Bispecific antibodies or antibody mixtures with a common light chain
阅读说明：本技术 具有共同轻链的双特异性抗体或抗体混合物 (Bispecific antibodies or antibody mixtures with a common light chain ) 是由 徐霆 汪皛皛 李倩 逄敏洁 韩莉 张庆青 于 2015-01-08 设计创作，主要内容包括：本发明涉及双特异性抗体或抗体混合物,具体涉及具有共同轻链的双特异性抗体或抗体混合物,以及所述双特异性抗体或抗体混合物的制备方法。本发明还涉及编码所述双特异性抗体或抗体混合物的核酸分子、含有该核酸分子的重组载体和重组细胞,以及所述双特异性抗体或抗体混合物的检测和定量方法。本发明通过共同轻链技术制备双特异性抗体或抗体混合物,其制备方法简单、可控,可以避免双特异性抗体中轻链的错配,对于抗体混合物,其可以在同一宿主细胞中表达,降低了混合细胞群培养的难度,更有利于放大生产。(The present invention relates to bispecific antibodies or antibody mixtures, in particular to bispecific antibodies or antibody mixtures having a common light chain, and methods for the preparation of said bispecific antibodies or antibody mixtures. The invention also relates to nucleic acid molecules encoding said bispecific antibodies or antibody mixtures, recombinant vectors and recombinant cells containing said nucleic acid molecules, and methods for the detection and quantification of said bispecific antibodies or antibody mixtures. The invention prepares the bispecific antibody or the antibody mixture by the common light chain technology, the preparation method is simple and controllable, the mismatching of the light chain in the bispecific antibody can be avoided, and the antibody mixture can be expressed in the same host cell, thereby reducing the difficulty of culturing mixed cell groups and being more beneficial to the amplification production.)
1. A method of detecting and/or quantifying whether an antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof, comprising the steps of:
1) preparing a specific antigen 1 capable of binding to the antigen-binding portion 1 of the bispecific antibody or antigen-binding portion thereof and not to the antigen-binding portion 2, and a specific antigen 2 capable of binding to the antigen-binding portion 2 and not to the antigen-binding portion 1, respectively;
2) taking the specific antigen 1 to coat the enzyme label plate, adding the antibody or the antigen binding part thereof, and then adding the labeled specific antigen 2; or, taking the specific antigen 2 to coat the enzyme label plate, adding the antibody to be detected, and then adding the marked specific antigen 1;
then adding a detection molecule capable of binding to the labeled specific antigen, said detection molecule carrying a detectable label;
3) when the result of step 2) is positive and the reaction of step 2) is concentration-dependent, the antibody or antigen-binding portion thereof is judged to be a bispecific antibody or antigen-binding portion thereof.
2. The method of claim 1, quantifying the bispecific antibody or antigen-binding portion thereof according to a positive value of step 3).
3. A method of detecting whether a mixture of antibodies or antigen-binding portions thereof is a homodimeric protein, the mixture comprising two antibodies, antibody 1 and antibody 2 or antigen-binding portions thereof, the method comprising the steps of:
1) preparing specific antigen 1 capable of binding to antibody 1 but not to antibody 2, and specific antigen 2 capable of binding to antibody 2 but not to antibody 1, respectively;
2) taking a specific antigen 1 coated enzyme label plate, adding a mixture to be detected, and adding a marked specific antigen 1; or, taking a specific antigen 2 coated enzyme label plate, adding the mixture to be detected, and then adding the marked specific antigen 2;
adding a detection molecule capable of being combined with the marked specific antigen, wherein the detection molecule is provided with a detectable mark;
3) taking a specific antigen 1 to coat an enzyme label plate, adding a mixture to be detected, and adding a marked specific antigen 2; or, taking a specific antigen 2 to coat the enzyme label plate, adding the mixture to be detected, and then adding the marked specific antigen 1;
adding a detection molecule capable of being combined with the labeled specific antigen, wherein the detection molecule is provided with a detectable label;
4) judging that the mixture contains homodimer protein and does not contain heterodimer protein when the result of step 2) is positive and the reaction of step 2) has concentration dependence while the result of step 3) is negative;
judging that the mixture contains homodimer protein and heterodimer protein when the result of the step 2) is positive and the result of the step 3) is positive.
4. The method of any one of claims 1-3, wherein the specific antigen 1 and/or specific antigen 2 is a variant protein of the extracellular region of the HER2 protein having a mutation selected from the group consisting of:
1) a mutation of glutamic acid at position 558 and a mutation of phenylalanine at position 573;
2) a mutation of serine at position 288 and a mutation of histidine at position 296.
5. A nucleic acid molecule encoding a variant protein of the extracellular region of HER2 protein of claim 4.
6. A recombinant vector comprising the nucleic acid molecule of claim 5.
7. A recombinant cell comprising the recombinant vector of claim 6 or the nucleic acid molecule of claim 5.
8. A method of making a bispecific antibody or antigen-binding portion thereof based on two monoclonal antibodies or antigen-binding portions thereof directed against different epitopes comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of the light chain of one of the monoclonal antibodies;
2) respectively constructing heavy chain sequences and the common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the bispecific antibody or the antigen binding part thereof.
9. A method of preparing a mixture comprising at least two monoclonal antibodies, or antigen binding portions thereof, comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of the light chain of one of the monoclonal antibodies;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the mixture.
10. A detection molecule that binds to the specific antigen of claim 1, the detection molecule being HRP-labeled streptavidin.
Technical Field
The present invention relates to bispecific antibodies or antibody mixtures and methods for making the bispecific antibodies or antibody mixtures. The invention also relates to nucleic acid molecules encoding said bispecific antibodies or antibody mixtures, recombinant vectors and recombinant cells containing said nucleic acid molecules, and methods for the detection and quantification of said bispecific antibodies or antibody mixtures.
Background
Monoclonal antibody drugs have grown rapidly in nearly fifteen years, becoming a growing point in the pharmaceutical industry. Since 1996, a total of about 30 monoclonal antibody drugs were approved for marketing. Nine of these monoclonal antibody drugs are sold annually in excess of one billion dollars. In 2010, the total sales of monoclonal antibody drugs exceed 300 billion dollars, and the annual growth rate exceeds 10%. Due to the strong target specificity of monoclonal antibodies, only a single target can be inhibited. In a variety of diseases, including tumors, autoimmunity, multiple signaling pathways need to be inhibited to avoid compensatory effects. For viral infectious diseases, inhibition of multiple antigenic sites is often required to prevent escape due to the high mutation rate of the virus. Therefore, there are several alternatives to solve such problems as follows. An alternative is to use polyclonal antibodies, or to obtain heterodimers such as bispecific antibodies by engineering the Fc region of the antibody, which may be active against at least two different antigens or different binding sites of the same antigen. Yet another approach is to use a mixture of antibodies to treat, which may comprise two or more antibodies directed against different epitopes on the same target, or a mixture of antibodies directed against different targets.
Bispecific antibodies (BsAbs) are immunoglobulin molecules that contain two distinct ligand binding sites. Instead of the same sequence in both arms of the classical antibody Fab, two different Fab sequences are used, so that the arms of the Y-form can bind different epitopes. The use of bispecific antibodies in cancer therapy has been reviewed in various publications (Carter 2001; Chames and Baty 2009; Chames and Baty 2009). One arm of BsAbs can be connected with related antigens on the surface of tumor cells, and the other arm can trigger immune effector cells to further kill cells, so that cancer tumor cells can be killed by the immune system.
For the preparation of bispecific antibodies, as early as 90 s, Carter et al successfully realized bispecific antibodies by engineering part of the amino acids of the antibody heavy chain using the "knob-hole" (knob-hole) model (Ridgway, Prestaet al 1996; Carter 2001). However, in their findings, the "pore-pore" model is still not sufficient to block homodimer formation, leaving behind approximately 5% homodimers. The group then further increased the heterodimer content by methods such as random mutation-phage display, but did not solve the underlying problem.
The inventors of the present invention successfully solved the 5% homodimer residual in the "handle-pore" model by modifying the CH 3-related amino acids of Fc through a charged amino acid interaction network to weaken the domain self-interaction (favoring the formation of homodimers) and enhance the interaction between domains (favoring the formation of heterodimers), and related methods have been patented (publication No.: CN102558355 a).
The development of the mixed antibody production platform was relatively early compared to the heterodimer platform technology. Of most interest is the antibody cocktail technique by Symphogen a/S, denmark. The technology firstly obtains a plurality of antibodies aiming at the same target through screening of an antibody screening platform, and then cell strain construction is carried out aiming at each antibody. And then mixing seed solutions cultured by different cells in a shake flask, finally carrying out gradual enlarged culture and amplification on the mixture, and optimizing a purification process to obtain a final product. Although the use of this method allows multiple antibodies to be obtained directly from a single recombinant production process by culturing a mixed population of cells, there are still some potential problems with this approach due to the difficulty of controlling the culture of the mixed population of cells and the resulting complexity of scale-up production.
The applicant has invented a method for producing a mixture comprising two or more homodimeric proteins or antibodies in a single recombinant cell by mutating the Fc part to alter the Fc direct interaction. The scheme avoids the potential difficulties of process control and amplification brought by mixed cell culture, and provides a more economical and effective preparation and production mode of the antibody mixture. This scheme has also been patented (publication No.: CN 103388013A).
However, in any of the above methods, when a bispecific antibody or a mixture of antibodies is prepared using a whole antibody framework, mismatches between light chain and heavy chain may occur, and thus the activity of the antibody is affected, and a currently well-established method in the art is crosssmab developed by roche (genentech), in which a light chain-heavy chain sequence is prevented from being mismatched with another light chain-heavy chain sequence by replacing one group of Fab with another (patent nos. US20090162359, US 20120164726). Although the method can solve most of heavy chain-light chain mismatching problems, new problems are brought by artificial modification of heavy and light chain sequences, such as dissociation of light chains, increase of polymer content, and influence on recognition of antigen epitopes by some Fab sequences.
Herceptin (Herceptin, also called Trastuzumab, Trastuzumab), the first therapeutic monoclonal antibody that showed substantial efficacy in breast cancer, was a human anti-human epidermal growth factor receptor 2(HER2) monoclonal antibody that acted on the HER2-Neu surface protein of breast cancer cells, interfering with the biological progression of cancer cells, and ultimately causing their death. The main suitable population of Herceptin (Herceptin) is breast cancer patients with over-expression of HER2 (immunohistochemistry 3+ or fluorescence in situ hybridization FISH positive), and this population accounts for about 20-30% of all breast cancer patients.
Pertuzumab (pertuzumab) is a recombinant monoclonal antibody that binds to the extracellular domain ii region of the HER-2 receptor, inhibits dimer formation, and inhibits the receptor-mediated signal transduction pathway (Agus DB, Gordon MS, TaylorC, et al 2005). This may explain, in part, why pertuzumab inhibits the growth of HER-2 overexpressing tumors, whereas trastuzumab binds to the extracellular iv region of the HER-2 receptor, and dimer formation does not involve the iv region, and therefore trastuzumab is effective only in breast cancer patients with HER-2 overexpression. Phase II clinical studies of pertuzumab on HER-2 low-expression advanced breast cancer are currently underway. Studies by Baselga (Baselga J, et al 2007) et al show that pertuzumab in combination with herceptin (trastuzumab) has demonstrated potent anti-tumor activity in patients with HER-2 positive breast cancer. This study showed that 1/5 patients were effective (tumor shrinkage or disappearance) for pertuzumab treatment and that 1/5 patients remained stable for more than 6 months. Results of phase III clinical trials of pertuzumab therapy for breast cancer show that the drug can greatly prolong the progression-free survival of ERBB2 positive metastatic breast cancer patients.
Previously, roche published the results of a recent experiment that was a phase II neoadjuvant therapy study evaluating the efficacy of pertuzumab and herceptin (trastuzumab) in combination with chemotherapy (docetaxel) for the treatment of female patients with early proto-oncogene human epidermal growth factor receptor 2(HER2) positive breast cancer. Data published by the american cancer research institute (CTRC-AACR) on the San Antonio Breast Cancer Symposium (SABCS) show that the complete breast tumor elimination rate (complete in 45.8% of cases) of the two-antibody combination docetaxel treatment in the pre-operative neoadjuvant therapy was significantly improved by more than 50% over the complete in 29.0 case ratio of herceptin combination docetaxel. Pertuzumab and pertuzumab in combination with docetaxel did not result in side effects or significant increase in cardiac risk compared to herceptin and chemotherapy.
The invention takes pertuzumab and trastuzumab as examples, prepares the bispecific antibody and the antibody mixture which have the functions of pertuzumab and trastuzumab simultaneously, and finds a new method for preparing the bispecific antibody or the antibody mixture with the right combination of the light chain and the heavy chain on the basis.
Disclosure of Invention
The inventors of the present invention have surprisingly found, by repeated experiments, that it is possible to replace the light chain in two original antibodies or antibody mixtures with a common light chain to obtain a bispecific antibody or antibody mixture with a common light chain, which is capable of achieving the correct combination of light chain and heavy chain and which has good binding properties, biological activity and stability compared to the two original antibodies, even being superior in biological activity to the original antibodies.
The first aspect of the present invention relates to a bispecific antibody or an antigen-binding portion thereof characterized in that the two light chains of said bispecific antibody or antigen-binding portion thereof have the same sequence.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, the heavy chain of which is capable of binding correctly to said light chain under physiological conditions or in vitro protein expression conditions, respectively.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the light chain of the bispecific antibody or antigen-binding portion thereof has been modified from two original monoclonal antibodies, said light chain having a sequence that differs from the light chain of at least one of the two original monoclonal antibodies.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, the heavy chain Fc fragment of which is engineered to favor the formation of heterodimeric proteins.
In an embodiment of the invention, the two original monoclonal antibodies are the antibody to pertuzumab and the antibody to herceptin.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the light chain is selected from the group consisting of the light chains of pertuzumab or trastuzumab, or a mutant thereof.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, the heavy chain (including the variable and constant regions) of which can be identical to, or engineered from, the two original monoclonal antibodies; such as the modification of the heavy chain Fc segment to favor the formation of heterodimeric proteins.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 6 at amino acids 1 to 107.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the sequence of the light chain constant region comprises SEQ ID NO:1, amino acids 108 to 214.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the heavy chain variable regions are those of pertuzumab and trastuzumab, respectively.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the sequences of the two heavy chain variable regions comprise the amino acid sequences as set forth in SEQ ID NOs: 23 and SEQ ID NO: 24, or a sequence shown in fig. 24.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, wherein the sequences of the two heavy chain Fc segments comprise the amino acid sequences as set forth in SEQ ID NOs: 25 and SEQ ID NO: 26, or a sequence shown in fig. 26.
The bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, the sequences of the two heavy chains of which comprise the amino acid sequences as set forth in SEQ ID NOs: 19 and SEQ ID NO: 20, or a sequence shown in fig. 20.
The second aspect of the present invention relates to a mixture of antibodies or antigen-binding portions thereof capable of being correctly produced in one cell, the mixture comprising at least two antibodies or antigen-binding portions thereof having a common light chain.
The mixture according to any of the second aspect of the invention, wherein the heavy chain of said antibody, or antigen binding portion thereof, is capable of binding correctly to said light chain under physiological conditions or in vitro protein expression status, respectively.
According to the mixture of any of the second aspects of the invention, the bispecific antibody or antigen-binding portion thereof is derived from two original monoclonal antibodies, the heavy chain variable region sequence and/or the CH1 domain sequence of the bispecific antibody or antigen-binding portion thereof being identical to the original monoclonal antibodies.
According to the mixture of any of the second aspects of the invention, the heavy chain (including the variable and constant regions) of the bispecific antibody or antigen-binding portion thereof may be identical to, or engineered from, the two original monoclonal antibodies; such as the modification of the heavy chain Fc segment to favor the formation of homodimeric proteins.
In an embodiment of the invention, the two original monoclonal antibodies are the antibody to pertuzumab and the antibody to herceptin.
The mixture according to any of the second aspect of the invention, wherein the light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
The mixture according to any of the second aspect of the invention, wherein the light chain is selected from the group consisting of the light chain of pertuzumab or trastuzumab, or a mutant thereof.
The mixture according to any of the second aspects of the invention, wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 6 at amino acids 1 to 107.
The mixture according to any of the second aspects of the invention, wherein the sequence of the light chain constant region comprises SEQ ID NO:1, amino acids 108 to 214.
The mixture according to any of the second aspect of the invention, wherein the heavy chain variable regions of the antibodies or antigen-binding portions thereof are the heavy chain variable regions of pertuzumab and trastuzumab, respectively.
The mixture according to any of the second aspects of the invention, wherein the sequences of the two heavy chain variable regions comprise the sequences as set forth in SEQ ID NOs: 23 and SEQ ID NO: 24, or a sequence shown in fig. 24.
The mixture according to any of the second aspects of the invention, wherein the sequences of the two heavy chain Fc-segments comprise the amino acid sequences as set forth in SEQ ID NOs: 27 and SEQ ID NO: 28, and (b) the sequence shown in figure 28.
The mixture according to any of the second aspect of the invention, wherein the heavy chain sequences of the antibodies or antigen binding portions thereof comprise the amino acid sequences as set forth in SEQ ID NOs: 21 and SEQ ID NO: 22, or a sequence shown in fig. 22.
A third aspect of the invention relates to a variant protein of the extracellular region of the HER2 protein, which has a mutation selected from the group consisting of:
1) a mutation of glutamic acid at position 558 and a mutation of phenylalanine at position 573;
2) a mutation of serine at position 288 and a mutation of histidine at position 296.
In one embodiment of the invention, the glutamic acid at position 558 is mutated to alanine.
In one embodiment of the invention, phenylalanine at position 573 is mutated to alanine.
In one embodiment of the invention, the serine at position 288 is mutated to an alanine.
In one embodiment of the invention, histidine at position 296 is mutated to alanine.
In one embodiment of the invention, the HER2 variant protein comprises a sequence selected from the group consisting of SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.
In an embodiment of the invention, the sequence of the extracellular region of the wild-type HER2 protein is as set forth in SEQ ID NO: 18, respectively.
A fourth aspect of the invention relates to a nucleic acid molecule encoding the bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention or the antibody or antigen-binding portion thereof described in the mixture according to any one of the second aspect, or a partial sequence of said antibody or antigen-binding portion thereof, or encoding the HER2 variant protein according to any one of the third aspect.
The fifth aspect of the present invention relates to a recombinant vector comprising the nucleic acid molecule of any one of the fourth aspects of the present invention.
The sixth aspect of the present invention relates to a recombinant cell comprising the recombinant vector of any one of the fifth aspects of the present invention or the nucleic acid molecule of any one of the fourth aspects.
The seventh aspect of the present invention relates to a method for producing a bispecific antibody or an antigen-binding portion thereof based on two monoclonal antibodies or antigen-binding portions thereof directed against different epitopes, comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of one of the monoclonal antibody light chains;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors; preferably, the heavy chain sequence, in particular the Fc-segment, is mutated to favor the binding of Fc-segments with different heavy chains;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the bispecific antibody or the antigen binding part thereof.
The method according to any one of the seventh aspects of the present invention, wherein the common light chain is obtained by determining the interfacial amino acids in contact between the two monoclonal antibodies and the antigen or the epitope of the antigen, determining the difference amino acids when the interfacial amino acids in contact between the common light chain and the antigen or the epitope of the antigen are compared with the amino acids at the corresponding positions of the light chains of the other monoclonal antibodies when the light chain of any one of the monoclonal antibodies is used as the candidate common light chain, and selecting the light chain with the smaller number of the difference amino acids as the common light chain; preferably, the common light chain is further mutated to obtain a more cohesive common light chain.
An eighth aspect of the present invention relates to a method of preparing a mixture comprising at least two monoclonal antibodies or antigen-binding portions thereof, said method comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of one of the monoclonal antibody light chains;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors; preferably, the heavy chain sequence, in particular the Fc-segment, is mutated to favor the binding of the Fc-segment with the same heavy chain;
3) the two recombinant expression vectors are transferred into the same host cell and expression is induced to obtain a mixture of antibodies or antigen-binding portions thereof.
The method according to any one of the eighth aspect of the present invention, wherein the common light chain is obtained by determining the interfacial amino acids in contact between the two monoclonal antibodies and the antigen or the epitope, then determining the difference amino acids when the interfacial amino acids in contact between the common light chain and the antigen or the epitope are compared with the amino acids at the corresponding positions of the light chain of the other monoclonal antibody when the light chain of any one of the monoclonal antibodies is used as the candidate common light chain, and selecting the light chain with less number of the difference amino acids as the common light chain; preferably, the common light chain is further mutated to obtain a more cohesive common light chain.
The present invention also relates to a method of detecting and/or quantifying whether an antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof, comprising the following steps (see schematic diagram of fig. 25):
1) preparing a specific antigen 1 capable of binding to the antigen-binding portion 1 of the bispecific antibody or antigen-binding portion thereof and not binding to the antigen-binding portion 2, and a specific antigen 2 capable of binding to the antigen-binding portion 2 and not binding to the antigen-binding portion 1, respectively;
2) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding an antibody to be detected, reacting for a period of time, adding a labeled specific antigen 2 (or the specific antigen 1), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive or negative according to the reading of a detection principle;
3) when the reaction is positive and the reaction is concentration dependent, determining that the antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof; optionally, the bispecific antibody or antigen-binding portion thereof is further quantified based on the resulting positive values.
In the present invention, the antigen-binding portions 1 and 2 refer to two portions of the bispecific antibody or antigen-binding portion thereof, respectively, that bind to different antigens or epitopes, respectively; in an embodiment of the invention, the antigen-binding portions 1 and 2 are engineered on the basis of two original antibodies, respectively, and the antigen-binding portions 1 and 2 bind to the same antigen or epitope as the two original antibodies, respectively.
In an embodiment of the invention, the specific antigen 1 and specific antigen 2 are hemm 1 and hemm 2.
In an embodiment of the invention, the labeled specific antigen is a specific antigen labeled with biotin.
In an embodiment of the invention, the detection molecule refers to a substrate molecule that can be used for detection, for example, HRP-labeled streptavidin.
The present invention also relates to a method of detecting whether a mixture of antibodies or antigen-binding portions thereof, which mixture comprises two antibodies (antibody 1 and antibody 2) or antigen-binding portions thereof, is a homodimeric protein, comprising the following steps (see schematic of fig. 26):
1) preparing specific antigen 1 capable of binding to antibody 1 but not to antibody 2, and specific antigen 2 capable of binding to antibody 2 but not to antibody 1, respectively;
2) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding a mixture to be detected, reacting for a period of time, adding a labeled specific antigen 1 (or a specific antigen 2), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive or negative according to the reading of a detection principle;
3) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding the enzyme label plate to be detected, reacting for a period of time, adding a labeled specific antigen 2 (or the specific antigen 1), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive reaction or negative reaction according to the reading of a detection principle;
4) when the reaction in the step 2) is positive and has concentration dependence, and the reaction in the step 3) is negative, judging that the mixture is homodimer protein and does not contain heterodimer protein; and when the reaction in the step 2) is positive and the reaction in the step 3) is positive, judging that the mixture contains both homodimer protein and heterodimer protein.
In an embodiment of the invention, specific antigen 1 and specific antigen 2 are hemm 1 and HERm 2.
In an embodiment of the invention, the labelled specific antigen is an antigen labelled with biotin.
In an embodiment of the invention, the detection molecule refers to a substrate molecule that can be used for detection, for example, HRP-labeled streptavidin.
The invention also relates to compositions (e.g., pharmaceutical compositions) comprising a bispecific antibody or antigen-binding portion thereof according to any one of the first aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
The invention also relates to a composition (e.g. a pharmaceutical composition) comprising a mixture according to any of the second aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
The invention also relates to a kit comprising a bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention, and optionally a buffer or instructions.
In an embodiment of the invention, the kit is for diagnosing a HER2 positive tumor (e.g. breast cancer, gastric cancer).
The invention also relates to a kit comprising a mixture according to any of the second aspects of the invention, and optionally a buffer or instructions.
In an embodiment of the invention, the kit is for diagnosing a HER2 positive tumor (e.g. breast cancer, gastric cancer).
The invention also relates to the use of a bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention in the manufacture of a medicament for the prevention and/or treatment of HER2 positive tumors (e.g. breast cancer, gastric cancer).
The invention also relates to the use of a mixture according to any of the second aspects of the invention for the preparation of a medicament for the prevention and/or treatment of HER2 positive tumors (e.g. breast cancer, gastric cancer).
The invention also relates to the use of a bispecific antibody or antigen-binding portion thereof according to any one of the first aspect of the invention in the manufacture of a reagent or kit for the diagnosis of HER2 positive tumors (e.g. breast cancer, gastric cancer).
The invention also relates to the use of a mixture according to any of the second aspects of the invention for the preparation of a reagent or kit for the diagnosis of HER2 positive tumors (e.g. breast cancer, gastric cancer).
The invention also relates to the use of a variant protein of the extracellular region of HER2 protein according to any one of the third aspect of the invention for the detection of a bispecific antibody or antigen-binding portion thereof according to any one of the first aspect or for the detection of a mixture according to any one of the second aspect.
The present invention also relates to a method of preventing and/or treating HER2 positive tumors (e.g. breast cancer, gastric cancer), said method comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of the bispecific antibody of any one of the first aspect of the invention, or an antigen-binding portion thereof.
The present invention also relates to a method for the prevention and/or treatment of HER2 positive tumors (e.g. breast cancer, gastric cancer), said method comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a mixture according to any of the second aspects of the present invention.
The present invention is further described below.
In the present invention, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.
In the present invention, the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., HER2) to which the antibody binds, competing with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, ch.7(Paul, w., e)d., 2 nd edition, Raven Press, n.y. (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen-binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. In some cases, the antigen binding portion includes Fab, Fab ', F (ab')2Fd, Fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capability on the polypeptide. The antigen-binding portion of an antibody (e.g., the antibody fragment described above) can be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and specifically screened for the antigen-binding portion of the antibody in the same manner as for an intact antibody.
In the present invention, the term "Fd fragment" means a fragment consisting of VHAnd CH1 domain; the term "Fv fragment" means a V consisting of a single arm of an antibodyLAnd VHAntibody fragments consisting of domains; the term "dAb fragment" means a fragment consisting of VHAntibody fragments consisting of domains (Ward et al, Nature 341:544-546 (1989)); the term "Fab fragment" means a fragment consisting of VL、VH、CLAnd CH1 domain; the term "F (ab')2By fragment "is meant an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.
In the present invention, the term "antibody Fc fragment" is a term well known to the skilled artisan and is defined based on the papain cleavage of antibodies and refers to the carboxy-terminal end of, or a portion of, a human immunoglobulin chain constant region, particularly an immunoglobulin heavy chain constant region. For example, an immunoglobulin Fc region may comprise two or more domains of heavy chains CH2, CH3, CH4 in combination with an immunoglobulin hinge region. Depending on the amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classes, mainly to the class 5 immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), such as IgG-l, IgG-2, IgG-3, IgG-4, IgA-l and IgA-2. The selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the purview of those skilled in the art.
In an embodiment of the invention, the antibody Fc fragment used in the present invention comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, in particular human IgG1 Fc.
In the present invention, the term "bispecific antibody" is capable of binding to two antigens or epitopes, respectively, which comprises a light chain and a heavy chain of an antibody specifically binding to a first antigen, and a light chain and a heavy chain of an antibody specifically binding to a second antigen.
In the present invention, the term "epitope" or "antigenic epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.
In the present invention, 20 kinds of conventional amino acids and abbreviations thereof follow conventional usage. See Immunology-A Synthesis (2 nd edition, E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
In the present invention, the light chain sequences of two monoclonal antibodies (i.e., original antibodies) against different antigens or epitopes are analyzed and verified to obtain a common light chain capable of binding to the heavy chains of the two monoclonal antibodies. After the common light chain and the common heavy chain are combined, the antigen or the antigen epitope of the original monoclonal antibody can be still specifically combined.
In the present invention, the common light chain can be used in expressing bispecific antibodies, as well as in expressing mixtures containing both antibodies; in a bispecific antibody, the antibody comprises a light chain and a heavy chain capable of binding to a first antigen, and a light chain and a heavy chain capable of binding to a second antigen, wherein the two light chain sequences are identical, i.e. the common light chain; in the antibody mixture, each antibody contains two light chains and two heavy chains, wherein the light chains have the same sequence, i.e., the common light chain.
In the present invention, the light chain types (κ or λ) of the two original antibodies may be the same or different, and when the types are the same, only the variable region of the light chain may be mutated to obtain a common light chain; when the types are different, it is necessary to mutate both the variable and constant regions of the light chain to obtain a common light chain.
In the present invention, the heavy chain types of the two original antibodies may be the same or different, and preferably the types are the same. In an embodiment of the invention, the variable region of the heavy chain sequence and the sequence of the CH1 domain are not changed when preparing the bispecific antibody and antibody mixture compared to the original antibody.
In the present invention, both arms of the bispecific antibody or the antibody in the mixture containing both antibodies are derived from two original monoclonal antibodies. In constructing a bispecific antibody or antibody mixture, only the sequence of the light chain variable region need be changed to obtain a common light chain, and not the sequence of the heavy chain variable region. That is, in the constructed bispecific antibodies and antibody mixtures, the heavy chain variable region sequence of the antibody may be identical to the original antibody, but the light chain variable region sequence is different from the original antibody. That requires a method of altering the sequence of the heavy chain variable region, which is identical to the original antibody, differs from the present invention even though the light chain in the bispecific antibody or antibody mixture obtained is identical.
In the present invention, two original monoclonal antibodies can be selected according to different needs or purposes, for example, two monoclonal antibodies directed to different epitopes of the same antigen can be selected, or one of the antibodies can be linked to a related antigen on the surface of a tumor cell, and the other antibody can trigger immune effector cells to further kill the cells.
In embodiments of the invention, in the preparation of bispecific antibodies, heavy chains such as Fc segments can be engineered using existing techniques to favor heterodimeric protein formation upon antibody expression.
In embodiments of the invention, the heavy chain, e.g., the Fc region, may be engineered using existing techniques to favor the formation of homodimeric proteins upon antibody expression when preparing the antibody mixture.
In the present invention, techniques for engineering the Fc portion of an antibody heavy chain to favor homodimeric or heterodimeric proteins are well known in the art, e.g., see Ridgway, Presta et al 1996; carter 2001, patent CN102558355A, patent CN 103388013 a.
In embodiments of the invention, techniques for fusing polypeptides having different antigen recognition epitopes include, but are not limited to, heterodimeric Fc fusion techniques as in the specific examples, but may also be "Fab" techniques, see fig. 1.
In the embodiment of the present invention, the heterodimeric Fc fusion technology used in the present invention can be a "handle" -hole "model, or a" charge repulsion "model, but is not limited to these two models.
In embodiments of the invention, the platform of the antibody mixture that can be produced in a single recombinant cell production for use in the invention may be, but is not limited to, a "charge repulsion" model.
In some embodiments of the invention, when used to prepare a bispecific antibody or mixture of antibodies, the nucleic acid molecule encodes the light and/or heavy chain of an antibody directed against a first antigen, or encodes the light and/or heavy chain of an antibody directed against a second antigen. In an embodiment of the invention, the light chain is a common light chain; in an embodiment of the invention, the Fc region of the heavy chain is engineered.
In some embodiments of the invention, the vector may be a cloning vector or an expression vector. The cloning vector is used for cloning relevant fragments of the antibody; the expression vector is used for expressing a bispecific antibody or an antibody mixture. Vectors suitable for antibody expression may be selected according to common general knowledge in the art. In a specific embodiment of the invention, the expression vector is pcDNA4m which is a vector obtained by modifying the vector pcDNA 4/myc-HisA.
In some embodiments of the invention, the expression vector comprises a nucleic acid molecule encoding a light chain and/or a heavy chain of an antibody directed against a first antigen, or comprises a nucleic acid molecule encoding a light chain and/or a heavy chain of an antibody directed against a second antigen.
In some embodiments of the invention, the host cell is a host cell suitable for expression of the antibody, e.g., a prokaryotic cell (e.g., e.coli) or a eukaryotic cell; the eukaryotic cell is, for example, a yeast cell, a plant cell, or a mammalian cell, such as a CHO cell, HEK293 cell, or myeloma cell, among others.
In some embodiments of the invention, the host cell contains both an expression vector expressing the light and/or heavy chain of an antibody directed to a first antigen and an expression vector expressing the light and/or heavy chain of an antibody directed to a second antigen; in a specific embodiment of the invention, the light chain is a common light chain; in an embodiment of the invention, the Fc region of the heavy chain is engineered. When used to express bispecific antibodies, the light and heavy chains of antibodies directed against different antigens are more easily combined by engineering of the Fc segment to form bispecific antibodies; when used to express a mixture of antibodies, the light and heavy chains of antibodies directed to the same antigen are more readily combined by engineering the Fc region to form a mixture of antibodies.
Bispecific antibodies or antibody mixtures can be purified from host cells using standard experimental means. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration. In an embodiment of the invention, the bispecific antibody and antibody mixture is purified by ProteinA affinity chromatography.
In the present invention, the bispecific antibody or antigen-binding portion thereof or mixture thereof of the present invention can also be used in combination with a chemotherapeutic agent and/or other antibody, and thus the composition of the present invention can also contain a chemotherapeutic agent and/or other antibody.
In the present invention, the chemotherapeutic agents include, but are not limited to: doxorubicin (Adriamycin), cyclophosphamide, and taxanes [ paclitaxel (Taxol) and docetaxel (Taxotere) ], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (runde, froron, arninoxin), 5-FU plus leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), and the like, or combinations thereof.
In the invention, a HER2 protein mutant which can only be specifically combined with one of pertuzumab and herceptin is prepared by mutating HER2 protein. In an embodiment of the invention, bispecific antibodies and antibody mixtures are identified using these several mutants.
In the present invention, the antibodies are identified as bispecific antibodies or whether the antibody mixtures contain homodimeric proteins by the double antigen sandwich ELISA (also called bridge ELISA) method in combination with the mutated HER2 protein and the homodimeric proteins in the bispecific antibodies or antibody mixtures are further quantified.
In the invention, the double-antigen sandwich ELISA is well known in the field, and the working principle is that an antigen and an enzyme-labeled antigen which are connected to a solid phase carrier are respectively combined with two antigen binding sites on a detected antibody molecule in a sample to form a solid phase antigen-antibody-enzyme-labeled antigen immune complex. The detection steps of the method include, for example: the technical scheme includes coating a solid phase carrier with a specific antigen. Incubating for a period of time to allow formation of solid phase antigen, and washing to remove unbound antigen and impurities. And adding a specimen to be detected, and incubating to ensure that the antibody in the specimen fully reacts with the antigen on the solid-phase carrier to form a solid-phase antigen-antibody compound. Other unbound material is washed away. And thirdly, adding the enzyme-labeled antigen, and incubating to form the solid phase antigen-antibody to be detected-enzyme-labeled antigen sandwich compound. Washing to remove unbound enzyme-labeled antigen. And fourthly, bottom adding object color development. The enzyme on the solid phase catalyzes the substrate to produce a colored product, and the amount of antibody in the sample is measured by colorimetry.
In the invention, the HER2 positive tumor comprises a tumor with over-expression of HER2 protein (such as breast cancer and gastric cancer) and a tumor with low expression of HER2 protein (such as breast cancer and gastric cancer).
The invention obtains the common light chain capable of being combined with the heavy chains of the two monoclonal antibodies respectively by analyzing the light chain sequences of the two different monoclonal antibodies, and prepares the bispecific antibody and the antibody mixture with the common light chain on the basis, and experiments prove that the bispecific antibody and the antibody mixture prepared by the method have good combination property, biological activity and stability, and are possibly superior to the original antibody in the aspect of biological activity.
The common light chain technology is simple and controllable, and the problem of heavy and light chain mismatching in the bispecific antibody is effectively solved under the condition of not influencing the stability, activity and purity of the antibody; the antibody mixture can be expressed in the same host cell, so that the difficulty of culturing a mixed cell population can be avoided, and the amplification production is facilitated.
Drawings
FIG. 1 shows a schematic diagram of heterodimeric protein fusion. Panel a represents heterodimeric FC fusion technique and panel b represents "fab" technique.
FIG. 2 shows identification of hypervariable regions of pertuzumab and trastuzumab light chains, where A is identification result of hypervariable region of pertuzumab light chain, B is identification result of hypervariable region of trastuzumab light chain, and C is comparison of sequences of pertuzumab and trastuzumab light chains and comprehensive analysis result of amino acids at antigen interface.
FIG. 3 structural diagrams of the trastuzumab Fab fragment and Her2 extracellular domain (ECD).
FIG. 4 results of SDS-PAGE electrophoretic analysis (18% SDS-PAGE non-reducing conditions) of Her2m1 and Her2m2 variant proteins.
1: HER2m 1; 2: HER2m 2; m: and (4) protein quality standard.
FIG. 5 detection of specific binding of HER2 variant protein to Trastuzumab or Pertuzumab by ELISA method
FIG. 6: the common light chain monoclonal antibody protein sample obtained by one-step affinity chromatography purification is preliminarily detected by non-reduced SDS-PAGE (12% SDS-PAGE reduction condition)
1-6: tmax bCLC 1-6; 7-12: PmaBCLC 1-6; m: and (4) protein quality standard.
FIG. 7: affinity of Trastuzumab with a common light chain for its specific antigen HER2m1
FIG. 8: affinity of Pertuzumab with a common light chain for its specific antigen HER2m2
FIG. 9 shows the primary detection of the protein sample of the KN026 antibody obtained by single-step affinity chromatography (12% SDS-PAGE reduction)
1: KN026 transient expression cell culture supernatant; 2: KN026 affinity chromatography flow-through; 3: purifying the protein sample (reducing) after KN026 one-step affinity chromatography; 4: purified protein sample after KN026 one-step affinity chromatography (non-reducing) M: and (4) protein quality standard.
FIG. 10 SE-HPLC DETECTION OF PROTEIN PURITY OF KN026 ANTIBODY
FIG. 11 affinity curves for the bispecific antibody KN026 recognition of two antigens
FIG. 12 preliminary detection of KN010 antibody protein sample obtained by one-step affinity chromatography purification by SDS-PAGE (12% SDS-PAGE reduction conditions)
FIG. 13 SE-HPLC DETECTION OF PURITY OF MIXED ANTIBODY PROTEIN KN026
FIG. 14 affinity curves for the recognition of two antigens by the mixed antibody protein KN026
FIG. 15 concentration dependence curves of Ptmab bispecific antibody (KN026) and binding of Pertuzumab, Trastuzumab to BT474 cells
FIG. 16 concentration dependence curves of Ptmab bispecific antibody (KN026) and binding of Pertuzumab and Trastuzumab to N-87 cells
FIG. 17 concentration dependence curves of Pmab, Tma antibody cocktail (KN010) and binding of Pertuzumab, Trastuzumab to BT474 cells
FIG. 18 shows the inhibition of human breast cancer BT474 cell proliferation by KN026, Trastuzumab, and Trastuzumab + Pertuzumab combination
FIG. 19 shows the inhibitory effect of the combination of KN026, Trastuzumab and Trastuzumab + Pertuzumab on the proliferation of human gastric cancer N-87 cells
FIG. 20 detection of the thermal stability (Tm value) of the Ptmab bispecific antibody KN026 (light curve) and Trastuzumab reference sample (dark curve)
FIG. 21 shows the pharmacokinetic profiles of KN026 and Trastuzumab
FIG. 22 Effect of Ptmab bispecific antibody on tumor volume of human ovarian carcinoma SKOV3 nude mouse transplantable tumor
FIG. 23 Effect of Ptmab bispecific antibody on tumor volume of human gastric carcinoma N-87 nude mouse transplantable tumors
FIG. 24 Effect of PTmab bispecific antibody on tumor volume of human gastric carcinoma N-87 nude mouse transplantable tumors
FIG. 25 is a schematic diagram of a method for detecting whether an antibody is a bispecific antibody and a method for quantification.
FIG. 26 is a schematic diagram of a method for detecting whether an antibody mixture is a homodimeric protein.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The present application provides the following embodiments:
1. a bispecific antibody or antigen-binding portion thereof, characterized in that the two light chains of said bispecific antibody or antigen-binding portion thereof have the same sequence.
2. The bispecific antibody of embodiment 1 or an antigen-binding portion thereof, wherein the heavy chain is capable of properly binding to said light chain under physiological conditions or in vitro protein expression conditions, respectively.
3. The bispecific antibody or antigen-binding portion thereof of embodiment 1 or 2, wherein the light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
4. A bispecific antibody or antigen-binding portion thereof of embodiment 3, wherein the light chain is selected from the group consisting of the light chains of pertuzumab or trastuzumab, or a mutant thereof.
5. The bispecific antibody or antigen-binding portion thereof of embodiment 4, wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 6 at amino acids 1 to 107.
6. The bispecific antibody or antigen-binding portion thereof of embodiment 3, wherein the heavy chain variable regions are those of pertuzumab and trastuzumab, respectively.
7. The bispecific antibody or antigen-binding portion thereof of any one of embodiment 3, wherein the sequences of the heavy chain Fc segments comprise the amino acid sequences as set forth in SEQ ID NOs: 25 and SEQ ID NO: 26, or a sequence shown in fig. 26.
8. The bispecific antibody or antigen-binding portion thereof of any one of embodiment 3, wherein the sequences of the two heavy chains comprise the amino acid sequences as set forth in SEQ ID NOs: 19 and SEQ ID NO: 20, or a sequence shown in fig. 20.
9. A mixture of antibodies, or antigen-binding portions thereof, capable of being correctly produced in a cell, said mixture comprising at least two antibodies, or antigen-binding portions thereof, having a common light chain.
10. The mixture of embodiment 9, wherein the heavy chain of said antibody, or antigen-binding portion thereof, is capable of properly binding to said light chain under physiological conditions or in vitro protein expression conditions, respectively.
11. The mixture of embodiments 9 or 10, wherein the light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
12. The mixture of embodiment 11, wherein the light chain is selected from the group consisting of pertuzumab or trastuzumab light chains or mutants thereof.
13. The mixture of embodiment 11, wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 6 at amino acids 1 to 107.
14. The mixture of embodiment 11, wherein the heavy chain variable regions of the antibodies or antigen-binding portions thereof are the heavy chain variable regions of pertuzumab and trastuzumab, respectively.
15. The mixture of embodiment 11, wherein the sequences of the heavy chain Fc fragments of the antibodies, or antigen binding portions thereof, comprise the amino acid sequences set forth in SEQ ID NOs: 27 and SEQ ID NO: 28, and (b) the sequence shown in figure 28.
16. The mixture of embodiment 11, wherein the heavy chain sequences of the antibodies, or antigen-binding portions thereof, comprise the amino acid sequences set forth in SEQ ID NOs: 21 and SEQ ID NO: 22, or a sequence shown in fig. 22.
A variant protein of the extracellular region of a HER2 protein having a mutation selected from the group consisting of:
1) a mutation of glutamic acid at position 558 and a mutation of phenylalanine at position 573;
2) a mutation of serine at position 288 and a mutation of histidine at position 296.
18. A variant protein of the extracellular region of HER2 protein according to embodiment 17, characterized by one or more of the following items (1) to (4):
(1) mutating the 558 th glutamic acid to alanine;
(2) phenylalanine at position 573 is mutated to alanine;
(3) the 288 th serine is mutated into alanine;
(4) histidine at position 296 was mutated to alanine.
19. A variant protein of the extracellular region of HER2 protein of embodiment 18, said variant protein of the extracellular region of HER2 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13. SEQ ID NO: 14 or SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.
20. A nucleic acid molecule encoding the bispecific antibody or antigen-binding portion thereof according to any one of embodiments 1 to 8 or the antibody or antigen-binding portion thereof or a partial sequence of said antibody or antigen-binding portion thereof as described in the mixture according to any one of embodiments 9 to 16 or encoding the HER2 variant protein according to any one of embodiments 17 to 19.
21. A recombinant vector comprising the nucleic acid molecule of embodiment 20.
22. A recombinant cell comprising the recombinant vector of embodiment 21 or the nucleic acid molecule of embodiment 20.
23. A method of making a bispecific antibody or antigen-binding portion thereof based on two monoclonal antibodies or antigen-binding portions thereof directed against different epitopes comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of one of the monoclonal antibody light chains;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors; preferably, the heavy chain sequence, in particular the Fc-segment, is mutated to favor the binding of Fc-segments with different heavy chains;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the bispecific antibody or the antigen binding part thereof.
24. The method of embodiment 23, wherein the common light chain is obtained by first determining the interfacial amino acids in contact between two monoclonal antibodies and the antigen or the epitope of the antigen, then determining the difference amino acids when the interfacial amino acids in contact between the common light chain and the antigen or the epitope of the antigen are compared with the amino acids at the corresponding positions of the light chain of another monoclonal antibody when the light chain of any one of the monoclonal antibodies is used as the candidate common light chain, and selecting the light chain with the smaller number of difference amino acids as the common light chain; preferably, the common light chain is further mutated to obtain a more cohesive common light chain.
25. A method of preparing a mixture comprising at least two monoclonal antibodies, or antigen binding portions thereof, comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of one of the monoclonal antibody light chains;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors; preferably, the heavy chain sequence, in particular the Fc-segment, is mutated to favor the binding of the Fc-segment with the same heavy chain;
3) the two recombinant expression vectors are transferred into the same host cell and expression is induced to obtain a mixture of antibodies or antigen-binding portions thereof.
26. The method of embodiment 25, wherein the common light chain is obtained by first determining the interfacial amino acids in contact between the two monoclonal antibodies and the antigen or the epitope of the antigen, then determining the difference amino acids when the light chain of any one of the monoclonal antibodies is the candidate common light chain, and comparing the interfacial amino acids in contact between the common light chain and the antigen or the epitope of the antigen with the amino acids at the corresponding positions of the light chain of the other monoclonal antibody, and selecting the light chain with the smaller number of difference amino acids as the common light chain; preferably, the common light chain is further mutated to obtain a more cohesive common light chain.
27. A method of detecting and/or quantifying whether an antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof, comprising the steps of:
1) preparing a specific antigen 1 capable of binding to the antigen-binding portion 1 of the bispecific antibody or antigen-binding portion thereof and not binding to the antigen-binding portion 2, and a specific antigen 2 capable of binding to the antigen-binding portion 2 and not binding to the antigen-binding portion 1, respectively;
2) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding an antibody to be detected, reacting for a period of time, adding a labeled specific antigen 2 (or the specific antigen 1), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive or negative according to the reading of a detection principle;
3) when the reaction is positive and the reaction is concentration dependent, determining that the antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof; optionally, the bispecific antibody or antigen-binding portion thereof is further quantified based on the resulting positive values.
28. A method of detecting whether a mixture of antibodies or antigen-binding portions thereof is a homodimeric protein, the mixture comprising two antibodies (antibody 1 and antibody 2) or antigen-binding portions thereof, the method comprising the steps of:
1) preparing specific antigen 1 capable of binding to antibody 1 but not to antibody 2, and specific antigen 2 capable of binding to antibody 2 but not to antibody 1, respectively;
2) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding a mixture to be detected, reacting for a period of time, adding a labeled specific antigen 1 (or a specific antigen 2), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive or negative according to the reading of a detection principle;
3) taking a specific antigen 1 (or a specific antigen 2) to coat an enzyme label plate, adding the enzyme label plate to be detected, reacting for a period of time, adding a labeled specific antigen 2 (or the specific antigen 1), reacting for a period of time, finally adding a detection molecule capable of being combined with the labeled molecule, reacting for a period of time, wherein the detection molecule is provided with a detectable label, and judging as positive reaction or negative reaction according to the reading of a detection principle;
4) when the reaction in the step 2) is positive and has concentration dependence, and the reaction in the step 3) is negative, judging that the mixture is homodimer protein and does not contain heterodimer protein; and when the reaction in the step 2) is positive and the reaction in the step 3) is positive, judging that the mixture contains both homodimer protein and heterodimer protein.
29. A composition (e.g., a pharmaceutical composition) comprising a bispecific antibody or antigen-binding portion thereof of any one of embodiments 1-8, or a mixture of any one of embodiments 9-16, and optionally a pharmaceutically acceptable carrier or excipient.
30. The composition of embodiment 29, further comprising a chemotherapeutic agent and/or other antibody.
31. A kit comprising a bispecific antibody or antigen-binding portion thereof of any one of embodiments 1-8, or a mixture of any one of embodiments 9-16, and optionally a buffer or instructions.
32. Use of a bispecific antibody or antigen-binding portion thereof according to any one of embodiments 3 to 8 for the preparation of a medicament for the prevention and/or treatment of HER2 positive tumors (e.g. breast cancer, gastric cancer).
33. Use of a mixture according to any of embodiments 11 to 16 for the preparation of a medicament for the prevention and/or treatment of HER2 positive tumors (e.g. breast cancer, gastric cancer).
34. Use of a bispecific antibody or antigen-binding portion thereof of any one of embodiments 3-8 in the preparation of an agent or kit for the diagnosis of a HER2 positive tumor (e.g. breast cancer, gastric cancer).
35. Use of a mixture according to any of embodiments 11 to 16 for the preparation of a reagent or kit for the diagnosis of HER2 positive tumors (e.g. breast cancer, gastric cancer).
36. Use of a variant protein of the extracellular region of HER2 protein according to any one of embodiments 17 to 19 for the detection of a bispecific antibody or antigen-binding portion thereof according to any one of embodiments 3 to 8 or for the detection of a mixture according to any one of embodiments 11 to 16.
37. A method of detecting and/or quantifying whether an antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof, comprising the steps of:
1) preparing a specific antigen 1 capable of binding to the antigen-binding portion 1 of the bispecific antibody or antigen-binding portion thereof and not to the antigen-binding portion 2, and a specific antigen 2 capable of binding to the antigen-binding portion 2 and not to the antigen-binding portion 1, respectively;
2) taking the specific antigen 1 to coat the enzyme label plate, adding the antibody or the antigen binding part thereof, and then adding the labeled specific antigen 2; or, taking the specific antigen 2 to coat the enzyme label plate, adding the antibody to be detected, and then adding the marked specific antigen 1;
then adding a detection molecule capable of binding to the labeled specific antigen, said detection molecule carrying a detectable label;
3) when the result of step 2) is positive and the reaction of step 2) is concentration-dependent, the antibody or antigen-binding portion thereof is judged to be a bispecific antibody or antigen-binding portion thereof.
38. The method of embodiment 37, quantifying the bispecific antibody or antigen-binding portion thereof according to a positive value of step 3).
39. The method of embodiment 37, wherein said quantifying comprises a bridge ELISA.
40. A method of detecting whether a mixture of antibodies or antigen-binding portions thereof is a homodimeric protein, the mixture comprising two antibodies, antibody 1 and antibody 2 or antigen-binding portions thereof, the method comprising the steps of:
1) preparing specific antigen 1 capable of binding to antibody 1 but not to antibody 2, and specific antigen 2 capable of binding to antibody 2 but not to antibody 1, respectively;
2) taking a specific antigen 1 coated enzyme label plate, adding a mixture to be detected, and adding a marked specific antigen 1; or, taking a specific antigen 2 coated enzyme label plate, adding the mixture to be detected, and then adding the marked specific antigen 2;
adding a detection molecule capable of being combined with the marked specific antigen, wherein the detection molecule is provided with a detectable mark;
3) taking a specific antigen 1 to coat an enzyme label plate, adding a mixture to be detected, and adding a marked specific antigen 2; or, taking a specific antigen 2 to coat the enzyme label plate, adding the mixture to be detected, and then adding the marked specific antigen 1;
adding a detection molecule capable of being combined with the labeled specific antigen, wherein the detection molecule is provided with a detectable label;
4) judging that the mixture contains homodimer protein and does not contain heterodimer protein when the result of step 2) is positive and the reaction of step 2) has concentration dependence while the result of step 3) is negative;
judging that the mixture contains homodimer protein and heterodimer protein when the result of the step 2) is positive and the result of the step 3) is positive.
41. The method of any one of embodiments 37-40, wherein said specific antigen 1 and/or specific antigen 2 is a variant protein of the extracellular region of the HER2 protein having a mutation selected from the group consisting of:
1) a mutation of glutamic acid at position 558 and a mutation of phenylalanine at position 573;
2) a mutation of serine at position 288 and a mutation of histidine at position 296.
42. The method of embodiment 41, wherein said variant protein of the extracellular region of the HER2 protein comprises a mutation in one or more of the following (1) to (4):
1) mutating the 558 th glutamic acid to alanine;
2) phenylalanine at position 573 is mutated to alanine;
3) the 288 th serine is mutated into alanine;
4) histidine at position 296 was mutated to alanine.
43. The method of embodiment 41, wherein said variant protein of the extracellular region of the HER2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 13-15.
44. The method of embodiment 37 or 40, wherein said specific antigen 1 comprises the amino acid sequence of SEQ ID NO: 13, and the specific antigen 2 comprises an amino acid sequence shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
45. A nucleic acid molecule encoding a variant protein of the extracellular region of HER2 protein according to any one of embodiments 41-44.
46. A recombinant vector comprising the nucleic acid molecule of embodiment 45.
47. A recombinant cell comprising the recombinant vector of embodiment 46 or the nucleic acid molecule of embodiment 45.
48. A method of making a bispecific antibody or antigen-binding portion thereof based on two monoclonal antibodies or antigen-binding portions thereof directed against different epitopes comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of the light chain of one of the monoclonal antibodies;
2) respectively constructing heavy chain sequences and the common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the bispecific antibody or the antigen binding part thereof.
49. The method of embodiment 48, wherein in step 2) the heavy chain sequence is mutated such that it binds to an Fc fragment having the same heavy chain.
50. The method of embodiment 48, wherein the Fc region of the heavy chain sequence in step 2) is mutated such that it binds to an Fc region having the same heavy chain.
51. The method of embodiment 48, wherein said common light chain is obtained by a method comprising,
firstly, determining the contact interface amino acid between two monoclonal antibodies and antigen or antigen epitope,
then determining the difference amino acid when the interface amino acid contacted between the common light chain and the antigen or the antigen epitope is compared with the amino acid at the corresponding position of the light chain of another monoclonal antibody when the light chain of any one monoclonal antibody is taken as a candidate common light chain,
the light chain with the smaller number of different amino acids is selected as the common light chain.
52. The method of embodiment 48, wherein the common light chain is further mutated to obtain a more cohesive common light chain.
53. A method of preparing a mixture comprising at least two monoclonal antibodies, or antigen binding portions thereof, comprising the steps of:
1) obtaining a common light chain sequence which can be combined with the heavy chains of the two monoclonal antibodies respectively according to the light chain sequences of the two monoclonal antibodies, wherein the common light chain is the light chain of one of the monoclonal antibodies or a mutant of the light chain of one of the monoclonal antibodies;
2) respectively constructing heavy chain sequences and common light chain sequences of the two monoclonal antibodies in expression vectors to obtain two recombinant expression vectors;
3) transferring the two recombinant expression vectors into the same host cell, and inducing expression to obtain the mixture.
54. The method of embodiment 53, wherein in step 2) the heavy chain sequence is mutated such that it binds to an Fc fragment having the same heavy chain.
55. The method of embodiment 53, wherein the Fc region of the heavy chain sequence in step 2) is mutated such that it binds to an Fc region having the same heavy chain.
56. The method of embodiment 53, wherein the common light chain is obtained by a method comprising,
firstly, determining the contact interface amino acid between two monoclonal antibodies and antigen or antigen epitope,
then determining the difference amino acid when the interface amino acid contacted between the common light chain and the antigen or the antigen epitope is compared with the amino acid at the corresponding position of the light chain of another monoclonal antibody when the light chain of any one monoclonal antibody is taken as a candidate common light chain,
the light chain with the smaller number of different amino acids is selected as the common light chain.
57. The method of embodiment 53, wherein the common light chain is further mutated to obtain a more cohesive common light chain.
58. The method of embodiment 48 or 53, wherein said two monoclonal antibodies are trastuzumab and pertuzumab, respectively.
59. A detection molecule that binds to the specific antigen of embodiment 37, the detection molecule being HRP-labeled streptavidin.
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