Patent Application
20220340677
The present disclosure relates to split inteins containing novel flanking sequence pairs, and recombinant polypeptides using the same, and the use of the inteins in the preparation of antibodies, in particular bispecific antibodies. The present disclosure also relates to a method of screening for the split inteins containing novel flanking sequence pairs.
Protein trans-splicing refers to a protein splicing reaction mediated by split inteins. During the splicing process, firstly the N-terminal fragment or N-terminal protein splicing region (In) and the C-terminal fragment or C-terminal protein splicing region (Ic) of the split intein recognize each other and are non-covalently bound; once the structure is correctly folded after binding, the split intein with a reconstructed active center completes the protein splicing reaction according to the typical protein splicing pathway, and connects the exteins at both sides (Saleh. L., Chemical Record. 6 (2006) 183-193).
In the technology of preparing recombinant proteins, a gene expressing a precursor protein can be split into two open reading frames, and a split intein consisting of two parts, N′fragment of intein (referred to as In) and C′fragment of intein (referred to as Ic), is used to catalyze the protein trans-splicing reaction, so that the two split exteins (En, Ec) that constitute the precursor protein are linked by a peptide bond, thereby obtaining a recombinant protein (Ozawa. T., Nat Biotechbol. 21 (2003) 287-93).
A bispecific antibody refers to an antibody molecule that can recognize two antigens or two epitopes, such as a bispecific or multispecific antibody capable of binding two or more antigens, which is known in the art and can be obtained in a eukaryotic expression system or in a prokaryotic expression system by a cell fusion method, chemical modification method, gene recombination method and other methods.
Currently, a wide variety of recombinant bispecific antibody formats have been developed, for example, a tetravalent bispecific antibody by fusing e.g. a IgG antibody format with a single chain domain (see e.g. Coloma, M J, et al., Nature Biotech. 15 (1997) 159-163; WO 2001077342; and Morrison, S., L., Nature Biotech. 25(2007) 1233-1234). However, due to the large difference from natural antibodies in structure, such antibodies will cause a strong immune response and a short half-life in vivo.
In addition, several other novel formats capable of binding two or more antigens have also been developed, e.g., small molecule antibodies such as minibodies, several single chain formats (scFv, bi-scFv), and the like. In these small molecule antibodies, the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer maintained (Holliger, P., et al., Nature Biotech. 23 (2005) 1126-1136; Fischer, N, and Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., J. Immunol. Methods. 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).
There are obvious advantages over bispecific antibodies by linking a core binding region of an antibody to a core binding region of other antibodies via a linker, however, there are also some problems in its application as a medicament, which greatly limits its use in preparation of medicine.
In fact, in terms of immunogenicity, these foreign proteins may elicit an immune response against the linker per se, or against the linker-containing protein, or even cause an immune storm. In addition, due to the flexibility, these linkers are prone to protein degradation, which can easily lead to poor stability, easy aggregation, shortened half-life of the antibody and may further enhance immunogenicity. For example, Blinatumomab of Amgen has a half-life of only 1.25 hours in blood, resulting in a 24-hour continuous administration via a syringe pump, which greatly limits its application (Bargou, R and Leo .E., Science. 321 (2008) 974-7).
In addition, it is desirable that in the engineering of bispecific antibodies, effector functions of antibody Fc fragment are retained, for example, CDC (complement-dependent cytotoxicity), or ADCC (cytotoxicity), and prolonged half-life of antibody binding to FcRn (Fc receptor) at blood vessel endothelium. These functions must be mediated by the Fc region, therefore, the Fc region should be retained in the engineered bispecific antibody.
Therefore, there is a need to develop bispecific antibodies that are structurally very similar to those of naturally occurring antibodies (e.g., IgA, IgD, IgE, IgG, IgM), and furthermore, humanized bispecific antibodies with minimal sequence differences from human antibodies and complete human bispecific antibodies are required.
At present, attempts have been made to prepare bispecific antibodies by the trans-splicing mechanism of Npu-PCC73102 DnaE (abbreviated as NpuDnaE) intein. There is not a linker peptide in the obtained spliced product by preparing a bispecific antibody via the intein trans-splicing mechanism, however, there still exist the following problems: in the bispecific antibody thus obtained, a free sulfhydryl group introduced by the Ic flanking sequence cannot be avoided, leading to a great risk of misfolding and instability, as well as undesirable splicing efficiency (Han L, Zong H, et al., Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies, Methods,. Vol 154, 2019 Feb 1;154:32-37).
The efficiency of split intein-mediated protein splicing is directly related to the intein sequence and flanking sequences of the intein.
In the NEB database (http://inteins.com/), more than 600 split inteins are listed, wherein the commonly used one is for example NpuDnaE with the In flanking sequence being AEY (En-AEY-In) and the Ic flanking sequence being CFNGT (Ic-CFNGT-Ec). The protein format of En-AEY-In and Ic-CFNGT-Ec after splicing is En-AEYCFNGT-Ec, which has a cysteine residue. Therefore, there is a free sulfhydryl group in the spliced product, which greatly increases the risk of misfolding and instability of the product.
In order to avoid the free sulfhydryl group in the spliced product, the existing flanking sequences pairs of split inteins need to be improved, and novel flanking sequences that maintain the good splicing efficiency of intein and do not contain a cystine residue are needed.
It has been reported that through amino acid mutation and screening of the flanking sequences of NpuDnaE, a In flanking sequence with sequence of MGG (En-MGG-In) and a Ic flanking sequence with sequence of SVY (Ic-SVY-Ec) are obtained. When trans-splicing is performed using an intein with such flanking sequences, En-MGGSVY-Ec is obtained after splicing of En-MGG-In and Ic-SVY-Ec (Cheriyan M., et al., J Mol Biol. 2014 Dec 12 ; 426(24): 4018-4029), without free sulfhydryl groups present in the final product.
In addition, amino acid mutations in the flanking sequence pairs of existing split inteins will affect the efficiency of trans-splicing. Therefore, a screening method is needed to screen an intein containing a novel flanking sequence pair with excellent trans-splicing efficiency and without introducing free sulfhydryl groups at the junction into the spliced product. Furthermore, there is a need for a split intein suitable for the preparation of antibodies, especially bispecific antibodies, which has excellent trans-splicing efficiency and does not introduce free sulfhydryl groups at the junction in the spliced product and contains novel flanking sequence pairs.
In the present disclosure, through performing regular amino acid mutations on the flanking sequences pairs of existing intein and screening for the flanking sequence pairs with excellent trans-splicing efficiency, a split intein with novel flanking sequence pairs is obtained, which has flanking sequences without cysteine residues, does not introduce free sulfhydryl groups at the junction in the spliced product, has an excellent trans-splicing efficiency, and is especially suitable for the preparation of antibodies (especially bispecific antibodies).
By using the split intein of the present disclosure, under relatively mild conditions (such as normal temperature, physiological salt concentration, neutral pH, etc.), polypeptide fragments from different proteins can be spliced together with high splicing efficiency to form a recombinant fusion polypeptide protein.
In addition, based on the screening of the above split inteins, the inventors established a method for preparing recombinant polypeptides (especially bispecific antibodies) by using the split inteins. The bispecific antibody thus prepared does not contain a non-natural domain, has a structure closely similar to that of natural antibody (IgA, IgD, IgE, IgG or IgM), and has a Fc domain. The bispecific antibody has a complete structure and good stability, and can retain or remove CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) or FcRn (Fc receptor)-binding activity according to different IgG subclasses.
The bispecific antibody prepared by the method of the present disclosure has the following advantages: the bispecific antibody has a long half-life in vivo and low immunogenicity, and does not introduce any form of linkers; has an improved stability, and reduced in vivo immune response.
The bispecific antibody prepared by the method of the present disclosure can be prepared by a mammalian cell expression system, so that it has the same glycosylation modification as that of wild-type IgG, has better biological function, is more stable, and has a long half-life in vivo; the in vitro splicing method by using inteins can completely avoid the problems of heavy chain mismatch and light chain mismatch commonly found in traditional methods.
The preparation method for bispecific antibodies of the present disclosure can also be used to produce humanized bispecific antibodies and bispecific antibodies with complete human sequences. The sequence of such an antibody prepared by the method of the present disclosure is more similar to that of a human antibody, which can effectively reduce the immune response.
The preparation method for bispecific antibodies of the present disclosure is a method for constructing universal bispecific antibodies, which is not limited by antibody subclasses (IgG, IgA, IgM, IgD, IgE, and light chain κ and λ types), and does not need to design different mutations according to a specific target and can be used to construct any bispecific antibody.
The present disclosure provides the following technical solutions.
A_3 is X or deletion; A_2, is selected from D, F, G, L, N, S or W; A_1 is selected from G, A, K, Q, R, W, T or S;
B1 is S; B2 is E; B3 is X or deletion, or preferably T, I, A, D, E, F, H, L, M, S, V, W or Y;
2. The flanking sequence pair for a split intein according to item 1, wherein the split intein together with the flanking sequence pair are used for trans-splicing,
3. A recombinant polypeptide obtained by trans-splicing via the flanking sequence pair for a split intein according to item 1 or 2.
4. The recombinant polypeptide according to item 3, wherein the recombinant polypeptide is obtained by a component A and a component B through trans-splicing;
in the component B, the C-terminus of the flanking sequence b is connected to the N-terminus of the C-terminal extein (Ec), and the N-terminus of the flanking sequence b is connected to the Ic, optionally a tag protein is connected to the N-terminus of the Ic;
5. The recombinant polypeptide according to item 3, wherein the recombinant polypeptide is obtained by a component A and a component B through trans-splicing;
6. The recombinant polypeptide according to item 4 or 5, wherein the recombinant polypeptide is a fluorescent protein, protease, signal peptide, antimicrobial peptide, antibody, or a polypeptide with biological toxicity.
7. The recombinant polypeptide according to item 4 or 5, wherein the same protein, or one or more of the different proteins is an antibody.
8. The recombinant polypeptide according to item 7, wherein the antibody is a natural immunoglobulin class IgG, IgM, IgA, IgD or IgE , or an immunoglobulin subclass: IgG1, IgG2, IgG3, IgG4, IgG5, or with light chains of different classes: kappa, lambda; or a single domain antibody; or
9. The recombinant polypeptide according to item 8, wherein the functional fragment of an antibody is selected from one or more of the group consisting of: antibody heavy chain variable region VH, antibody light chain variable region VL, antibody heavy chain constant region fragment Fc, antibody heavy chain constant region 1 CHL antibody heavy chain constant region 2 CH2, antibody heavy chain constant region 3 CH3, antibody light chain constant region CL or single domain antibody variable region VHH.
10. The recombinant polypeptide according to item 7, wherein, the same protein or one or more of the different proteins is specific to an antigen or epitope A,
11. The recombinant polypeptide according to item 10, wherein, the same protein or one or more of the different proteins is specific to an antigen or epitope B different from the antigen or epitope A,
12. The recombinant polypeptide according to item 11, which is a bispecific antibody that can simultaneously bind to both the antigen or epitope A and the antigen or epitope B, preferably a humanized bispecific antibody or a bispecific antibody of complete human sequence.
13. The recombinant polypeptide according to any one of items 7 to 11, wherein,
14. The recombinant polypeptide according to any one of items 3 to 13, wherein,
15. A composition comprising the recombinant polypeptide according to any one of items 3 to 14.
16. A composition further comprising, in addition to the recombinant polypeptide according to any one of items 3 to 14, a carrier.
17. The composition according to item 16, which is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier.
18. A carrier, which is connected with the recombinant polypeptide according to any one of items 3 to 14, preferably for purification including chromatography.
19. A kit comprising the recombinant polypeptide according to any one of items 3 to 14, for the detection of the presence of the antigen or epitope A and/or the antigen or epitope B in a sample, wherein preferably the recombinant polypeptide is stored in a liquid or in a form of lyophilized powder, optionally can be present separately or in a state of being fixed to a carrier by linking, complexing, associating or chelating.
20. An expression vector, which is an expression vector for preparing the recombinant polypeptide according to any one of items 3 to 14.
21. A method for preparing recombinant polypeptides, comprising:
(1) providing a component A and a component B, wherein, the component A comprises a flanking sequence a, an N-terminal extein En and an In; the N-terminus of the flanking sequence a is connected to the C-terminus of the N-terminal extein En, and the C-terminus of the flanking sequence a is connected to the In, optionally a tag protein is further connected to the C-terminus of In;
(2) performing an in vitro trans-splicing on the component A and the component B to obtain a recombinant polypeptide;
22. The method for preparing recombinant polypeptides according to item 21, further comprising:
23. The method for preparing recombinant polypeptides according to item 21, wherein the recombinant polypeptide is a bispecific antibody, and the coding sequences of the bispecific antibody are derived from two different antibodies P and R, respectively;
1) spliting the antibody P into a EnP and a EcP, and designing the sequences of component A and component B; spliting the antibody R into a EnR and a EcR, and designing the component A′ and the component B′; wherein,
2) performing a trans-splicing on the component A and the component B′, and/or the component A′ and the component B, to obtain the bispecific antibody.
24. A method of screening for a flanking sequence pair for a split intein, comprising:
1) splitting the amino acid sequence of protein P;
2) a flanking sequence a is an independently designed combination of 2-3 amino acids, denoted as flanking sequence a1-an, and a flanking sequence b is an independently designed combination of 2-3 amino acids, denoted as flanking sequence b1-bn; wherein, the amino acid is any amino acid selected from the group consisting of G, A, V, L, M, I, S, T, P, N, Q, F, Y, W, K, R, H, D, E, C;
3) for the split intein, expression sequences of components A1-An and components B1-Bn that contain the sequences split from protein P are designed by using the flanking sequences a1-an and b1-bn designed in step 2);
4) the expression sequences are linked to a vector respectively, and the components A and B are co-transfected in a manner of one-to-one correspondence and then intracellularly trans-spliced to obtain spliced products F1 to Fn;
5) detecting the spliced products F1 to Fn, and selecting the flanking sequence pair with a splicing efficiency more than 20%;
6) the flanking sequence pairs selected in 5) are analyzed, and the flanking sequences that can lead to free sulfhydryl group after splicing are removed to optimize the flanking sequence pair selected in 5);
7) the steps 1) to 5) are repeated to select the flanking sequence pairs 1 to m that have a splicing efficiency of top 20% in all candidate sequence pairs, and do not have free sulfhydryl groups in the recombinant polypeptide as the spliced product,
wherein, n is 2 or 3 and m is a positive integer.
25. The method of screening for a flanking sequence pair for a split intein according to item 24, further comprising:
1) splitting a protein R which is different from the protein P;
2) expression sequences of components A′1 to A′m and components B′1 to B′m are designed by using the flanking sequence pairs 1 to m;
3) the expression sequences are linked to a vector, and then a transfection, expression and purification are performed to obtain components A′1 to A′m and components B′1 to B′m,
4) the components A1-Am and the components B′1-B′m, and/or the components A′1-A′m and the components B1-Bm obtained by the flanking sequence pairs 1˜m are in vitro trans-spliced respectively in a manner of one-to-one correspondence; the spliced products are detected and multiple flanking sequence pairs with a splicing efficiency of more than 50% are selected.
26. A method for producing a recombinant polypeptide, characterized by performing a trans-splicing by using the flanking sequence pair for a split intein according to item 1 or 2.
27. Use of the flanking sequence pair for a split intein according to item 1 or 2 for the preparation of a recombinant polypeptide, preferably for the trans-splicing together with the split intein.
The advantages of recombinant polypeptides (such as, bispecific antibodies) prepared by the flanking sequences pair for a split intein of the present disclosure include: (1) no free sulfhydryl groups; (2) high-throughput and high-efficiency; and (3) the target product and impurities are easy to be distinguished and identified.
It should be noted that the term “a” or “an” entity refers to one or more of that entity (entities); for example, “bispecific antibody” shall be understood to refer to one or more of bispecific antibody (antibodies). Likewise, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
The term “polypeptide” as used herein includes the singular “polypeptide” as well as plural “polypeptides”, and also refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be achieved by combining polynucleotides or polypeptides that would not normally occur together.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.
A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, such percentage of bases (or amino acids) are the same in comparing the two sequences.
Biologically equivalent polynucleotides are polynucleotides that have the above-mentioned specified percentage of homology and encode polypeptides with the same or similar biologically activity.
The term “split intein” refers to a split intein consisting of two parts: an N-terminal protein splicing region or N-terminal fragment (i.e., In, or N′ fragment of intein) and a C-terminal protein splicing region or C-terminal fragment (i.e., Ic, or C fragment of intein). A gene expressing a precursor protein is split into two open reading frames, and the splitting site is internal to the intein sequence.
“N-terminal precursor protein” refers to a fusion protein translated by a fusion gene formed by a N-terminal extein (En)-encoding gene and a N-terminal fragment (In)-encoding gene.
“C-terminal precursor protein” refers to a fusion protein translated by a fusion gene formed by a C-terminal fragment (Ic)-encoding gene and a C-terminal extein (Ec) -encoding gene.
The N-terminal fragment (In) or the C-terminal fragment (Ic) of the split intein alone does not have a protein splicing function. After protein translation, the In in the N-terminal precursor protein and the Ic in the C-terminal precursor protein bind to each other by a non-covalent bond to form a functional intein, which can catalyze protein trans-splicing reaction, thus two separate protein exons are connected by peptide bonds (the N-terminal protein exon or N-terminal extein can be referred to as En, and the C-terminal protein exon or C-terminal extein can be referred to as Ec) (Ozawa.T. Nat Biotechbol .21 (2003) 287 93).
Protein trans-splicing refers to a protein splicing reaction mediated by split inteins. During the trans-splicing process, firstly, the N-terminal fragment (In) and the C-terminal fragment (Ic) of the split intein recognize each other and are non-covalently bound. Once bound, the structure is correctly folded and the split intein has a reconstructed active center, and then the protein splicing reaction is completed according to the typical protein splicing pathway, thereby linking the exteins at both sides.
The term “In” refers to a separate N-terminal portion of the split-intein, and also can be referred to herein as the N-terminal fragment or N-terminal protein splicing region of the split-intein.
The term “Ic” refers to a separate C-terminal portion of the split intein, and also can be referred to herein as the C-terminal fragment or C-terminal protein splicing region of the split intein.
The term “flanking sequence a” refers to an amino acid sequence flanking both the N-terminus of In and the C-terminus of En and linking the In and the En. As shown in
The term “flanking sequence b” refers to an amino acid sequence flanking both the C-terminus of Ic and the N-terminus of Ec and linking the Ic and the Ec. As shown in
During the split intein-mediated trans-splicing, for example as shown in
In the present disclosure, 20 common amino acids (hereinafter referred to as 20 amino acids) are used for the screening of flanking sequences, that is, glycine (G), alanine (A), valine (V), leucine (L), methionine (M), isoleucine (I), serine (S), threonine (T), proline (P), asparagine (N), glutamine (Q), phenylalanine (F), tyrosine (Y), tryptophan (W), lysine (K), arginine (R), histidine (H), aspartic acid (D), glutamic acid (E) and cysteine (C).
As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen or immunogenic epitope.
An antibody can be an intact antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing a specific molecule, wherein the specific molecule comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen or immunogenic epitope. Examples of such include, but are not limited to a complementary determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.
The term “antibody fragment” or “antigen-binding fragment”, as used herein, refers a portion of an antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen or immunogenic epitope to form a complex.
A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins.
The term “antibody” encompasses a wide variety of polypeptides that can be biochemically recognized. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ϵ) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and functionally specific. Modified versions of each of these classes and isotypes are readily discernable to those skilled in the art in view of the present disclosure and, accordingly, are within the scope of the present disclosure.
All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 joined by disulfide bonds in a “Y” configuration.
Antibodies, antigen-binding polypeptides, variants or derivatives thereof in the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, antigen-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass of immunoglobulin molecule.
In some examples, for example, certain immunoglobulins derived from camelid species or engineered based on camelid immunoglobulins, an intact immunoglobulin molecule thereof may consist of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).
Both the light and heavy chains are divided into structural regions and functional homology regions. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain determine the antigen recognition and specificity. Generally, the number of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
Regarding the antigen-binding site, those skilled in the art can easily identify the amino acids of the CDR and framework regions for any given heavy chain or light chain variable region since they have been clearly defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987), the full text of which is incorporated herein by reference).
In the case where there are two or more definitions of a term that are used and/or accepted within the art, the definitions of the term as used herein are intended to include all meanings, unless explicitly stated to the contrary.
The term “complementarity determining region” (“CDR”) refers to the non-contiguous antigen binding sites present in the variable regions of both heavy chain and light chain polypeptides. This specific region has been described by Kabat et al., U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol. 196:901-917 (1987), the full text of which is incorporated herein by reference. Those skilled in the art can routinely determine which residues comprise a particular CDR if the amino acid sequence of the variable region of the antibody is provided .
The “Kabat numbering” as used herein refers to the numbering system described by Kabat et al., U.S. Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).
The term “heavy chain constant region” as used herein includes amino acid sequences derived from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (for example, upper hinge region, middle hinge region, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide for use in the present disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide comprising a CH1 domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. In addition, the antibodies used in the present disclosure may lack at least a portion of a CH2 domain (for example, all or a portion of the CH2 domain). As set forth above, it will be understood by those skilled in the art that the heavy chain constant regions may be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
The heavy chain constant regions of the antibody disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region that is partly derived from an IgG1 molecule and partly from an IgG3 molecule. In another example, the heavy chain portion may comprise a chimeric hinge that is partly derived from an IgG1 molecule and partly derived from an IgG4 molecule.
The term “light chain constant region” as used herein includes an amino acid sequence derived from the light chain of an antibody. Preferably, the light chain constant region includes at least one of a constant kappa domain and a constant lambda domain.
The term “VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term “CH1 domain” includes a first (mostly amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.
The term “CH2 domain” as used herein includes a portion of a heavy chain molecule that ranges, for example, from a residue at about position 244 to a residue at position 360 of an antibody according to a conventional numbering system (residues at position 244 to 360, according to Kabat numbering system; and residues at position 231-340, according to EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique because it does not pair with another domain tightly. On the contrary, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact natural IgG molecule. It is documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, and comprises about 108 residues.
By “specifically binding” or “specific to”, it generally means that when the antibody binds to the antigen epitope, the binding via the antigen-binding domain is easier than that via binding to a random, unrelated antigen epitope. The term “specificity” is used herein to determine the affinity of a certain antibody to bind to a particular antigen epitope.
The term “treating” (“treat” or “treatment”) as used herein refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as cancer progression. Beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, diminishing the degree of disease, stabilizing (for example, preventing it from worsening) disease state, delaying or slowing the disease progression, alleviating or palliating the disease state, and alleviating (whether partial or total), regardless of whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival without receiving treatment.
Any of the aforementioned antibodies or polypeptides may further include additional polypeptides, for example, an encoded polypeptide as described herein, a signal peptide at the N-terminus of the antibody used to direct secretion, or other heterologous polypeptides as described herein.
In other embodiments, the polypeptide of the present disclosure may comprise conservative amino acid substitutions.
A “conservative amino acid substitution” is one in which an amino acid residue is substituted by an amino acid residue having a similar side chain Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), (3-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably substituted by other amino acid residues from the same side chain family. In another embodiment, a string of amino acids may be substituted by a structurally similar string of amino acids that differs in sequence and/or composition of the side chain family
Transient transfection is a technical means of introducing DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into a highly infectious cell line to obtain transient but high-level expression of the gene of interest. The transfected DNA does not have to be integrated into the host chromosome, and the transfected cells can be harvested in a shorter time than stable transfection, and the target product in the expression supernatant can be detected.
“SPLICING 2” are both incubated at 37° C. overnight, and the other groups are stored at 4° C. (MW, molecular weight; RD, reduced)
The present disclosure relates to a preparation method of a bispecific antibody, which includes: splitting the DNA sequence of the target antibody, constructing a mammalian cell expression vector through whole gene synthesis, purifying the vector, and then the purified vector can be transiently transfected or stably transfected into mammalian cells such as HEK293 or CHO, respectively. The fermentation broth is collected separately, and the component A and the component B are purified by methods such as protein A, protein L, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity chromatography; the purified component A and component B are subjected to in vitro trans-splicing, and the spliced product is subjected to affinity chromatography for tag proteins such as nickel column to obtain a bispecific antibody with high-purity. The process flow is shown in
The antibodies described herein can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, the variable region may be derived from a condricthoid (e.g., from a shark).
In some embodiments, the antibody may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
The antibody may be linked or fused to a therapeutic agent, which may include detectable labels, such as radioactive labels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxicity agents, which can be drugs or toxins, ultrasound enhancers, non-radioactive labels, a combination thereof and other such components known in the art.
The antibody can be detectably labeled by coupling it to chemiluminescent compounds. Then, the presence of the chemiluminescent-labeled antigen-binding polypeptide is determined by detecting the luminescence produced during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
The antibodies can also be detectably labeled by using fluorescence emitting metals such as 152Eu, or other lanthanide labels. These metals can be attached to the antibody by using the following metal chelating groups, such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The binding specificity of the antigen-binding polypeptides of the present disclosure can be measured by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
Cell lines for production of recombinant polypeptides can be selected and cultured by using techniques well known to those skilled in the art.
Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the antibodies of the present disclosure, including, but not limited to, site-directed mutagenesis and PCR-mediated mutations which result in amino acid substitutions. Preferably, the variants (including derivatives), relative to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2 or CDR- L3, encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, and less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions. Alternatively, mutations can be randomly introduced along all or part of the encoding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutations that retain activity.
The tag protein used in the present disclosure may be Fc, oligo-histidine (His-tag), Strep-tag, Flag, HA, or maltose-binding protein (MBP) or the like.
The transfection used in the present disclosure may be transient transfection or stable transfection.
Mammalian cells such as HEK293 or CHO are used in the present disclosure, but are not limited thereto.
Liquids containing expression products from mammalian cells, such as fermentation broth and culture medium supernatant, can be purified by methods such as protein A, protein G, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity chromatography.
The spliced product can be subjected to affinity chromatography for the tag protein to remove unspliced components.
The gene fragment used for constructing the vector of the present disclosure can be constructed by whole gene synthesis, but is not limited thereto.
The vector used in the present disclosure is pcDNA3.1 or pCH01.0, but is not limited thereto.
The restriction enzymes used in the present disclosure include, but are not limited to, Notl, Nrul, or BamHI-HF, for example.
BLAST is an alignment program that uses default parameters. Specifically, the programs are BLASTN and BLASTP. Detailed information of these programs is available at the following Internet address:
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
In a specific embodiment of the present disclosure, as shown in
In another specific embodiment of the present disclosure, as shown in
In another specific embodiment of the present disclosure, the component B expression plasmids may include three types of expression plasmids, pB-L, pB-H, and pB-FcIc.
In the present disclosure, Pa also refers to the N-terminal protein exon or N-terminal extein of protein P, also referred to as Enp; Pb also refers to the C-terminal protein exon or C-terminal extein of protein P, also referred to as Ecp. Ra also refers to the N-terminal protein exon or N-terminal extein of protein R, also referred to as EnR; Rb also refers to the C-terminal protein exon or C-terminal extein of protein R, also referred to as EcR.
NSYVSWFAY
WGQGTLVTVSS
NSYVSWAAY
WGQGTLVTVSS
V
WGAGTTVTVSS
H
WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDF
FDV
WGQGTMVTVSS
MWSED
WGQGTLVTVSS
WG
WIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVT
GLFDY
WGQGTLVTVSS
KENLLA
WYQQKPGQSPKLLIYYASIRFTGVPDRF
FDS
WGQGTLVTVSS
T
FGDGTKLEIK
I
WGQGTMVTVSS
WFDP
WGQGTLVTVSS
VS
WYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSK
FDY
WGQGTLVTVSS
SYLH
WYQQKPGQAPRLLIYLASYLESGVPARFSG
GFDY
WGQGTTVTVSS
GSTYYADSVKG
RFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQ
T
FGQGTKVEIK
GKAHYAQKFQG
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGM
DV
WGQGTTVTVSS
STI
NYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWG
T
FGQGTKVEIK
GEATYVEEFKG
RFVFSLDTSVSTAYLQISSLKADDTAVYYCARWDFYDYVEAMDY
FT
FGQGTKVEIK
GEPTYAADFKR
RFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYF
DV
WGQGTLVTVSS
T
FGQGTKVEIK
GYTH
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDY
T
FGQGTKVEIK
GYTYYADSVKG
RFTISADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYW
T
FGQGTKVEIK
DIANYAQ
RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCASTLGLVLDAMDYW
IT
FGQGTRLEIK
DIANYAQ
RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYW
IT
FGQGTRLEIK
STDYSAAFMS
RLSITKDNSKSQVFFKMNSLQADDTAIYFCAKQAYGHYMDYWGQG
SHVPWT
FGGGTKLEIK
STDYSAAFMS
RLTISKDNSKNTVYLQMNSLRAEDTAVYFCAKQAYGHYMDYWGQG
SHVPWT
FGQGTKVEIK
GDTDYNGKFKG
RVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG
LELPYT
FGGGTKVEIK
STDYSAAFMS
RLSITKDNSKSQVFFKMNSLQADDTAIYFCAKQAYGHYMDYWGQG
DYSYPFT
FGSGTKLEIK
GYTKYNRKFRD
KATLTADKSSSTAYMQLTSLTYEDSAVYYCARGGNGYYFDYWGQ
GYTKYNQKFKV
KATLTADKSSSTAYMQLSSLTDEDSAVYYCANGNLGYFFDYWGQ
GYTKYNRKFRDKATLTADKSSSTAYMQLTSLTYEDSAVYYCARGGNGYYLDYWGQ
GTNDYNPSLKNRISITRDTSENQFFLKLNSVTTEDTATYYCARGPPCTYWGQGTL
DGTKYNEKFKG
KATLTSDKSSSTAYMELSSLTSEDSAVYYCARGNRYDVGSYAMD
Y
WGQGTSVTVSS
YYRFPYT
FGGGTKLEIK
GGSIYNQKFKG
KATLTIDKSSSTAYMELRSLTSEDTAVYYCARRRSYGYYFDYWG
T
FGAGTKLEIK
GYTRYADSVKG
RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW
T
FGQGTKVEIK
GGSIYNQRFKG
RFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWG
T
FGQGTKVEIK
YTYATFYAESLEGR
VTISRDDSESSVYLQVSSLRAEDTAIYYCTRSLTGGDYFDY
LLIYRASNLAS
GIPARFSGSGSGTDFTLTINPVQADDIATYFCQQSRK
SPWT
FAGGTKLELR
YTYATFYAASVKG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSLTGGDYFDY
RLLIYRASNLAS
GIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQSR
KSPWT
FGQGTKVEIK
YNYETYYSDSVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQ
THVPWT
FGGGTKLEIK
The DNA sequences in the Examples of the present disclosure were all obtained by reverse translation based on the amino acid sequences, and were synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd.
The recombinant polypeptides involved in the Examples were all prepared by the following method: in the presence of recombinase, the DNA sequence and a vector pcDNA3.1 digested by a restriction enzyme EcoRI were ligated at 37° C. for 30 minutes, and then transformed into a Trans10 competent cell by heat shock method, and then transiently transfected into 293E cells (purchased from Thermo Fisher) after verified by sequencing (Wuhan GeneCreate Biological Engineering Co., Ltd.). After expression, the recombinant polypeptides were purified.
2. The Co-Transfected Plasmids Involved in the Examples were Shown as Follows:
1) To express the component A and component B shown in
2) To express the component A and component B′ shown in
3) To express the component A shown in
In general, if two plasmids were co-transfected and expressed, the molar ratio of the two plasmids was 1:1 or any other ratio. If three plasmids were co-transfected and expressed, the molar ratio of the three plasmids was 1:1:1, or any other ratio.
3. Purification of Ppolypeptides with Tag Proteins
(1) When the tag protein was Fc, the polypeptide was purified by affinity chromatography, for example, MabSelect SuRe (GE, Cat. No. 17-5438-01), 18m1 column.
(2) When the tag protein was His-tag, the polypeptide was purified by affinity chromatography, for example, Ni-NTA (Jiangsu Qianchun, product number: A41002-06).
(3) When the tag protein was Strep-tag, Flag, HA or MBP, etc., the polypeptide was purified by Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography, or cross-linked starch affinity chromatography by selecting corresponding packings and buffers.
(4) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by an ion exchange chromatography based on the difference in isoelectric point. The chromatography packing can be a cation exchange chromatography packing or an anion exchange chromatography packing, such as Hitrap SP-HP (GE Company).
(5) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by a hydrophobic chromatography based on the difference in hydrophobicity by using a chromatography packing such as Capto phenyl ImpRes packing (GE Company).
(6) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by a molecular sieve chromatography based on the difference in molecular weight by using a chromatography packing such as HiLoad Superdex 200pg (GE Company).
Construction of Expression Plasmids pA-HIn, pA-L and Plasmid (pTag-Ic-FSb-Pb)
In this Example, the amino acid selected from any one of the 19 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y) defined in the present disclosure is denoted as X amino acid. H refers to a portion of heavy chain of the antibody and L refers to a portion of light chain of the antibody. For the flanking sequence pairs of the original NpuDnaE, each amino acid is mutated by degenerate primer design to be one of the 19 amino acids described in the present disclosure.
For the A-HIn expression plasmid (pA-HIn) with the mutated flanking sequence pair, for the sake of simplicity, a plasmid with the X amino acid at position -1 of the flanking sequence a is referred to as pA-HIn(X), and a plasmid with the X amino acids independently at positions −2 and −1 (wherein the X amino acids may be the same or different) is referred to as pA-HIn(XX).
The sequences involving X amino acid in pA-HIn(XX) are all obtained by degenerate primer design.
The expression plasmid of A-Fab 1 contains two polypeptides, A-HIn and AL, wherein the plasmid for polypeptide A-HIn is denoted as pA-HIn(1), and the plasmid for polypeptide A-L is denoted as pA-L; and so on.
The B-FcIc expression plasmid with the mutated flanking sequence pair is similarly designated as the expression plasmid pTag-Ic-FSb-(B-FcIc). Similarly, the expression plasmid pTag-Ic-FSb-(B-FcIc) with the amino acids at positions +1, +2 and +3 of the flanking sequence b being independently the same or different X amino acids is denoted as plasmid pTag-Ic-FSb(XXX)-(B-FcIc), wherein the sequences involving X amino acids are all obtained by degenerate primer design.
According to the steps and conditions described in “Preparation of Recombinant Polypeptides”, as shown in
The expression plasmid pTag-Ic-FSb-(B-FcIc) was divided into 19 groups, wherein the plasmids with the same amino acid residue at position +1 of the flanking sequence b were divided into one group. Accordingly, for each group, the amino acid X at position +1 of the flanking sequence b was definite, and the amino acids at positions +2 and +3 were X (that is, any one of the 19 amino acids).
293E cells were co-transfected with the above 19 groups of pTag-Ic-FSb(XXX)-(B-FcIc) plasmids and the corresponding expression plasmids pA-HIn(XX) and pA-L, respectively.
The molar ratio of transfection was pTag-Ic-FSb(XXX)-(B-FcIc):pA-HIn(XX):pA-L=3:1:1.
At the same time, a positive control group (the flanking sequence a was AEY, and the flanking sequence b was CFN) was set up, and the molar ratio of positive control plasmid and co-transfected plasmids thereof was pTag-Ic-FSb-(B -FcIc1):pA-HIn(1):pA-L=3:1:1.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot, wherein the molecular weight marker protein was PageRuler Prestained Protein Ladder (purchased from Thermo Company, Cat. No. 26616). The results showed that at the position of 50 kD (corresponding to the complete heavy chain), there was a clear band in the positive control group (intein+natural flanking sequence pair); however, for other groups, only the group with serine (S) at position +1 of the flanking sequence b showed an obvious band.
The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S.
The above screened expression plasmid pTag-Ic-FSb(SXX)-(B-Fcic) with Serine (S) at position +1 was further grouped into 19 subgroups, wherein the plasmids with the same amino acid residue at position +2 of the flanking sequence b were divided into one group. Accordingly, for each subgroup, the amino acid at position +1 of the flanking sequence b was S, and the amino acid at position +2 was definite.
The expression plasmid pA-HIn(XX) was divided into 19 groups, wherein the plasmids with the same amino acid residue at position −1 of the flanking sequence a were divided into one group. Accordingly, for each group, the amino acid at position +1 of the flanking sequence a was definite.
293E cells were co-transfected with the above 19 subgroups of expression plasmids pTag-Ic-FSb(SXX)-(B-FcIc) and the 19 groups of expression plasmids pA-HIn(XX) and pA-L, respectively.
In order to reduce the number of experiments in the cross-pairing test, the above 19 groups of expression plasmids pA-HIn(XX) were divided into groups Al—A6 according to Table 34 below, and the above 19 subgroups of expression plasmids pTag-Ic-FSb(SXX)-(B-Fcic) were divided into groups B1˜B6 according to Table 35 below.
Transfection was performed in the same manner as above based on the pairings in Table 36 with a molar ratio of pTag-Ic-FSb(SXX)-(B-FcIc):pA-HIn(XX):pA-L=3:1:1. A positive control monoclonal antibody was also set in the same manner as above, and 36 groups of transfections (i.e., groups 1-1 to groups 6-6) were obtained, respectively.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent β-mercaptoethanol, and the detection antibody was HRP-labeled goat anti-human IGG antibody purchased from Sigma). The results were shown in
The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position −1 is A, V or G, and the amino acid at position +2 is R, K, E or D.
All plasmids in groups 1-6 were selected for rescreening.
All plasmids in groups 1-6 in which the amino acid residue at position −1 was A, V or G and the amino acid residue at position +2 was R, K, E or D were paired and co-transfected according to Table 37.
Transfection was performed in the same manner as above based on the pairings in Table 37 with a molar ratio of pTag-Ic-FSb(SRX or SKX or SEX or SDX)-(B-FcIc):pA-HIn(XA or XV or XG):pA-L=3:1:1. A positive control was also set in the same manner as above.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot ((SDS-PAGE plus reducing agent). The results were shown in
The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S, the amino acid at position +2 is E and the amino acid at position −1 is G.
Therefore, all plasmids of the pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(XG) group were selected for the following screening of amino acids at positions −2 and +3 .
The pA-HIn(XG) plasmids were divided into 19 groups based on the amino acid X at position −2, and these 19 groups were co-transfected with all the plasmids of the group pTag-Ic-FSb(SEX)-(B-FcIc) and the plasmid pA-L, respectively for preliminary screening.
Transfection was performed in the same manner as above according to the pairings in Table 38, and 19 transfection groups (i.e., group AG-SE to group YG-SE) were obtained, respectively. The molar ratio of plasmids for transfection was pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(XG):pA-L=3:1:1. A positive control was also set in the same manner as above.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot ((SDS-PAGE plus reducing agent). The results were shown in
The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position −1 is G and the amino acid at position −2 is selected from D, F, G, L, N, G, S, W, and in particular, has the highest splicing efficiency when the amino acid at position −1 is G and the amino acid at position −2 is G or S.
Other solutions of amino acids at positions −1 and −2
Eight expression plasmids pA-HIn (GA or GE or GK or GQ or GR or GW or GT or GP) were co-transfected with expression plasmid pTag-Ic-FSb(SEX)-(B-FcIc) and plasmid pA-L respectively.
Transfection was performed in the same manner as above based on the pairings in Table 39 with a molar ratio of pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(GA or GE or GK or GQ or GR or GW or GT or GP):pA-L=3:1:1. A positive control was also set in the same manner as above.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent). The results were shown in
The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position −2 is G and the amino acid at position −1 is selected from A, K, S, Q, R, W, T.
The expression plasmids pTag-Ic-FSb(SEX)-(B-FcIc) were divided into 19 groups based on the amino acid X at position +3, and were respectively co-transfected with all the plasmids of group pA-HIn(GX) and the plasmid pA-L for primary screening.
Transfection was performed in the same manner as above according to the pairings in Table 40, and 19 transfection groups (i.e., group G-SEA to group G-SEY) were obtained, respectively. The molar ratio of plasmids for transfection was pTag-Ic-FSb(SEA—SEY)-(B-FcIc):pA-HIn(GX):pA-L=3:1:1. A positive control was also set in the same manner as above.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent). The results were shown in
The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S, the amino acid at position +2 is E and the amino acid at position +3 is selected from A, D, E, F, H, I, L, M, S, T, V, W, Y, and in particular, has a very high splicing efficiency when the amino acid at position +3 is selected from H, I, S, T.
In summary, Table 41 showed the novel flanking sequence pairs of split intein NpuDnaE of the present disclosure with an efficient splicing.
Construction of expression plasmids A-Hln, pA-L, plasmid (pTag-Ic-FSb-Pb)
Under the conditions as described above in “Preparation of Recombinant Polypeptides”, as shown in
The molar ratio of plasmids for transfection was pTag-Ic-FSb-(B-FcIc6):pA-HIn(2):pA-L=3:1:1, and the expression product was A61. Positive controls Al and A10 were also set, wherein the plasmids corresponding to Al were pA-HIn(4), pTag-Ic-FSb-(B-FcIc2) and pA-L, and the plasmids corresponding to A10 were pA-HIn(3), pTag-Ic-FSb-(B-FcIcI) and pA-L, with the same molar ratio of plasmids for transfection as described above.
The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were subjected to protein A affinity chromatography and then detected by coomassie brilliant blue staining via SDS-PAGE (with a reducing agent).
The results in
The inteins and flanking sequences corresponding to groups A1, A10 and A61 were shown in Table 42.
Under the same condition as that in Example 1, component expression plasmids of intein NpuDnaE were respectively constructed by pcDNA3.1 based on the structure as shown in Tables 31 and 33.
The expression plasmids of component A in this Example were pA-L and pA-HIn(x), wherein the x represented different numbers.
The expression plasmids of component B′ in this Example were divided into three types: B′-L expression plasmid (pB′-L), B′-H expression plasmid (pB′-H) and B′-FcIc expression plasmid (pB′-FcIcx), wherein, the x represented different numbers. Each component B′ shared the same pB′-L and B′-H expression plasmids.
For the intein NpuDnaE, plasmids pB′-FcIc(1)˜B′-FcIc(7) corresponding to B′-HAb1˜B′-HAb7 were constructed.
Each plasmid pA-HIn(x) and the plasmid pA-L were co-transfected into CHO cells and cultured at 37° C. , with a plasmid molar ratio of pA-HIn:pA-L=1:1, and the cell supernatant was harvested at 10 day after transfection. The supernatant was purified by nickel column chromatography (Jiangsu Qianchun, cat no. A41002-06) to obtain a purified polypeptide fragment of component A.
The plasmid pB′-L, plasmid pB′-H and each plasmid pB+-FcIc were co-transfected into 293E cells and cultured at 37° C. , with a plasmid molar ratio of pB′-L:pB′-H:pB′-FcIc=1:1:3, and the cell supernatant was harvested at 10 day after transfection. The supernatant was purified by nickel column chromatography to obtain a purified polypeptide fragment of component B′.
As shown in Table 43, the obtained polypeptide fragments of component A and component B′ were referred to as Fab4 and HAb4, respectively.
The obtained purified polypeptide fragments of component A and component B′ were subjected to non-reducing SDS-PAGE and coomassie brilliant blue staining, and the results were shown in
E1, E2, and E3 represent elution fractions eluted with different imidazole concentrations (from low to high concentration) during nickel column chromatography. It can be seen from
The obtained purified polypeptide fragments of component A and component B′, Fab4 and HAb4, were dialyzed into a buffer at 4° C. with a 3 kD dialysis bag (purchased from Sigma) with a concentration of 1 to 10 micromolar/L. The buffer included 10 to 50 mM Tris/HCl (pH 7.0-8.0), 100 to 500 mM NaCl, and 0 to 0.5 mM EDTA. Then, the components A (Fab4) and B′ (Hab4) were respectively mixed according to corresponding serial numbers in a molar ratio of 1:10 to 10:1, and DTT was added to be 0.5 to 5 mM, then the mixture was incubated overnight at 37° C.
The obtained spliced product polypeptides were subjected to SDS-PAGE and coomassie brilliant blue staining, and the results were shown in
In
It can be seen from
The biological activity detection based on double antigen sandwich ELISA was performed for the recombinant polypeptide “SPLICING 2”.
1) Preparation of antigen: for the proteins PD-L1 and CD38, only the extracellular domain was selected for construction, and an expression plasmid with His-tag was constructed by using the vector pcDNA3.1.
After construction, 293E cells were used for transient transfection, and a two-step purification including nickel column purification and molecular sieve purification was carried out. After purification, an antigen protein with a purity of no less than 95% detected by SDS-PAGE was obtained.
PD-L1 protein was labeled with horseradish peroxidase (HRP).
2) Coating of the first antigen: the concentration of CD38 protein was adjusted to 2 μg/ml, and an microtiter plate was coated with the CD38 protein-containing liquid at 100 μl/well, 4° C. overnight; the supernatant was discarded and 250 μl blocking solution (3% BSA in PBS) was added to each well;
3) addition of antibody: according to the experimental design, the operation was performed at room temperature. The antibody was diluted in a gradient with 1% BSA in PBS. For example, the initial concentration of antibody for dilution was 20 μg/mL, and the antibody was diluted by 2-fold with 5 gradients. The diluted antibody was added into wells of microtiter plate at 200 μl/well, incubated at room temperature for 2 hours, and then the supernatant was discarded;
4) washing: the plate was washed by 200 μl/well PBST (PBS containing 0.1% Tween20) for 3 times;
5) incubation of secondary antigen: a diluted secondary antigen (HRP-labeled PD-L1 protein) was added with a volume of 100 μl/well and incubated at room temperature for 1 hour, wherein the secondary antigen was diluted at 1:1000 and the diluent was 1% BSA in PBS ;
6) washing: the plate was washed with 200 μl/well PBST for 5 times;
7) color-developing: TMB color-developing solution (prepared from A and B color-developing solutions purchased from Wuhan Boster Company, and mixed according to A:B=1:1, ready to use) was added at 100 μl/well, and the color-developing was performed at 37° C. for 5 min;
8) 2M HCl stopping solution was added at 100 μl/well, and then the microplate reader should be read at 450 nm within 30 minutes.
It can be seen from
The results prove that the spliced product Fab4+HAb4 obtained by using the intein and the novel flanking sequence pair contained therein of the present disclosure has a good bispecific antibody activity.
Based on the splicing principle of intein, according to the molecular weight of spliced product obtained in the present disclosure and the results of double-antigen sandwich ELISA, it can be speculated that an effective bispecific antibody with a natural IgG-like structure was obtained in the present disclosure. The test results confirmed that the structure of the bispecific antibody was a heterodimeric IgG structure composed of two different heavy chains and two different light chains, rather than a mixture of homodimeric IgG structure composed of two identical heavy chains and two identical light chains.
The present disclosure provides methods for preparing recombinant polypeptides, particularly bispecific antibodies, by using split inteins with novel flanking sequence pairs. The split inteins with novel flanking sequence pairs of the present disclosure can be widely used in the preparation of recombinant polypeptides in the fields of medicine and bioengineering, especially in the field of antibodies, especially in the preparation of bispecific antibodies. The bispecific antibody prepared by using the split inteins with novel flanking sequence pairs of the present disclosure does not have a non-natural domain, has a structure closely similar to that of natural antibody (IgA, IgD, IgE, IgG or IgM), and has a Fc domain. The bispecific antibody has a complete structure and good stability, and can retain or remove CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) or FcRn (Fc receptor)-binding activity according to different IgG subclasses.
The bispecific antibody prepared by the method of the present disclosure has the following advantages: the bispecific antibody has a long half-life in vivo and low immunogenicity, and does not introduce any form of linkers; has an improved stability, and a reduced in vivo immune response. The bispecific antibody prepared by the method of the present disclosure has the same glycosylation modification as that of wild-type IgG, has better biological function, is more stable, and has a long half-life in vivo; the in vitro splicing method by using inteins can completely avoid the problems of heavy chain mismatch and light chain mismatch commonly found in traditional methods.
The preparation method for bispecific antibodies of the present disclosure can also be used to produce humanized bispecific antibodies and bispecific antibodies with complete human sequences. The sequence of such an antibody prepared by the method of the present disclosure is more similar to that of a human antibody, which can effectively reduce the immune response. The preparation method for bispecific antibodies of the present disclosure is not limited by antibody subclasses (IgG, IgA, IgM, IgD, IgE, and light chain κ and λ types) and can be used to construct any bispecific antibody.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910850928.1 | Sep 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/114274 | 9/9/2020 | WO |
