of human epidermal growth factor receptor (uniprot, P00533-1, F481-S645).
Term “antibody” as used herein includes monoclonal antibodies (including full-length antibodies with immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, especially antigen binding fragments, (e.g., Fab, F(ab′)2, and FV). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.
The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibody consists of 5 basic heterotetramer units and another polypeptide called J chain, which contains 10 antigen binding sites; IgA antibody contains 2-5 basic 4 chain units, which can polymerize with J chain to form a multivalent assemblages. In the case of IgGs, the 4-chain unit is typically about 150,000 daltons. Each light chain is connected to a heavy chain through a covalent disulfide bond, while the two heavy chains are connected to each other through one or more disulfide bonds, and the number of disulfide bonds depends on the isotype of the heavy chain. Each heavy and light chain also has an interchain disulfide bridge with regular spacing. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three constant domains (for each α and γ chain, CH1, CH2, and CH3) and four constant domains (for μ and ε isoforms, CH1, CH2, CH3, and CH4) and the hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is aligned with VH, while CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form an interface between light and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, ed, Appleton & Lange, Norwalk, CT, 1994, page 71, and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
“Heavy chain antibody” described herein is an antibody derived from camelidae or sharks. Compared with the above 4-chain antibody, the heavy chain antibody lacks light chain and heavy chain constant region 1 (CH1), and only contains two heavy chains composed of variable region (VHH) and other constant regions, wherein the variable region is connected to the constant region through a hinge region like structure. Each heavy chain of camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of shark heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies may have CH2 and CH3 of human IgG Fc by fusing with the constant region of human IgG Fc.
As used herein, the terms “single domain antibody”, “anti-EGFR single domain antibody”, “heavy chain variable region domain of heavy chain antibody”, “VHH” and “nanobody” can be used interchangeably, and all refer to single domain antibodies that specifically recognize and bind to human epidermal growth factor receptor. Single domain antibodies are variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Preferably, the single domain antibody of the description has CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3. Single domain antibodies are the smallest functional antigen binding fragments. Generally, after obtaining an antibody which naturally lacks light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
Binding molecules comprising two or more single domain antibodies are multivalent single domain antibodies; and binding molecules comprising two or more single domain antibodies with different specificities are multispecific single domain antibodies. Multivalent single domain antibodies or multispecific single domain antibodies are connected to multiple single domain antibodies through linkers. The linker usually consists of 1-15 amino acids selected from G and S.
The terms “heavy chain antibody” and “antibody” herein are intended to distinguish different composition forms of antibodies. Due to the similarity of their structures, the following descriptions on structures of antibodies except for light chains also apply to heavy chain antibodies.
The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively.
These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains, namely HCDR1, HCDR2 and HCDR3 of heavy chain variable region (CDR1, CDR2 and CDR3 in heavy chain antibodies for short) and LCDR1, LCDR2 and LCDR3 of light chain variable region. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. Generally, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
An “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc” refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from CH2 and CH3 constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 to the carboxy-terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat. As used herein, the Fc region may be a native sequence Fc or a variant Fc.
An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. The antibody fragment is preferably an antigen binding fragment of the antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, scFv-Fc fragment; multispecific antibodies formed from antibody fragments; and any fragment that should be able to increase the half-life by chemical modification or by incorporation into liposomes. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector function functions of an antibody is antibodies are determined by the sequence sequences in the Fc region, the region which is also recognized by Fc receptors (FcRs) found on some certain types of cells.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method, phage-display technologies, recombinant DNA methods, and technologies for producing human or humanlike antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.
The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Therefore, “humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies are well known in the art, such as using mice with genetically engineered immune systems. In the description, antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of the antibodies.
A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries.
In some embodiments, the description further provides a single domain antibody, heavy chain antibody, antibody or antigen binding fragment thereof that binds to the same epitope of human epidermal growth factor receptor as any anti-EGFR single domain antibody of the description (For example, human epidermal growth factor receptor membrane proximal region III and IV), that is, a single domain antibody, heavy chain antibody, antibody or antigen binding fragment thereof that can cross-compete with any single domain antibody of the description for binding to human epidermal growth factor receptor.
In the present invention, the CDR1 of the anti-EGFR single domain antibody comprises the sequence shown in SEQ ID NO: 1, and SEQ ID NO: 1 is GX1X2X3X4X5X6X7X8, wherein X1 is G, X2 is S, F, G, L or R, X3 is G, T, V, P, A, S or I, X4 is F, L or D, X5 is T, S, E, D, L or I, X6 is I, D, S or T, X7 is Q, Y, H or F, and X8 is A or T. Preferably, X1 is G, X2 is F or L, X3 is T, P, or A, X4 is F or L, X5 is T, S, E, D, L or I, X6 is D or T, X7 is Y, and X8 is A or T.
In one or more embodiments, CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-12.
In the present invention, The CDR2 of the anti-EGFR single domain antibody comprises the sequence shown in SEQ ID NO: 2, SEQ ID NO: 2 is X1X2X3X4X5X6X7X8, wherein X1 is I, L or V, X2 is H, T, F, A or S, X3 is Q, G, S, P, T, I or N, X4 is G, T, D, A, S or Y, X5 is G, H, E or N, X6 is S, E, K, D, G or A, X7 is T, H, I, K, S or null, and X8 is S, T, I, P or null. Preferably, X1 is I or L, X2 is T, F, A or S, X3 is G, S, P, T or I, X4 is T, D, A, or S, X5 is G or E, X6 is E, K, D or G, X7 is T, H, I, K, S or null, and X8 is S, T, I or P.
In one or more embodiments, CDR2 comprises the sequence shown in any one of SEQ ID NOs:13-21.
The CDR3 of the anti-EGFR single domain antibody comprises the sequence shown in SEQ ID NO: 3, SEQ ID NO: 3 is X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22, wherein X1 is N, S, A, K or H, X2 is V, I, R, K, A or L, X3 is V, Y, S, D, L, T or N, X4 is P, H, L, V, S, K or Y, X5 is P, Y, S, R, T or A, X6 is L, P, T, F, A, S or D, X7 is R, P, F, S, A, I or D, X8 is V, D, G, A or Y, X9 is Y, N, T, R, F or null, X10 is P, A, W, E or null, X11 is S, H, L, Y or null, X12 is F, D, N, L or null, X13 is Y, V or null, X14 is I, G or null, X15 is G or null, X16 is Y, A or null, X17 is G or null, X18 is G or null, X19 is G or null, X20 is E or null, X21 is V or null, X22 is R or null, X23 is Y or null, X24 is E or null, X25 is Y or null. Preferably, X1 is N, S, A or K, X2 is I, K or A, X3 is Y, D, L or T, X4 is H, L, V, S or K, X5 is P, S, R or T, X6 is P, F, A, S or D, X7 is R, P, F, S or I, X8 is V, D, G or A, X9 is Y, N, T, R or F, X10 is P, A, W or null, X11 is S, H, L or null, X12 is F, D, N, L or null, X13 is Y, V or null, X14 is I, G or null, X15 is G or null, X16 is Y, A or null, X17 is G or null, X18 is G or null, X19 is G or null, X20 is E or null, X21 is V or null, X22 is R or null, X23 is Y or null, X24 is E or null, X25 is Y or null.
In one or more embodiments, CDR3 comprises the sequence shown in any one of SEQ ID NOs: 22-30.
In one or more embodiments, The CDR1 of the anti-EGFR single domain antibody comprises the sequence shown in any one of SEQ ID NOs:5, 7, 8, 9 or 11. CDR2 comprises the sequence shown in any one of SEQ ID NOs: 14, 16, 17, 18, or 20. CDR3 comprises the sequence shown in any one of SEQ ID NOs: 23, 25, 26, 27, or 29.
In one or more embodiments, the single domain antibody comprises CDR1, CDR2, and CDR3 shown in any one of the following groups a1 to a9:
Preferably, containing CDR1, CDR2, and CDR3 selected from any of the following groups: a2, a4, a5, a6, or a8.
In one or more embodiments, FR1 region of the single domain antibody VHH is the FR1 region of any VHH selected from SEQ ID NOs:31-82, FR2 region of VHH is the FR2 region of any VHH selected from SEQ ID NOs: 31-82, FR3 region of VHH is the FR3 region of any VHH selected from SEQ ID NOs: 31-82, and FR4 region of VHH is the FR4 region of any VHH selected from SEQ ID NOs: 31-82.
In a preferred embodiment, the FR region of the single domain antibody VHH of the invention is the FR region of any VHH selected from SEQ ID NOs: 31-82. More preferably, the CDR of such antibodies is selected from any of the aforementioned groups a1 to a9. In one or more embodiments, the single domain antibody VHH is as shown in any one of SEQ ID NOs: 31-38, 40-46, 57-82.
The human epidermal growth factor receptor binding molecule described herein may be a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, or an antigen binding fragment thereof, an antibody, or an antigen binding fragment thereof, comprising one, two, or more anti-EGFR single domain antibodies described herein. The heavy chain antibody further comprises a heavy chain constant region, such as a constant region of camelid heavy chain antibody or shark heavy chain antibody. Preferably, the heavy chain constant region is shown in SEQ ID NO: 83.
The description also comprises the antibody derivatives and analogues. “Derivatives” and “analogues” refer to polypeptides that basically maintain the same biological function or activity of the antibody of the present description. The derivatives or analogues of the present description may be polypeptides formed from (i) a polypeptide with a substituent group in one or more amino acid residues, or (ii) a polypeptide formed from fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol, or (iii) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretory sequence, or a sequence or prokaryotic sequence used for purifying this polypeptide, or a fusion protein formed with a 6His tag). According to the teaching herein, these derivatives and analogues belong to common sense known to those skilled in the art.
Without substantially affecting the activity of the antibody, those skilled in the art may change one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequence of the description to obtain the variant of the antibody or the functional fragment sequence thereof. These variants include (but are not limited to): deletion, insertion, and/or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually less than 20, preferably less than 10, and more preferably less than 5) amino acids at the C-terminus and/or N-terminus. In this field, conservative substitution with amino acids with similar or similar properties usually does not change the function of the protein. For example, substituting with amino acids having similar properties may be performed in the FR and/or CDR regions of the variable region. Amino acid residues available for conservative substitution are well known in the art. Such substituted amino acid residues may or may not be encoded by a genetic code. For another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the scope of the present description.
The variant forms of the antibody described herein include: homologous sequence, conservative variant, allelic variant, natural mutant, induced mutant, protein encoded by DNA that can hybridize with the coding DNA of the antibody of the description under high or low strictness conditions, and polypeptide or protein obtained by using the antiserum of the antibody of the description.
In some embodiments, the sequence of the variant of the present description may have at least 95%, 96%, 97%, 98% or 99% identity with its source sequence. The sequence identity described in the description can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The description also comprises those molecules with variable regions of antibody heavy chain with CDRs, if their CDRs have more than 90% homology (preferably more than 95%, more preferably more than 98%) with the CDRs identified here.
The antibody of the description can be prepared by conventional methods in the art, such as hybridoma technology well known in the art. The heavy chain antibody of the description can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, the antibodies or heavy chain antibodies of the present description may be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the present description. Transformation can be carried out using any known method, including, for example, packaging polynucleotides in viruses (or viral vectors) and transducing host cells with the viruses (or vectors). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran mediated transfection, calcium phosphate precipitation, Polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Typical Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial human epidermal growth factor receptor binding properties.
The description also provides polynucleotides encoding the above antibody or fragments thereof. Polynucleotides encoding heavy chain variable region, light chain variable region, heavy chain, light chain and CDRs are provided. The polynucleotide of the description can be in the form of DNAs or RNAs. DNAs include cDNAs, genomic DNAs, or synthetic DNAs. DNAs can be single stranded DNAs or double stranded DNAs. DNAs can be coding or noncoding strand DNAs.
As those skilled in the art will understand, due to the degeneracy of the genetic code, an extremely large number of nucleic acids can be prepared, all of which encode the antibody of the description or antigen binding fragment thereof. Therefore, when a specific amino acid sequence has been identified, those skilled in the art can simply modify the sequence of one or more codons without changing the amino acid sequence of the encoded protein to produce any number of different nucleic acids. Therefore, the present description also relates to polynucleotides that hybridize with the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The description particularly relates to polynucleotides that can hybridize with the polynucleotides of the description under strict conditions. In the present description, “strict conditions” refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) addition of denaturants during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42° C., etc; or (3) hybridization that occurs only when the identity between two sequences is at least more than 90%, or preferably, more than 95%. Moreover, the polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as mature polypeptides.
The nucleotide full-length sequence of the antibody of the description or fragment thereof can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. A feasible method is to synthesize relevant sequences by artificial synthesis, especially with short fragment length. Usually, fragments with very long sequence can be obtained by synthesizing several small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
Once the relevant sequences are obtained, they can be obtained in large quantities by recombination. They are related sequences usually cloned into vectors, transferred into cells, and then isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) according to the present description include biomolecules in isolated form. At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the description can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the description through chemical synthesis.
Therefore, the present description also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above appropriate DNA sequence and the appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as “flanking sequence” in some embodiments) generally comprises one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication origin, transcription termination sequence, complete intronic sequence comprising donor and receptor splice sites, sequence encoding leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, a multi-linker region for inserting nucleic acids encoding antibodies to be expressed and an optional marker element.
Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
In some embodiments, host cells may be various functional cells well known in the art, such as various killer cells, including but not limited to cytokine induced killer cells (CIK), dendritic cell stimulated cytokine induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL) γδ T cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 mAb), and car-t/tcr-t cells. In some embodiments, the killer cells are T cells or NK cells. Exemplary NK cells include, but are not limited to, primary NK cells, NK cell strains (such as NK92), and NKT cells. In some embodiments, the NK cells are primary NK cells. Exemplary T cells include, but are not limited to, peripheral blood T lymphocytes, cytotoxic T cells (CTLs), helper T cells, inhibitory/regulatory T cells γδ T cells and T cells with mixed cell populations such as cytokine induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL). In some embodiments, the T cells are selected from peripheral blood T lymphocytes and til derived T cells.
Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth period and treated with CaCl2 method. The steps used are well known in the art. Another method is to use MgCl2. If necessary, the transformation can also be carried out by electroporation. When the host is eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the description. According to the host cells used, the medium used in the culture can be selected from various conventional media. Culture is performed under conditions suitable for host cell growth. When the host cells grow to the appropriate cell density, the selected promoters are induced by appropriate methods (such as temperature conversion or chemical induction), and the cells are cultured for another period of time.
The polypeptide in the above method can be expressed inside the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other characteristics. These methods are familiar to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitant (salting-out method), centrifugation, permeation, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC) and other various liquid chromatography technologies and combinations of these methods.
By constructing a nanobody library, the inventors discovered, expressed and purified a number of nanobodies that can bind to EGFR protein. The ability of binding to antigens and cells, and drug safety of these antibodies were verified by protein level affinity detection, protein binding epitope analysis, ligand competition assay, cross-species reactivity assay, cell level affinity detection, tumor cell EGFR binding detection, Membrane Proteome Array screening and tissue cross reaction.
Therefore, all aspects of the antibodies described herein can be used to prepare drugs to prevent or treat various conditions and diseases described herein, especially those related to cells expressing human epidermal growth factor receptor. In some embodiments, the condition and disease are cancers, including but not limited to head and neck cancer, breast cancer, bladder cancer, ovarian cancer, renal carcinoma, colon cancer, non-small cell lung cancer, and the like.
The pharmaceutical composition herein comprises the binding molecules described herein, as well as pharmaceutically acceptable excipients, including but not limited to diluents, vehicles, solubilizers, emulsifiers, preservatives, and/or adjuvants. The excipients are preferably non-toxic to the recipient at the dose and concentration used. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and their combinations. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined according to the expected route of administration, mode of delivery, and required dose.
Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization and rehydration. The pharmaceutical composition of the present description may be selected for parenteral delivery. Compositions for parenteral administration may be in lyophilized form or stored in solution. For example, it is prepared by conventional methods with normal saline or aqueous solution comprising glucose and other adjuvants. Parenteral compositions are usually placed in containers with sterile access holes, such as intravenous solution strips or vials with plugs that can be pierced by subcutaneous injection needles. Alternatively, compositions may be selected for inhalation or delivery through the digestive tract, such as oral. The preparation of the pharmaceutically acceptable composition is within the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising antibodies in sustained or controlled release delivery formulations. The techniques used to prepare a variety of other sustained or controllable delivery modes (such as liposome carriers, bioerodible particles or porous beads, and deposit injection) are also known to those skilled in the art.
Once the pharmaceutical composition is prepared, it is stored in sterile vials in the form of solution, suspension, gel, emulsion, solid, crystal or dehydrated or lyophilized powder. The formulation may be stored in ready to use form or rehydrated before administration (e.g., lyophilized). The description also provides a kit for generating a single dose administration unit. The kit of the description can each contain a first container with dried protein and a second container with aqueous formulation. In some embodiments of the present description, a kit comprising single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.
The present description also provides a method for treating a patient (especially a patient's EGFR related disease) by administering a binding molecule or a pharmaceutical composition thereof according to any embodiment of the present description. Terms “patient”, “subject”, “individual” and “object” are used interchangeably herein, including any organism, preferably animals, more preferably mammals (such as rats, mice, dogs, cats, rabbits, etc.), and most preferably humans. “Treatment” refers to the subject accepting the treatment scheme described herein to achieve at least one positive treatment effect (for example, a reduced number of cancer cells, a reduced tumor volume, a reduced rate of cancer cells infiltrating into peripheral organs, or a reduced rate of tumor metastasis or tumor growth). The treatment scheme for effectively treating patients can vary according to many factors, such as the patient's disease status, age, weight, and the ability of the therapy to stimulate the subject's anti-cancer response.
The therapeutically effective amount of the pharmaceutical composition comprising the binding molecule of the present description to be used will depend on, for example, the degree and the target of treatment. Those skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health condition) of the patient. In some embodiments, clinicians may titrate the dose and change the route of administration to obtain the optimal therapeutic effect. For example, about 10 micrograms/kg body weight per day to about 50 mg/kg body weight.
The dosing frequency will depend on the pharmacokinetic parameters of the bound molecules in the formulation used. Clinicians typically administer the compositions until a dosage that achieves the desired effect. The composition may therefore be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.
The route of administration of the pharmaceutical composition is according to known methods, such as oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intra-arterial, portal vein or intralesional injection; by continuous release system or by implantable device.
The binding molecule of the present description can be used in assays due to its high avidity with EGFR, such as binding assays to detect and/or quantify EGFR expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in further studies investigating the function of EGFR in disease. The methods for detecting EGFR are roughly as follows: obtaining cell and/or tissue samples; and detecting the level of EGFR in the sample.
The human epidermal growth factor receptor binding molecule of the invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions related to human epidermal growth factor receptor. The invention provides a method for detecting the presence of human epidermal growth factor receptor in a sample using a classical immunohistological method known to those skilled in the art. Human epidermal growth factor receptor can be detected in vivo or in vitro. Examples of methods suitable for detecting the presence of human epidermal growth factor receptor include ELISA, FACS, RIA, etc.
For diagnostic uses, binding molecules such as single domain antibodies are usually labeled with detectable labeling groups. Suitable labeling groups include (but are not limited to): radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used to carry out the present description.
Another aspect of the present description provides a method for detecting the presence of a test molecule that competes with an antibody of the present description to bind EGFR. An example of one such assay would involve detecting the amount of free antibody in a solution comprising an amount of EGFR in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind EGFR) will indicate that the test molecule can compete with the antibody for binding to EGFR. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled, and the amount of free test molecule is monitored in the presence or absence of the antibody.
The description also provides a detection kit for detecting EGFR level. The kit comprises an antibody that recognizes EGFR protein, a lysis medium for dissolving samples, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.
The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present description. Unless otherwise specified, the methods and materials used in the examples are conventional materials and methods in the art.
The sequence of EGFR protein (L25-S645, P00533-1) was obtained from uniprot, and was fused with the sequence of human IgG Fc fragment, and the eukaryotic expression vector of pCDNA3.4 (Thermo) plasmid was synthesized and constructed by Nanjing Genscript Company. The synthesized plasmid was expressed using ExpiCHO™ (Thermo Fisher) expression system. After expression, 5 ml of protein A pre-packed column (GE) was used for one-step affinity purification, and the purified sample was replaced into PBS buffer. After the purity was identified by SDS-PAGE electrophoresis gel and HPLC, and the activity was identified by ELISA, the sample was split and frozen in −80° C. refrigerator for subsequent immunization.
For first immunization, 400 μg of antigen was mixed with the adjuvant (GERBU FAMA). An alpaca was immunized subcutaneously at four sites on the back with an immune amount of 1 mL at each site. The second to sixth the immunization: the amount of immune antigen was 200 μg, and the alpaca was immunized subcutaneously at four sites on the back with an immune amount of 1 mL at each site, the interval of each immunization being one week.
EGFR.His antigen (Acro, Cat #EGR-H522a), EGFR-V3-his tag antigen (Acro, Cat #EGI-H52H4) were coated overnight at 4° C. After blocking and washing, the gradient diluted serum was added to the ELISA plate for incubation, and then incubated with the anti-llama IgG HRP (Abcam) antibody. After washing, TMB chromogenic solution was added for development, and the reaction was terminated with 2 M HCl. Then the absorbance value at OD450 nm was detected with a microplate reader. As shown in
(1) After six immunizations, 100 mL of camelid peripheral blood lymphocytes were extracted, and total RNA was extracted. RNA was extracted according to the instructions of RNAiso reagent of Takara.
(2) The first strand of cDNA was synthesized with RNA as template and oligo dT as primer according to the instructions of reverse transcriptase of Takara.
(3) The variable region coding gene of heavy chain antibody was obtained by nested PCR using PrimeSTAR high fidelity DNA polymerase. The variable region fragment of the heavy chain antibody was amplified by nested PCR:
The fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified, annealed at 55° C. for 30 cycles; a DNA fragment of about 600 bp was recovered as a template for the second round of PCR.
The fragment (long fragment and short fragment) between the FR1 region and the long and short hinge regions of the heavy chain antibody was amplified, annealed at 55° C. for 30 cycles, and the target fragment was recovered. The result showed that the size of the fragment was about 500 bp, that is, the nanobody gene electrophoresis band was about 500 bp.
(4) The phage pME207 and PCR amplification products were digested with Sfi I and Not I (NEB), respectively. After recovery and quantification, the two fragments were ligated with T4 DNA ligase (Takara) at a molar ratio of 1:3 and ligated overnight at 16° C.
(5) After ethanol precipitation, the ligated product was dissolved in 100 μL sterile water and electroporated into Escherichia coli TG1 in ten times. 100 μL of the bacterial solution was taken after electric shock and culture, diluted by multiple ratio, coated on an ampicillin LB culture plate, the storage capacity was calculated, and the rest was coated with ampicillin 2×YT culture plate, at 37° C., invertly cultured for 13-16 h. After scraping and washing the colonies on the culture plate with 10 ml, 2×YT medium, 25% glycerol at the final concentration was added, split, and stored at −80° C. for further use. The size of the storage capacity is 4.3×109. To detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached more than 90%.
(6) According to the calculated library capacity results, viable cells with 10 times the library capacity were seeded in 200 ml of 2×YT (comprising 2% glucose, 100 μg/ml ampicillin), cultured at 37° C. for 200 r/min until the OD600 reached 0.5, auxiliary phage was added according to the multiplicity of infection of 20:1, and left for 30 min at 37° C., 200 r/min for 30 min. The culture was centrifuged, and the pellet was resuspended with 200 ml of 2×YT (comprising 100 μg/ml ampicillin and 50 μg/ml kanamycin), incubated overnight at 37° C., 250 r/min, centrifuged at 8000 rpm to obtain the supernatant, added with 5×PEG/NaCl solution, placed on ice for 60 min, centrifuged at 8000 rpm for 30 min. The pellet was resuspended in 5 ml of PBS to obtain the single domain antibody (VHH) immune library against EGFR, and 10 μL was taken to determine the titer, and the rest were split at −80° C. for storage.
(7) EGFR.His protein was used for phage library panning. The protein was coated on an ELISA plate at 5 μg/ml, 100 μl per well, and placed overnight at 4° C. The next day, 200 μL, 3% BSA were added to the five wells and blocked for 2 hours at room temperature. Two hours later, they were washed three times with PBST (comprising 0.05% Tween 20 in PBS). After the plate was washed, 100 μL of phage pre-blocked with 5% skim milk (2-3×1011 tfu immunized camelid nanobody phage display gene library) was added, left for 1.5 hours at room temperature, and then the supernatant after negative screening was transferred to the target antigen coated well for 1.5 hours at room temperature. It was washed with PBST (comprising 0.05% Tween 20 in PBS) for 12 times to wash out the unbound phage. The phage specifically bound to EGFR was dissociated with Glycine (SIGMA), and the eluted phage was neutralized by Tris (Invitrogen, 1 M, pH 8.0) and infected with TG1 in logarithmic phase. After propagation and expansion, the next round of “adsorption-elution” was carried out. Finally, the eluted phage was impregnated with TG1, and the TG1 was induced by IPTG (Thermo) to express nanobody. The ELISA plate was coated with EGFR protein and EGFR-DIII-AlpFc protein. The supernatant was taken for ELISA detection, and the clones with OD450>0.5 were selected for sequencing.
(8) After sequence analysis, a total of 182 clones that could bind EGFR protein were obtained.
Nine nanobody VHH sequences with high binding activity to EGFR (see Tables 6 and 7 for their numbers and CDR1, CDR2, CDR3 and VHH sequences) were selected and constructed on pCDNA3.4-IgG4 vector to form VHH-IgG4, and then expressed by ExpiCHO™ (Thermo Fisher) expression system. After one week of expression, the supernatant was collected for protein A (GE) purification. Then Nanodrop was used to detect the protein quality, and HPLC was used to detect the protein purity. The purity and yield of the obtained protein met the requirement of subsequent tests.
The binding kinetics and affinity of candidate antibody to human EGFR.His antigen were determined using surface plasmon resonance (SPR). The purified antibody was flowed through the sensor chips pre-immobilized with protein A, and the antibody was captured by protein A. Then five different concentrations of EGFR.His protein were used as the mobile phase, and the binding time and dissociation time were 30 min and 60 min, respectively. The binding rate (kon), dissociation rate (koff), and equilibrium constant (kD) were analyzed using Biacore Evaluation Software 2.0 (GE). EC225, an antibody that binds to region III of human EGFR, was selected as a positive control (sequence source https.//go.drugbank.com/drugs/DB00002). The SPR detection results are shown in Table 1.
(2) Protein Binding Epitope Analysis: ELISA was used to determine the binding of VHH-IgG4 antibody to each domain of EGFR extracellular protein. Briefly, EGFR his tag protein, and EGFR-V3-his tag antigen protein were diluted to 1 μg/mL, EGFR-D1D2 (L25-A310, P00533-1 (uniprot)), and EGFR-D4 (F481-S645, P00533-1 (uniprot)) his tag antigen were diluted to 0.5 μg/mL, and each well of an ELISA plate was coated with 100 L antigen and incubated overnight at 4° C. The next day, the plates were washed twice with PBST (added with 0.5% TWEEN-20), then blocked with 3% BSA at 37° C. for 1.5 h, and then washed twice with PBST. Then, the antibody to be tested was diluted to 50 nM, 5 nM, 0.5 nM, 0.05 nM, and added to the wells coated with EGFR his tag protein or EGFR-V3-his tag antigen at 100 L/well, respectively. The antibody was diluted to 2000 nM, 200 nM, 20 nM, 2 nM, and added to the wells coated with EGFR his tag protein or EGFR-V3-his tag antigen at 100 L/well, respectively. The ELISA plates were incubated at 37° C. for 1 h. The plates were then washed three times with PBST, and then 100 μL HRP labeled Goat anti human IgG Fc antibody (1:10000, Bethyl) was added into each well, and incubated at 37° C. for another 40 min. After washing the plates four times with PBST, 100 μL of TMB substrate was added to each well, and the reaction was allowed to proceed for 3-5 min. The reaction was stopped by adding 100 μL of 3N hydrochloric acid per well. After stopping the reaction, the optical density at 450/650 nm was measured using a Tecan microplate reader. The results are shown in
ELISA was used to identify whether the VHH-IgG4 antibody competed with the ligand EGF for binding to EGFR extracellular protein. Briefly, EGFR his tag protein was diluted with PBS to 2 μg/mL, and 100 μL of the antigen was added to each well of an ELISA plate and coated overnight at 4° C. The next day, the plates were washed twice with PBST (containing 0.5% TWEEN-20), and then blocked with 3% BSA at 37° C. for 1.5 h, followed by two washes with PBST. Human EGF Protein, Mouse IgG2a Fc Tag (Acro, Cat #EGF-H525b, 100 g) was diluted to 10 μg/mL. VHH-IgG4 antibodies were diluted to 2000 nM and then serially diluted 4-fold for 8 gradients. 50 μL of EGF protein and 50 μL of diluted antibody were added to each well, and the ELISA plate was incubated at 37° C. for 1 h. After washing 3 times, HRP goat anti-mouse IgG (Goat anti-Mouse IgG) (H+L) antibody (Abcam, cat #ab205719) diluted at 1:3000 was added to each well, and the plate was incubated at 37° C. for 40 min. The plates were then washed 3 times with PBST, and 100 μL of TMB substrate was added to each well and allowed to react for 3-5 min. The reaction was stopped by adding 100 μL of 3N hydrochloric acid per well. After stopping the reaction, the optical density at 450/650 nm was measured using a Tecan microplate reader. The dose-response curve was plotted, and the IC50 was calculated using GraphPad Prism 8.0 software. The Isotype 2 was used as a negative control, and its amino acid sequence is shown as SEQ ID NO: 2 in CN106046152A. The results are shown in
ELISA was used to identify the binding of VHH-IgG4 antibodies to the extracellular domains of mouse EGFR, monkey EGFR, and human EGFR proteins. The detection steps were as follows: Mouse EGFR Protein, His Tag (Acro, Cat #EGR-M5224) and Rhesus macaque EGFR Protein, His Tag (Acro, Cat #EGR-C52H1) were diluted to 1 μg/mL with phosphate buffer, and 100 μL of each dilution was added to a 96-well ELISA plate and coated overnight at 4° C. The next day, the plates were washed twice with PBST (containing 0.5% TWEEN-20) and then blocked with 3% BSA at 37° C. for 1.5 h, followed by two washes with PBST. VHH-IgG4 antibodies were diluted to 50 nM, and 100 μL of the dilution was added to each well and incubated at 37° C. for 1 h. The plates were then washed three times with PBST, and 100 μL of HRP-labeled goat anti-human IgG Fc antibody (1:10000, Bethyl) was added to each well and incubated at 37° C. for another 40 min. After washing the plates four times with PBST, 100 μL of TMB substrate was added to each well, and the reaction was allowed to proceed for 3-5 min. The reaction was stopped by adding 100 μL of 3N hydrochloric acid per well. After stopping the reaction, the optical density at 450/650 nm was measured using a Tecan microplate reader. The results are shown in Table 3, indicating that the proteins binding to domain III also bind to mouse EGFR protein.
HEK293T cells that express Region III of EGFR (i.e., HEK293T EGFR-II cells) were plated in a 96-well plate with 3×105 cells/well, and then HEK293T EGFR-11I cells were incubated with gradient diluted VHH-IgG4 antibody. After incubation for half an hour, anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot:145501) as the secondary antibody for detection was added for incubation, and then CytoFLEX flow cytometer was used for detection. The EC50 of the antibody was calculated by curve fitting with Graphprism8.0 software. The results are shown in
Two kinds of tumor cells, SKOV3 and Aspc-1, expressing EGFR were respectively plated in 96 well plates, with 3×105 cells per well, and then the tumor cells were incubated with gradient diluted VHH-IgG4 antibody. After incubation on ice for half an hour, anti human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot:145501) was added for incubation, and then CytoFLEX flow cytometry was used for detection. The EC50 of the antibody was calculated by curve fitting. The results are shown in
Membrane Proteome Array (MPA) is a membrane protein array screening platform developed by American Integral Molecular company. They display 5300 different human membrane proteins on the cell surface by transfecting HEK293 cells. The binding signal of antibody on these proteins is detected by FACS, so as to evaluate the specificity of the antibody to be detected. The MPA screening results (
This assay was mainly completed by the clinical trial testing center under Shanghai Cell Therapy Group. The purpose of this study is to evaluate whether several preferred antibodies cross-react with 12 or 34 types of normal human frozen tissues (each tissue is derived from 3 different individuals) by using the immunohistochemical staining method of streptavidin-biotin binding. The process was as follows. 12 or 34 types of tissues were selected for frozen section, dried at room temperature, and fixed with acetone. Blocking was performed using Reagent A and Reagent B of IHC Biotin Block Kit (Sangon, E674001). Biotin labeled antibody samples were incubated for 30 min, and horseradish peroxidase labeled streptavidin (Abcam, ab7403) was added for incubation for 15 min after washing. DAB development, hematoxylin counterstaining, neutral plastic sealing, natural air drying, and then microscopic examination were performed. The positive control was anti-EGFR antibody (Biotin)(Abcam), and the negative control was biotin labeled IgG4 isotype.
Table 4 shows the results of tissue cross reactivity of 9 EGFR targeting VHH-IgG4 antibodies with 12 types of normal human tissues. The 9 EGFR nanobodies and the purchased commercial EGFR antibody showed obvious typical positive localization in tonsils, cervical squamous epithelium, skin, and placenta. The E008 antibody exhibited the relatively smallest range and weakest tissue cross-reactivity; while the other antibodies, in addition to cross-reacting with tonsils, cervical squamous epithelium, skin, and placenta tissues, also cross-reacted with liver, kidney, gallbladder, and other tissues to varying degrees, among which the E007 antibody showed the relatively broadest and strongest tissue cross-reactivity.
Table 5 shows the tissue cross-reactivity results of 3 EGFR-targeting VHH-IgG4 antibodies with 32 types of normal human tissues. The tissue cross-reactivity results of the 3 antibodies were almost similar, with no significant differences. In the endothelial cells or squamous epithelial cells where the positive control antibody reacted, there were clear positive localizations. Compared to the other 2 antibodies, E008 exhibited relatively fewer tissue cross-reactivity.
The CDR sequences of 9 VHH antibodies are shown in Table 6, and the amino acid sequences of 9 VHH antibodies and EC255 control antibody are shown in Table 7.
Referring to the method of humanizing nanobodies (J. Biol. Chem. 2009; 284: 3273-3284), four EGFR-binding VHH antibodies, E002, E003, E005, E006 and E008 were humanized using the method of CDR region grafting. Germline sequences with high homology to E002, E003, E005, E006, and E008 were screened in the IgBLAST database and used as templates. The sequences of the humanized antibody variants for each antibody are shown in Table 8. The binding kinetics and affinities of the humanized VHH antibodies against the human EGFR.His antigen were determined by surface plasmon resonance (SPR) technology. The results shown in Table 9 indicate that the humanized antibodies have good affinities.
| Number | Date | Country | Kind |
|---|---|---|---|
| CN202110705939.8 | Jun 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/100888 | 6/23/2022 | WO |
