CN117645667A - Bispecific antibodies and uses thereof - Google Patents
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- CN117645667A CN117645667A CN202311357530.7A CN202311357530A CN117645667A CN 117645667 A CN117645667 A CN 117645667A CN 202311357530 A CN202311357530 A CN 202311357530A CN 117645667 A CN117645667 A CN 117645667A
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Classifications
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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- C07K2317/622—Single chain antibody (scFv)
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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Abstract
The invention provides a bispecific antibody and application thereof, wherein the antibody consists of a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody: the amino acid sequence of the light chain CDR is shown as SEQ ID NO. 1-3, and the amino acid sequence of the heavy chain CDR is shown as SEQ ID NO. 4-6; the second polypeptide chain comprises the variable region CDR sequences of a CD155 antibody: the amino acid sequence of the light chain CDR is shown as SEQ ID NO 7-9, and the amino acid sequence of the heavy chain CDR is shown as SEQ ID NO 10-12. The bispecific antibody prepared by the invention can target CD3 and CD155 simultaneously, thereby mediating the killing of T cells on tumor cells and having stronger tumor inhibition capability.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a bispecific antibody and application thereof.
Background
CD155, also known as NECL-5 or PVR, is a type I transmembrane protein, the extracellular segment of which contains 1 IgV domain, 2 IgC domains and the intracellular segment of which contains the ITIM motif. CD155 expression is on the surface of tissue cells such as monocytes, dendritic cells, etc., and up-regulates expression in various tumor tissues. Studies have shown that CD155 promotes tumor invasion, metastasis, and that high expression of CD155 correlates with poor tumor prognosis. To date, only one CD155 monoclonal antibody has entered the clinical trial phase, i.e., the NTX 1088 antibody from the Nectin company, and no CD 155-related bispecific antibody has entered the clinical trial.
Among bispecific antibodies based on T cell redirection, this target for CD3 is widely selected. Unlike immune checkpoint blocking antibodies, CD 3-related bispecific antibodies can mediate T cell activation across TCR and peptide-major histocompatibility complex (pMHC), but the molecules in the synapse function much like classical TCR-pMHC interactions. The activity of bispecific antibodies is affected by the affinity of CD3, and CD3 high affinity bispecific antibodies have better killing effects in vitro experiments, but are at higher risk of factor release syndrome in vivo. Also, very low affinity CD3 antibody sequences have been found to be effective in stimulating T cell activation after bispecific antibody construction. When the CD3 antibody affinity is in the appropriate range and the tumor target-associated antibody is of high affinity, the bispecific antibody will promote selective localization of T cells to tumor sites rather than to the peripheral circulation, avoiding systemic activation.
Bispecific antibodies are antibodies that can specifically bind to two antigenic sites simultaneously. Bispecific antibodies for tumor therapy can be classified into three classes according to their mechanism of action: redirecting effector cells; immunomodulation; targeting tumor cell receptor dual binding. Among them, antibodies for the redirecting function occupy the majority thereof, and a plurality of bispecific antibodies for tumor treatment, which have been currently marketed, are also based on cell redirecting.
Based on these findings, the present invention contemplates novel recombinant bispecific fusion proteins that can specifically bind to CD3 and specifically bind to CD155. The fusion protein can bring CD155 molecules near tumor cells in a targeting way, and promote the T cells and the tumor cells to form immune synapses by combining with the CD155 molecules on the tumor cells, so that the T cells activate and kill the CD155+ tumor cells, and a new inspiration and thinking are provided for antibody therapy of tumor immunity.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
the first aspect of the present invention provides a recombinant antibody comprising a first polypeptide chain and a second polypeptide chain:
the first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody, wherein the variable region CDR sequence of the CD3 antibody is shown in SEQ ID NO. 1-6 or has at least 85% of the amino acid sequence identical to SEQ ID NO. 1-6;
the second polypeptide chain comprises the variable region CDR sequence of a CD155 antibody, wherein the variable region CDR sequence of the CD3 antibody is as shown in SEQ ID NO. 7-12 or an amino acid sequence having at least 85% identity to SEQ ID NO. 7-12.
The present invention is based on a bispecific cell bridging method employing a bispecific binding protein having a binding arm that binds to CD3 on T cells and a binding arm that binds to CD155 on the cell surface of tumor cells. By promoting simultaneous binding of T cells and tumor cells, the bispecific protein promotes the formation of a cellular synapse between the two cells and thus selectively redirects T cell activity to the targeted tumor cells.
According to an embodiment of the invention, the recombinant antibody consists of a first polypeptide chain and a second polypeptide chain:
the first polypeptide chain comprises the variable region CDR sequences of a CD3 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 5, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 6,
the second polypeptide chain comprises the variable region CDR sequences of a CD155 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 8, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 12. The recombinant antibody according to the embodiment of the invention can be combined with CD3 and CD155 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.
According to an embodiment of the invention, the light chain variable region of the CD3 antibody has an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 13, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 14 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 14; and
The light chain variable region of the CD155 antibody has an amino acid sequence shown as SEQ ID NO. 15 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 15, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 16 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 16.
According to an embodiment of the invention, the first polypeptide chain comprises a scFab region and a first Fc region of a CD3 antibody, and the second polypeptide chain comprises a scFv region and a second Fc region of a CD155 antibody.
According to an embodiment of the invention, the first polypeptide chain comprises an scFv region and a first Fc region of a CD3 antibody and the second polypeptide chain comprises an scFv region and a second Fc region of a CD155 antibody.
According to an embodiment of the invention, the first polypeptide chain comprises a scFab region and a first Fc region of a CD3 antibody, and the second polypeptide chain comprises a scFab region and a second Fc region of a CD155 antibody.
According to an embodiment of the invention, the first and second Fc regions are linked by a knob-intoo-hole structure.
According to an embodiment of the invention, the first Fc region has at least one of the L234A, L235A mutations compared to the wild-type IgG1 Fc region and the second Fc region has at least one of the L234A, L235A mutations compared to the wild-type IgG1 Fc region.
According to an embodiment of the invention, at least a portion of the constant region of the CD3 antibody, the constant region of the CD155 antibody, the first Fc region and the second Fc region is derived from at least one of a murine antibody, a primate-origin antibody or a mutant thereof.
According to an embodiment of the invention, at least a portion of the constant region of the CD3 antibody, the constant region of the CD155 antibody, the first Fc region and the second Fc region are derived from human IgG1 or a mutant thereof.
According to an embodiment of the invention, the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 20, and the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 21.
According to an embodiment of the invention, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO. 24 or an amino acid sequence having at least 85% identity to an amino acid shown in SEQ ID NO. 24, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO. 25 or an amino acid sequence having at least 85% identity to an amino acid shown in SEQ ID NO. 25.
According to an embodiment of the invention, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO. 28 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 28, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO. 29 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 29.
According to an embodiment of the invention, the recombinant antibody further comprises a signal peptide added to the N-terminus of the first polypeptide chain and/or the second polypeptide chain, the signal peptide having the amino acid sequence shown as SEQ ID NO. 34.
In a second aspect the invention provides an isolated polynucleotide encoding the recombinant antibody of the first aspect.
In a third aspect the invention provides an expression vector carrying a polynucleotide according to the second aspect. According to an embodiment of the invention, the nucleic acid according to the second aspect is carried. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the invention can efficiently express the recombinant antibody in a proper host cell, and the recombinant antibody can be combined with CD3 and CD155 at the same time, so that the killing effect of T cells on tumor cells is effectively mediated, and the recombinant antibody has stronger tumor inhibition capability.
In a fourth aspect, the present invention provides a method for producing a recombinant antibody according to the first aspect, comprising:
introducing the expression vector of the third aspect into a cell;
culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibodies;
according to an embodiment of the invention, the cell is a eukaryotic cell.
In a fifth aspect, the invention provides a recombinant cell carrying a polynucleotide according to the second aspect or an expression vector according to the third aspect.
In a sixth aspect, the invention provides a composition comprising: at least one of the recombinant antibody of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. The composition of the invention comprises a food composition, a pharmaceutical composition and the like.
The seventh aspect of the present invention provides the use of the recombinant antibody according to the first aspect, the polynucleotide according to the second aspect, the expression vector according to the third aspect, the recombinant cell according to the fifth aspect or the composition according to the sixth aspect in the manufacture of a medicament for the treatment or prevention of cancer, the cancer having positive CD155 on the surface of cancer cells.
According to an embodiment of the invention, the cancer comprises at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.
According to an eighth aspect of the invention there is provided the use of a recombinant antibody according to the first aspect, a polynucleotide according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect in the manufacture of a kit for the detection of CD3 and/or CD155.
In a ninth aspect the invention provides a kit comprising a recombinant antibody according to the first aspect for the detection of CD3 and/or CD155. The recombinant antibodies can bind to CD3 and/or CD155 proteins, and thus kits comprising the recombinant antibodies can be used to efficiently detect CD3 and/or CD155. The kit can be used in scientific research, such as qualitative or quantitative detection of CD3 and/or CD155 protein in biological samples, and can also be used for judging the state of an individual, such as judging whether the level of CD155 of the individual is excessively higher or lower than the normal level after the level of CD155 of the individual is obtained.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a schematic structure of a CD3-CD155 bispecific antibody selected in example 1 of the present invention;
FIG. 2 shows a schematic structural diagram of the 3-155-scfv bispecific antibodies selected in example 1 of the present invention;
FIG. 3 shows a schematic structural diagram of the 3-155-fab bispecific antibody selected in example 1 of the present invention;
FIG. 4 is a diagram showing the binding of CD3-CD155 bispecific binding protein to CD3 protein in example 3 of the present invention;
FIG. 5 is a diagram showing the binding of CD3-CD155 bispecific binding protein to CD155 protein in example 3 of the present invention;
FIG. 6 is a graph showing the analysis result of chemiluminescence values detected after a reaction for adding a substrate after 6 hours in a gradient concentration of CD3-CD155 recombinant bispecific antibody molecules to jurkat-NFAT-luc cells mixed with non-small cell lung cancer HCC827 cells in example 4 of the present invention;
FIG. 7 is a graph showing the results of analysis of chemiluminescence values detected after a reaction for adding a substrate after 6 hours in a gradient concentration of CD3-CD155 recombinant bispecific antibody molecules to jurkat-NFAT-luc cells mixed with hepatoma HepG2 cells in example 4 of the present invention;
FIG. 8 is a graph showing the results of analysis of chemiluminescence values detected after a 6-hour post substrate addition reaction of a graded concentration of CD3-CD155 recombinant bispecific antibody molecules added after mixing jurkat-NFAT-luc cells with pancreatic cancer HPAF-II cells in example 4 of the present invention;
FIG. 9 is a graph showing the results of analysis of chemiluminescence values detected after a 6-hour post substrate addition reaction of a graded concentration of CD3-CD155 recombinant bispecific antibody molecules added after mixing jurkat-NFAT-luc cells with colorectal cancer HCT-15 cells in example 4 of the present invention;
FIG. 10 is a graph showing the results of in vitro cytotoxicity of CD3-CD155 bispecific binding protein and PBMC of example 5 of the present invention against liver cancer HepG2 cells;
FIG. 11 shows the results of an in vitro cytotoxicity of CD3-CD155 bispecific binding protein and PBMC against non-small cell lung cancer HCC827 cells of example 5 of the present invention;
FIG. 12 is a graph showing the results of an in vitro cytotoxicity of CD3-CD155 bispecific binding protein and PBMC against pancreatic cancer HPAF-II cells in example 5 of the present invention;
FIG. 13 shows the results of an in vitro cytotoxicity assay of CD3-CD155, 3-155-scfv, 3-155-fab bispecific binding protein against PBMC against colorectal cancer HCT-15 cells in example 5 of the invention;
FIG. 14 is a graph showing the effect of CD3-CD155 bispecific binding protein on IL-6 secretion by PBMC in the absence of target cells according to example 6 of the present invention;
FIG. 15 is a graph showing the effect of CD3-CD155 bispecific binding protein on PBMC secretion of TNF in the absence of target cells according to example 6 of the present invention;
FIG. 16 is a graph showing the effect of CD3-CD155 bispecific binding protein on secretion of IFN-gamma by PBMC in the absence of target cells in example 6 of the present invention;
FIG. 17 is a graph showing the effect of CD3-CD155 bispecific binding protein on secretion of IL-6 by PBMC in the presence of colorectal cancer NCI-H716 cells according to example 6 of the present invention;
FIG. 18 is a graph showing the effect of CD3-CD155 bispecific binding protein of example 6 of the present invention on the secretion of TNF by PBMC in the presence of colorectal cancer NCI-H716 cells;
FIG. 19 shows a graph of the effect of CD3-CD155 bispecific binding protein on secretion of IFN-gamma by PBMC in the presence of colorectal cancer NCI-H716 cells according to example 6 of the present invention;
FIG. 20 is a graph showing the effect of the CD3-CD155 bispecific binding protein of example 6 of the present invention on secretion of IL-6 by PBMC in the presence of melanoma A375 cells;
FIG. 21 is a graph showing the effect of CD3-CD155 bispecific binding protein on PBMC secretion of TNF in the presence of melanoma A375 cells according to example 6 of the present invention;
FIG. 22 shows a graph of the effect of the CD3-CD155 bispecific binding protein of example 6 of the present invention on secretion of IFN-gamma by PBMC in the presence of melanoma A375 cells.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system. Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprising two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively. Certain regions of amino acid composition and arrangement in the variable region have a higher degree of variation, known as hypervariable regions (Hypervariable region, HVR), which are the sites of antigen and antibody binding and are therefore also known as determinant-complementary-determining region (CDR). The heavy chain variable region and the light chain variable region each have three CDR regions.
Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
Herein, "diabody" means a peptide chain capable of specifically recognizing different protein molecules, which is obtained by linking two chains of an Fc region, respectively, wherein the two chains of the Fc region are linked by a knob intohole structure.
As used herein, the term "Knob intonation hole structure" refers to the formation of a Knob (Knob) button (hole) mutation in the CH3 region of an antibody heavy chain Fc, which facilitates heavy chain occlusion to form a heterodimer.
As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. It will be appreciated by those skilled in the art that the nucleic acid molecules encoding the antibodies or antigen binding fragments may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like. Such as Plasmid-X.
The term "identity" is used herein to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and the ALIGN program (Dayhoff (1978), atlas of Protein Sequence and Structure 5: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs of Pearson et al (1988) Proc.Natl.Acad.Sci.85:2444, the Smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.Mol.biol.215:403-410), and include but are also available in the programs of ALIGN or Megalign (DNASTAR), or the programs of BLAST-2, and the programs of Abelson.G.35:266, and the programs of Abelson.35:266, respectively.
anti-CD 3/CD155 antibodies or antigen binding fragments
The invention proposes a recombinant antibody consisting of a first polypeptide chain and a second polypeptide chain:
the first polypeptide chain comprises the variable region CDR sequences of a CD3 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 2, the light chain CDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 6;
the second polypeptide chain comprises the variable region CDR sequences of a CD155 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 8, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 12.
The present invention is based on a bispecific cell bridging method employing a bispecific binding protein having a binding arm that binds to CD3 on T cells and a binding arm that binds to CD155 on the cell surface of tumor cells. By promoting simultaneous binding of T cells and tumor cells, the bispecific protein promotes the formation of a cellular synapse between the two cells and thus selectively redirects T cell activity to the targeted tumor cells.
According to one embodiment of the invention, the invention provides a binding protein (CD 3-CD 155) comprising two polypeptide chains: the first polypeptide chain comprises a CD3 light chain variable domain (SEQ ID NO: 13), a CD3 light chain constant domain (SEQ ID NO: 17), a connecting peptide 1 (SEQ ID NO: 32), a CD3 heavy chain variable domain (SEQ ID NO: 14) and a CD3 heavy chain constant domain (SEQ ID NO: 18) in sequence from the N-terminus to the C-terminus; the second polypeptide chain comprises, in order from the N-terminus to the C-terminus, a CD155 light chain variable region (SEQ ID NO: 15), a connecting peptide 2 (SEQ ID NO: 33), a CD155 heavy chain variable domain (SEQ ID NO: 16) and a CD155 heavy chain constant domain (SEQ ID NO: 19). In some embodiments of the invention, the connecting peptide 1 and the connecting peptide 2 are flexible linkers, the first polypeptide chain having the amino acid sequence shown as SEQ ID NO. 20, and the second polypeptide chain having the amino acid sequence shown as SEQ ID NO. 21.
According to a specific embodiment of the present invention, there is provided a binding protein (3-155-scfv) comprising two polypeptide chains: the first polypeptide chain comprises a CD3 light chain variable domain (SEQ ID NO: 13), a connecting peptide 2 (SEQ ID NO: 33), a CD3 heavy chain variable domain (SEQ ID NO: 14) and a CD3 heavy chain constant domain from the N-terminus to the C-terminus in sequence; the second polypeptide chain comprises, in order from the N-terminus to the C-terminus, a CD155 light chain variable region (SEQ ID NO: 15), a connecting peptide 2 (SEQ ID NO: 33), a CD155 heavy chain variable domain (SEQ ID NO: 16) and a CD155 heavy chain constant domain (SEQ ID NO: 19). In some embodiments of the invention, the linker peptide 2 is a flexible linker, the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 24, and the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 25.
According to one embodiment of the invention, the invention provides a binding protein (3-155-fab) comprising two polypeptide chains: the first polypeptide chain comprises a CD3 light chain variable domain (SEQ ID NO: 13), a CD3 light chain constant domain (SEQ ID NO: 17), a connecting peptide 1 (SEQ ID NO: 32), a CD3 heavy chain variable domain (SEQ ID NO: 14) and a CD3 heavy chain constant domain (SEQ ID NO: 18) in sequence from the N-terminus to the C-terminus; the second polypeptide chain comprises, in order from the N-terminus to the C-terminus, a CD155 light chain variable region (SEQ ID NO: 15), a CD155 light chain constant domain, a connecting peptide 1 (SEQ ID NO: 32), a CD155 heavy chain variable domain (SEQ ID NO: 16) and a CD155 heavy chain constant domain. In some embodiments of the invention, the linker peptide 1 is a flexible linker, the first polypeptide chain has the amino acid sequence shown in SEQ ID NO. 28, and the second polypeptide chain has the amino acid sequence shown in SEQ ID NO. 29.
According to a specific embodiment of the present invention, there is provided a binding protein comprising two polypeptide chains, wherein a signal peptide is added to the N-terminus of the first peptide chain of the binding protein and/or the second peptide chain of the binding protein, so that an antibody can be efficiently expressed in a cell and secreted into a culture medium.
According to embodiments of the present disclosure, the 3 different configurations of CD3/CD155 bispecific antibodies screened in the present invention each have a binding arm that binds to CD3 on T cells and a binding arm that binds to CD155 on the cell surface of tumor cells. By promoting simultaneous binding of T cells and tumor cells, the bispecific protein promotes the formation of a cellular synapse between the two cells and thus selectively redirects T cell activity to the targeted tumor cells.
According to the embodiment of the disclosure, 3 kinds of CD3/CD155 bispecific antibodies with different configurations are screened, even though antigen binding domains are the same, the antigen binding capacity is different among the antibodies with different configurations, and a set of CD3-CD155 bispecific antibodies with better effect of promoting immune cells to kill tumors is screened from a plurality of CD3/CD155 bispecific antibodies, which comprises binding proteins of two polypeptide chains: the first polypeptide chain comprises a CD3 light chain variable domain, a connecting peptide 1, a CD3 heavy chain variable domain and a CD3 heavy chain constant domain from the N end to the C end in sequence; the second polypeptide chain comprises a CD155 light chain variable region, a connecting peptide 2, a CD155 heavy chain variable domain and a CD155 heavy chain constant domain from the N end to the C end in sequence, and the antibody not only has better capability of promoting immune cells to kill HCT-15 colorectal cancer, but also has better effect in killing other CD155 positive cells. In some embodiments of the invention, the connecting peptide 1 and the connecting peptide 2 are flexible linkers.
According to particular embodiments of the invention, one skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The sequences of the variants of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion. It will be appreciated by those skilled in the art that the CDR sequences analyzed by different databases may vary, but that such variations are intended to be within the scope of the present invention.
Nucleic acid molecules, recombinant vectors, recombinant cells, immunoconjugates
In preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding antibodies.
Thus, the present invention also provides an isolated nucleic acid encoding the above antibody or antigen-binding fragment thereof, and recombinant vectors and transformants containing the nucleic acid. The nucleic acid molecule encodes the antibody or antigen binding fragment thereof described above, preferably the nucleic acid is an expression cassette obtained by genetic engineering means.
The present invention provides isolated nucleic acid molecules encoding a first antigen binding unit and/or a second antigen binding unit of a protein of the invention. Other aspects provided herein are expression vectors comprising the nucleic acid molecules of the invention, host cells transfected with such expression vectors, and methods of making the proteins of the invention.
The present invention provides novel binding proteins that can more efficiently treat cancers that express CD155, such as colorectal cancer, melanoma, non-small cell lung cancer, pancreatic cancer, liver cancer, and the like that express CD 155.
Recombinant vectors may be referred to as cloning vectors, or as expression vectors, and may be obtained by operably linking the nucleic acids to commercially available vectors (e.g., plasmid or viral vectors), commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.
In some preferred embodiments, the nucleic acid molecule is species optimized for expression in mammalian cells.
The invention also provides an expression vector comprising the isolated nucleic acid molecule described above. In the case of ligating the above isolated polynucleotide to a vector, the polynucleotide may be directly or indirectly ligated to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the polynucleotide. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the polynucleotide may be operably linked to a control element.
The invention also provides a recombinant cell, which comprises the expression vector. The expression vector may be introduced into mammalian cells, constructed to obtain recombinant cells, and these recombinant cells may be used to express the antibodies or antigen-binding fragments provided by the present invention. The recombinant cells are cultured to obtain the corresponding antibodies. The host cell of the invention may be a prokaryotic host cell, a eukaryotic host cell or a phage. The prokaryotic host cell can be escherichia coli, bacillus subtilis, streptomycete, proteus mirabilis or the like. The eukaryotic host cell can be fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the host cell of the invention is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell or COS cell.
The immunoconjugates provided herein comprise a therapeutic agent coupled to an antibody or antigen-binding fragment thereof as described previously. The manner in which the antibody or antigen-binding fragment thereof is conjugated to the therapeutic agent may be in a conventional manner.
The present invention provides compositions comprising an antibody or antigen-binding fragment thereof as described above, and/or an immunoconjugate as described above, and a pharmaceutically acceptable carrier. In certain embodiments, the compositions comprise combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.
Medicine, kit and pharmaceutical application and application in preparation of kit
The invention also provides a medicine which comprises the antibody or the antigen binding fragment thereof and a pharmaceutically acceptable carrier, and can also comprise the immunoconjugate, the nucleic acid molecule, the expression carrier and the recombinant cell.
In some embodiments, these pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, including any solvents, solid excipients, diluents, binders, disintegrants, or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, glidants or lubricants, and the like, suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.
The compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies, or antigen binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit CD155 activity (including other antibodies or antigen binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with CD155 upstream or downstream signaling.
Typically, the antibody or antigen binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and/or prophylactic effect, e.g., an amount that causes prevention or alleviation of symptoms associated with a disease being treated, such as a disease associated with abnormal CD155 expression. The effective amount of the composition administered to a subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health, age, sex, weight and tolerance to drugs; the skilled artisan will be able to determine the appropriate dosage based on these factors, etc., will also depend on the severity and type of disease.
The effective amount of the antibodies or antigen binding fragments of the invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.
In some embodiments of the invention, the invention provides a kit for detecting CD3 and/or CD155 in a sample comprising an antibody or antigen binding fragment thereof as described above, a pharmaceutically acceptable carrier, an immunoconjugate, a nucleic acid molecule, an expression vector, a recombinant cell. In some embodiments, the sample may be tissue of a patient suffering from a CD155 mediated disease. The kit may also include reagents conventionally used for detecting CD3 and/or CD155, such as coating solutions and the like.
Nucleic acids encoding the heavy and/or light chains of the antibodies of the invention are within the scope of the invention, and corresponding nucleic acid sequences can be readily obtained by the skilled artisan based on the amino acid sequences of the heavy and/or light chains, as shown in table 1.
TABLE 1
The embodiments will be described in detail below. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of bispecific antibody molecules
The monovalent unit of the CD3 antibody and the monovalent unit of the CD155 antibody are cloned into the expression vector pcdna3.4 respectively by using common molecular biology technology, meanwhile, in order to make the monovalent unit of the CD3 antibody efficiently expressed and secreted into a culture medium in CHO cells, a leader peptide of a heavy chain of a murine antibody is selected to be inserted into the expression vector as a secretion signal peptide, the signal peptide is positioned at the N-terminal of a variable region of each polypeptide chain of the antibody, and the amino acid sequence is as follows: MGWSCIILFLVATATGVHS (SEQ ID NO: 34).
In this example, a plurality of CD3/CD155 bispecific antibodies were prepared and 3 different configurations of CD3/CD155 bispecific antibodies were selected as shown in FIG. 1, FIG. 2, and FIG. 3, respectively, wherein FIG. 1 is a heterodimeric configuration of bispecific antibodies designated CD3-CD155 comprising two monovalent units, wherein one monovalent unit is the scFab-Fc form of anti-CD 3 and the other monovalent unit is the scFv-Fc form variable region amino acid of anti-CD 155; FIG. 2 is a double scFv (3-155-scFv) configuration; FIG. 3 shows a double scFab (3-155-fab) configuration.
In the above-mentioned antibody, the first polypeptide chain and the second polypeptide chain contain constant regions, and the constant regions are derived from human antibody IgG1, so that different amino acid mutations are introduced into the constant regions of the first polypeptide chain and the second polypeptide chain to form a knob-in-hole structure in order to reduce the generation of homodimer. Also, in order to prevent activation of crosslinking by the Fcγ receptor, a (L234A/L235A) mutation was introduced into the first and second polypeptide chain constant regions.
Example 2 bispecific binding protein expression and purification
Bispecific molecules binding to CD3 and CD155 were prepared by transient transfection of expcho-S cells (Gibco, cat No. a 29127) with pcdna3.4 vector carrying the gene encoding the CD3-CD155 chain. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4). Times.10 6 Individual/ml, 37 ℃,8% CO 2 The culture was continued overnight with shaking at 120 rpm. On the day of transfection, cells grew to 7X 10 6 -1×10 7 Each ml, when the viability was greater than 95%, transfection was prepared and the cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) 6 Taking plasmids containing the CD3 polypeptide chain and the CD155 polypeptide chain according to the mass ratio of 1:1 transfection into ExpiCHO-S cells with ExpiFectamine CHO transfection reagent (Gibco, cat. No. A29129), 37℃and 8% CO 2 Shaking culture at 120 rpm. Mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed for 18-22 hr, immediately adding transfected cells, mixing, and mixing at 32deg.C with 5% CO 2 Shaking culture at 120 rpm. On day 5 after transfection, 8ml of ExpiCHO Feed was added to the cells again, and culture was continued after mixing. The change of cell number and cell viability was observed daily, and cells were harvested by centrifugation after the cell viability was reduced to less than 80% or after 10-14 days of culture, and the supernatant was purified or frozen at-80 ℃.
The expressed supernatant was filtered with a 0.22 μm filter membrane, an antibody having an Fc domain was captured from the expressed supernatant by using a Mabselect prism A affinity column (cytova, cat# 17549854), the column was equilibrated with phosphate buffer at pH7.2, the supernatant was passed through the affinity column, eluted with elution buffer (100 mM citric acid, pH 2.7), and finally concentrated and displaced with PBS buffer, and the purified antibody was identified to have a purity of 95% or more by SDS-PAGE, thereby obtaining a CD3-CD155 bispecific binding protein.
Example 3 determination of binding Activity of bispecific antibodies to antigens (ELISA)
Human CD3 antigen (ACRO biosystems, cat. No. CDD-H52W 1) or CD155 antigen (ACRO biosystems, cat. No. CD 5-H5223) was coated with a coating buffer (35 mM NaHCO) 3 ，15mM Na 2 CO 3 pH 9.6) was diluted to 2. Mu.g/mL and 100. Mu.L per well was added to the ELISA plate overnight at 4 ℃. Thereafter, the cells were washed 3 times with PBST (0.05% Tween20-PBS, pH 7.2). Direction boardTo this, 300. Mu.L of blocking buffer (1% BSA,0.05% Tween20-PBS, pH 7.2) was added, and the mixture was allowed to stand at room temperature for 2 hours. The mixture was washed 3 times with PBST. The corresponding bispecific antibody was added to each well and incubated for 1 hour at room temperature. The mixture was washed 3 times with PBST. Mu.l of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-097) diluted with blocking buffer was added to each well and incubated for 1 hour at room temperature. Washing with PBST for 3 times, adding TMB into each hole, reacting at room temperature in a dark place for 2-5 minutes, stopping the reaction with 2M sulfuric acid in each hole, and finally reading the OD450 value with an ELISA reader. FIG. 4 shows that the CD3-CD155 bispecific antibody of the present invention can bind to CD3, and FIG. 5 shows that the CD3-CD155 bispecific antibody of the present invention can bind to CD155.
Example 4 reporter Gene assay for bispecific binding proteins
The adherent tumor cells are transferred to a centrifuge tube for centrifugation after being digested, the suspension cells are directly transferred, 200g is centrifuged for 5min, the supernatant is discarded, and 1640 complete culture medium is added for cell resuspension counting. Target cells were diluted and then 2X 10 cells per well 4 Cells were added to 96-well plates. The jurkat cells (jurkat-NFAT-luc) carrying the luciferase gene were diluted in cell count and then 2X 10 per well was used 4 Cells were added to 96-well plates with little luciferase expression at the jurkat-NFAT-luc cell background and only after cell activation was the downstream NFAT signaling pathway stimulated to express luciferase. Adding bispecific binding protein CD3-CD155 in gradient concentration, mixing, and adding 5% CO 2 Incubate for 6h at 37 ℃. Then, 50. Mu.L of a luciferase substrate (Promega Co., E6120) per well was added thereto, and after mixing, the mixture was allowed to stand for 3 minutes, and then the luminescence value was read.
Specific experimental results show that as the concentration of CD3-CD155 bispecific antibody added increases, it detects a gradual increase in the chemiluminescent values of non-small cell lung cancer HCC827 cells (fig. 6), liver cancer HepG2 cells (fig. 7), pancreatic cancer HPAF-II cells (fig. 8) and colorectal cancer HCT-15 cells (fig. 9), indicating that bispecific binding proteins can significantly promote the activation of jurkat-NFAT-luc cells and the production of luciferase in the presence of CD155 positive tumor cells.
Example 5 in vitro killing assay of bispecific binding proteins
Digesting the tumor cells in the adherent cultureCounting, and adjusting cell density to 2×10 5 And each ml. The RTCA instrument (agilent) was turned on, the experimental mode was selected, and the cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 mu L of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plates, putting the plates into an instrument, closing the plates, and clicking the plates to start in the first step. After Done, the plate was removed, 100 μl of cell suspension was added, and left standing at room temperature for 15-30min to prevent edge effects, and placed into the instrument and clicked on. After growth to log phase, suspension was continued, 50. Mu.L of PBMC (1X 10) 6 And/ml) and add a gradient of bispecific antibody, click on, analyze after a period of time.
As the concentration of CD3-CD155 bispecific antibody increased, the killing efficiency of PBMC against liver cancer HepG2 cells (fig. 10), non-small cell lung cancer HCC827 cells (fig. 11) and pancreatic cancer HPAF-II cells (fig. 12) gradually increased, indicating that CD3-CD155 can well promote PBMC killing of cd155+ tumor cells.
Meanwhile, the scfv (3-155-scfv) and scfab (3-155-fab) diabodies constructed in example 1 were assayed for their ability to kill HCT-15 colorectal cancer cells in the same manner as described above, and the results show that CD3-CD155 bispecific antibodies employing scfab-scfv format have better ability to promote PBMC killing of HCT-15 colorectal cancer than other scfv (3-155-scfv) or scfab configuration (3-155-fab) as shown in FIG. 13.
Example 6 cytokine secretion assay
1. Effect of CD3-CD155 on PBMC cytokine secretion in the absence of target cells
Taking 5×10 4 Individual PBMCs were added to 96-well plates in a total volume of 200 μl. Then adding CD3-CD155 bispecific binding protein with gradient concentration as shown in the figure, and mixing. After 48 hours incubation at 37℃the supernatants were collected by centrifugation, the concentration of each cytokine in the supernatant was detected using the CBA human Th1/Th2 cytokine detection kit (BD, cat. No. 551809), and finally by flow cytometry.
FIGS. 14-16 show that the concentration of IL-6, TNF and IFN-gamma cytokines in the supernatant incubated in the absence of target cells, with a small increase in the level of the 3 cytokines secreted by PBMC with increasing CD3-CD155 antibody concentration, but with a small overall increase (up to 100 pg/mL) suggesting a potentially very good safety for the bispecific antibody.
2. Effect of CD3-CD155 on PBMC cytokine secretion in the presence of target cells
Taking 5×10 4 The PBMCs were added to 96-well plates, to which 1E4 colorectal cancer NCI-H716 cells or melanoma a375 cells were added in a total volume of 200 μl. Then adding CD3-CD155 bispecific binding protein with gradient concentration as shown in the figure, and mixing. After 48 hours incubation at 37℃the supernatants were collected by centrifugation, the concentration of each cytokine in the supernatant was detected using the CBA human Th1/Th2 cytokine detection kit (BD, cat. No. 551809), and finally by flow cytometry.
FIGS. 17-19 and 20-22 show the concentration of IL-6, TNF and IFN-gamma cytokines in the supernatant co-incubated in the presence of colorectal cancer NCI-H716 cells or melanoma A375 cells, with increasing concentrations of CD3-CD155 antibodies when target cells were present, the levels of these 3 cytokines secreted by PBMC were evident compared to non-target cells, but of a controlled magnitude, especially with levels of IL-6 associated with clinical side effects up to 100pg/mL, suggesting potentially good safety for the bispecific antibody.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (16)
1. A recombinant antibody comprising a first polypeptide chain and a second polypeptide chain:
The first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody, wherein the variable region CDR sequence of the CD3 antibody is shown in SEQ ID NO. 1-6 or has at least 85% of the amino acid sequence identical to SEQ ID NO. 1-6;
the second polypeptide chain comprises the variable region CDR sequence of a CD155 antibody, wherein the variable region CDR sequence of the CD3 antibody is as shown in SEQ ID NO. 7-12 or an amino acid sequence having at least 85% identity to SEQ ID NO. 7-12.
2. The recombinant antibody according to claim 1, wherein the recombinant antibody consists of a first polypeptide chain and a second polypeptide chain:
the first polypeptide chain comprises the variable region CDR sequences of a CD3 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 5, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 6,
the second polypeptide chain comprises the variable region CDR sequences of a CD155 antibody: the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 8, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 12;
Optionally, the light chain variable region of the CD3 antibody has an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 13, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 14 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 14; and
The light chain variable region of the CD155 antibody has an amino acid sequence shown as SEQ ID NO. 15 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 15, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 16 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 16.
3. The recombinant antibody according to claim 1, wherein said first polypeptide chain comprises a scFab region and a first Fc region of a CD3 antibody and said second polypeptide chain comprises a scFv region and a second Fc region of a CD155 antibody;
optionally, the first polypeptide chain comprises an scFv region and a first Fc region of a CD3 antibody and the second polypeptide chain comprises an scFv region and a second Fc region of a CD155 antibody;
optionally, the first polypeptide chain comprises a scFab region and a first Fc region of a CD3 antibody, and the second polypeptide chain comprises a scFab region and a second Fc region of a CD155 antibody.
4. The recombinant antibody according to claim 3, wherein the first and second Fc regions are linked by a knob-intoo-hole structure.
5. The recombinant antibody according to claim 3, wherein at least a portion of the constant region of the CD3 antibody, the constant region of the CD155 antibody, the first Fc region and the second Fc region is derived from at least one of a murine antibody, a primate-origin antibody, or a mutant thereof;
optionally, at least a portion of the constant region of the CD3 antibody, the constant region of the CD155 antibody, the first Fc region, and the second Fc region is from human IgG1 or a mutant thereof.
6. The recombinant antibody according to claim 3, wherein said first polypeptide chain has an amino acid sequence as shown in SEQ ID No. 20 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID No. 20 and said second polypeptide chain has an amino acid sequence as shown in SEQ ID No. 21 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID No. 21;
optionally, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO. 24 or an amino acid sequence having at least 85% identity to an amino acid set forth in SEQ ID NO. 24, and the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO. 25 or an amino acid sequence having at least 85% identity to an amino acid set forth in SEQ ID NO. 25;
Optionally, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO. 28 or an amino acid sequence having at least 85% identity to the amino acid set forth in SEQ ID NO. 28, and the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 85% identity to the amino acid set forth in SEQ ID NO. 29.
7. The recombinant antibody according to claim 1, further comprising a signal peptide added to the N-terminus of the first polypeptide chain and/or the second polypeptide chain, the signal peptide having an amino acid sequence as shown in SEQ ID No. 34.
8. An isolated polynucleotide encoding the recombinant antibody of any one of claims 1-7.
9. An expression vector carrying the polynucleotide of claim 8.
10. A method of producing the recombinant antibody of any one of claims 1-7, comprising:
introducing the expression vector of claim 9 into a cell;
culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibodies;
Optionally, the cell is a eukaryotic cell.
11. A recombinant cell carrying the polynucleotide of claim 8 or the expression vector of claim 9.
12. A composition, comprising:
at least one of the recombinant antibodies of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, or the recombinant cell of claim 11.
13. Use of the recombinant antibody of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, the recombinant cell of claim 11, or the composition of claim 12 in the manufacture of a medicament for treating or preventing cancer whose cancer cell surface is positive for CD155.
14. The use according to claim 13, wherein the cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.
15. Use of the recombinant antibody of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, the recombinant cell of claim 11, the composition of claim 12 in the preparation of a kit for detecting CD3 and/or CD155.
16. A kit comprising the recombinant antibody of any one of claims 1-7 for detecting CD3 and/or CD155.
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