CN117186223A - anti-PD-L1 antibody, nucleic acid encoding same, preparation method and application - Google Patents

anti-PD-L1 antibody, nucleic acid encoding same, preparation method and application Download PDF

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CN117186223A
CN117186223A CN202210616273.3A CN202210616273A CN117186223A CN 117186223 A CN117186223 A CN 117186223A CN 202210616273 A CN202210616273 A CN 202210616273A CN 117186223 A CN117186223 A CN 117186223A
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amino acid
acid sequence
antibody
ser
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杨亚平
赵安
张丽娜
李芸
靳照宇
霍耐凡
杨袭
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Mingji Biopharmaceutical Beijing Co ltd
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Mingji Biopharmaceutical Beijing Co ltd
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Abstract

The invention discloses an anti-PD-L1 antibody, nucleic acid for encoding the same, a preparation method and application, wherein the antibody at least comprises a VHH, the VHH comprises complementarity determining regions CDR1, CDR2 and CDR3 in an amino acid sequence shown as SEQ ID NO. 30, the CDR1 comprises an amino acid sequence shown as SEQ ID NO. 20, the CDR2 comprises an amino acid sequence shown as SEQ ID NO. 21, and the CDR3 comprises an amino acid sequence shown as SEQ ID NO. 22. The anti-PD-L1 antibody has stronger PD-L1/PD-1 blocking activity, smaller anti-PD-L1 fragments and better tumor infiltration capacity and drug effect.

Description

anti-PD-L1 antibody, nucleic acid encoding same, preparation method and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-PD-L1 antibody, nucleic acid for encoding the same, a preparation method and application.
Background
In recent years, the field of tumor treatment has developed rapidly, and more advanced treatment schemes such as targeted treatment, immunotherapy and the like have been developed from classical treatment means mainly including surgery, radiotherapy and chemotherapy. In particular, tumor immunotherapy, represented by PD-L1/PD-1 inhibitors, significantly extends the survival of tumor patients in a number of tumor indications and has also been targeted for first-line therapy in a number of tumor indications (Nat Rev immunol.; 2020 nov;20 (11): 651-668). Tumor immunotherapy is the central focus in the field of development of new antitumor drugs as the most probable cure regimen for tumors.
The PD-L1 is found to be highly expressed on the surface of various tumors in tumor research, including melanoma, lung cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, intestinal cancer and the like. The tumor cells can combine with PD-1 on T cells through the high-expression PD-L1 to transmit immunosuppressive signals, so that the body can be immune tolerant to the tumor cells, and the growth and the metastasis of the tumor cells are facilitated. PD-L1 high expression is closely related to poor prognosis and drug resistance in tumor patients (Hematol Oncol Stem Cell Ther.,2014 Mar;7 (1): 1-17). anti-PD-L1 antibodies and anti-PD-1 antibodies exert a durable anti-tumor effect by blocking PD-L1/PD-1 signaling pathways. However, current anti-PD-L1 antibodies and anti-PD-1 antibodies have innate or secondary resistance in most tumor patients, with only a small fraction of patients benefiting.
Disclosure of Invention
In order to overcome the defects that the anti-PD-L1 antibody in the prior art has weaker activity of blocking PD-L1/PD-1 and has larger volume, so that tumor infiltration capacity is poor and the drug effect is influenced, the invention provides the anti-PD-L1 antibody, nucleic acid for encoding the same, a preparation method and application thereof, and the antibody has stronger PD-L1/PD-1 blocking activity, smaller volume and better tumor infiltration capacity and drug effect.
One of the technical schemes provided by the invention is as follows: an anti-PD-L1 antibody comprising at least one VHH comprising complementarity determining regions CDR1, CDR2 and CDR3 in the amino acid sequence depicted as SEQ ID No. 30.
In some embodiments, the CDR1, CDR2, and CDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering system, and in some specific embodiments, CDR1, CDR2, and CDR3 are determined according to Kabat numbering rules. In some preferred embodiments, the anti-PD-L1 antibody, the CDR1 comprises the amino acid sequence shown as SEQ ID NO:20, the CDR2 comprises the amino acid sequence shown as SEQ ID NO:21, and the CDR3 comprises the amino acid sequence shown as SEQ ID NO: 22.
The antibody according to one of the claims, wherein the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence as shown in SEQ ID NO:18, SEQ ID NO:17, SEQ ID NO:16 or SEQ ID NO: 15. In some embodiments, the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence as shown in SEQ ID NO. 18. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering system, and in some specific embodiments, CDR1, CDR2, and CDR3 are determined according to Kabat numbering rules.
In a preferred embodiment of the invention, the CDR1 comprises an amino acid sequence as shown in SEQ ID NO. 3, SEQ ID NO. 2 or SEQ ID NO. 1, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO. 6, SEQ ID NO. 5 or SEQ ID NO. 4, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO. 8 or SEQ ID NO. 7.
In some embodiments, the VHH comprises a CDR1 as shown in SEQ ID NO. 3, SEQ ID NO. 2 or SEQ ID NO. 3, a CDR2 as shown in SEQ ID NO. 6, SEQ ID NO. 5 or SEQ ID NO. 4, and a CDR3 as shown in SEQ ID NO. 8 or SEQ ID NO. 7.
In a more preferred embodiment of the present invention, the CDR1 comprises an amino acid sequence as shown in SEQ ID NO. 3, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO. 6, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO. 8; or, the CDR1 comprises an amino acid sequence shown as SEQ ID NO. 1, the CDR2 comprises an amino acid sequence shown as SEQ ID NO. 4, and the CDR3 comprises an amino acid sequence shown as SEQ ID NO. 7; alternatively, CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, CDR2 comprises the amino acid sequence shown as SEQ ID NO. 5, and CDR3 comprises the amino acid sequence shown as SEQ ID NO. 8.
In some embodiments, the VHH comprises CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO 3, SEQ ID NO 6 and SEQ ID NO 8, respectively; or, the VHH comprises a CDR1, a CDR2 and a CDR3 with amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 7 respectively; or, the VHH comprises CDR1, CDR2 and CDR3 with amino acid sequences shown as SEQ ID NO. 2, SEQ ID NO. 5 and SEQ ID NO. 8 respectively.
The antibody of one of the claims, wherein the VHH is a humanized VHH.
In a preferred embodiment of the invention, the VHH comprises the framework regions FR1, FR2, FR3 and FR4 in the amino acid sequence shown as SEQ ID NO. 30.
In a more preferred embodiment of the invention said FR1 comprises the amino acid sequence shown as SEQ ID NO. 23, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 24 and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14.
Preferably, the amino acid sequence of FR1 is shown as SEQ ID NO. 23, the amino acid sequence of FR2 is shown as SEQ ID NO. 11, the amino acid sequence of FR3 is shown as SEQ ID NO. 24, and the amino acid sequence of FR4 is shown as SEQ ID NO. 14.
In a still more preferred embodiment of the present invention, said FR1 comprises the amino acid sequence shown as SEQ ID NO. 10 or SEQ ID NO. 9, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 13 or SEQ ID NO. 12, and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14;
preferably, the amino acid sequence of FR1 is shown as SEQ ID NO. 10 or SEQ ID NO. 9, the amino acid sequence of FR2 is shown as SEQ ID NO. 11, the amino acid sequence of FR3 is shown as SEQ ID NO. 13 or SEQ ID NO. 12, and the amino acid sequence of FR4 is shown as SEQ ID NO. 14.
In a preferred embodiment of the invention, said FR1 comprises the amino acid sequence shown as SEQ ID NO. 10, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 13, and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14; or, the FR1 comprises an amino acid sequence as shown in SEQ ID NO. 9, the FR2 comprises an amino acid sequence as shown in SEQ ID NO. 11, the FR3 comprises an amino acid sequence as shown in SEQ ID NO. 12, and the FR4 comprises an amino acid sequence as shown in SEQ ID NO. 14; alternatively, the FR1 comprises the amino acid sequence shown as SEQ ID NO. 10, the FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, the FR3 comprises the amino acid sequence shown as SEQ ID NO. 12, and the FR4 comprises the amino acid sequence shown as SEQ ID NO. 14.
Preferably, the amino acid sequence of the FR1 is shown as SEQ ID NO. 10, the amino acid sequence of the FR2 is shown as SEQ ID NO. 11, the amino acid sequence of the FR3 is shown as SEQ ID NO. 13, and the amino acid sequence of the FR4 is shown as SEQ ID NO. 14; or the amino acid sequence of the FR1 is shown as SEQ ID NO. 9, the amino acid sequence of the FR2 is shown as SEQ ID NO. 11, the amino acid sequence of the FR3 is shown as SEQ ID NO. 12, and the amino acid sequence of the FR4 is shown as SEQ ID NO. 14; or the amino acid sequence of the FR1 is shown as SEQ ID NO. 10, the amino acid sequence of the FR2 is shown as SEQ ID NO. 11, the amino acid sequence of the FR3 is shown as SEQ ID NO. 12, and the amino acid sequence of the FR4 is shown as SEQ ID NO. 14.
The antibody according to one of the claims, wherein the amino acid sequence of the VHH is shown as SEQ ID NO. 18, SEQ ID NO. 17, SEQ ID NO. 16 or SEQ ID NO. 15, or has at least 90%, at least 95% or at least 99% sequence identity with the amino acid sequence shown as SEQ ID NO. 18, SEQ ID NO. 17, SEQ ID NO. 16 or SEQ ID NO. 15.
In a preferred embodiment of the invention, the amino acid sequence of the VHH is shown as SEQ ID NO. 18, SEQ ID NO. 17, SEQ ID NO. 16 or SEQ ID NO. 15.
The antibody of one of the claims, further comprising a heavy chain constant region.
In a preferred embodiment of the invention, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, or IgG 4.
Preferably, the heavy chain constant region is an Fc region of human IgG1, e.g., the heavy chain constant region comprises the amino acid sequence shown as SEQ ID NO. 19; in some embodiments, the constant region includes a variant constant region that does not alter the structure and function of the variable region of the antibody. A variety of such constant region variants have been disclosed in the art, such as an Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (U.S. Pat. No.6,737,056) in 238, 265, 270, 297, 327 and 329 (using the EU numbering system), or an Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (234, 235, 265, 329 (using the EU numbering system), or an Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (see U.S. Pat. No.7,371,826) in 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (using the EU numbering system), and the like. These mutations have been shown to confer new properties to antibodies without altering the function of the variable regions of the antibodies.
In a preferred embodiment of the invention, the VHH is linked to the heavy chain constant region by a linker; the linker is preferably a linker having a structure as shown in (G) 4 S) x G y A linker of the amino acid sequence shown, wherein x is independently selected from integers from 1 to 20 and y is independently selected from integers from 0 to 4; more preferably, the amino acid of the linkerThe sequence is shown as SEQ ID NO. 25.
In a still more preferred embodiment of the invention, the amino acid sequence of the antibody is as shown in SEQ ID NO. 29, SEQ ID NO. 28, SEQ ID NO. 27 or SEQ ID NO. 26, or has at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 29, SEQ ID NO. 28, SEQ ID NO. 27 or SEQ ID NO. 26; and/or, the antibody has 2 polypeptide chains.
In a preferred embodiment of the invention, the full length amino acid sequence of the antibody is shown as SEQ ID NO. 29, SEQ ID NO. 28, SEQ ID NO. 27 or SEQ ID NO. 26. In some embodiments, the antibody has 2 polypeptide chains; and the amino acid sequence of the polypeptide chain is shown as SEQ ID NO. 29.
The second technical scheme provided by the invention is as follows: an isolated nucleic acid encoding an antibody according to one of the claims.
The third technical scheme provided by the invention is as follows: a recombinant expression vector comprising an isolated nucleic acid according to claim two.
In a preferred embodiment of the invention, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; for example, the backbone of the plasmid is pcDNA3.4.
The technical scheme provided by the invention is as follows: a transformant comprising the recombinant expression vector according to claim III.
In a preferred embodiment of the invention, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.
In a more preferred embodiment of the invention, the eukaryotic cell is a yeast cell or a mammalian cell.
In still more preferred embodiments of the invention, the mammalian cells are, for example, HEK293 cells.
The technical scheme provided by the invention is as follows: a method of making an anti-PD-L1 antibody, the method comprising the steps of:
culturing the transformant according to the fourth aspect, and obtaining the anti-PD-L1 antibody from the culture.
The technical scheme provided by the invention is as follows: a multispecific antibody comprising an anti-PD-L1 antibody according to one of the claims, and one or more additional antigen-binding properties of the antibody linked to the anti-PD-L1 antibody.
The seventh technical scheme provided by the invention is as follows: a pharmaceutical composition comprising an anti-PD-L1 antibody according to one of the claims, and a pharmaceutically acceptable carrier.
In a preferred embodiment of the invention, the pharmaceutical composition further comprises other agents; the other agent is preferably one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
The eighth technical scheme provided by the invention is as follows: the use of an anti-PD-L1 antibody according to one of the claims, a nucleic acid according to the second of the claims, a recombinant expression vector according to the third of the claims, a transformant according to the fourth of the claims, a multispecific antibody according to the sixth of the claims, or a pharmaceutical composition according to the seventh of the claims for the preparation of a medicament for the prevention and/or treatment of a tumor.
Preferably, the tumor is, but is not limited to, lymphoma, breast, ovarian, prostate, pancreatic, renal, lung, liver, stomach, colorectal, bladder, rhabdomyosarcoma, esophageal, cervical, multiple myeloma, leukemia, gall bladder, glioblastoma, or melanoma.
The technical scheme provided by the invention is as follows: a kit comprising an anti-PD-L1 antibody according to one of the claims, a nucleic acid according to the second of the claims, a recombinant expression vector according to the third of the claims, a transformant according to the fourth of the claims, a multispecific antibody according to the sixth of the claims, or a pharmaceutical composition according to the seventh of the claims.
In a preferred embodiment of the invention, the kit further comprises (i) a device for administering an antibody or pharmaceutical composition; and/or (ii) instructions for use.
The tenth technical scheme provided by the invention is as follows: a packaged medicine box, which comprises a medicine box A and a medicine box B, wherein,
the kit A contains the anti-PD-L1 antibody according to one of the technical schemes, the multispecific antibody according to the sixth technical scheme or the pharmaceutical composition according to the seventh technical scheme.
The kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
The eleventh technical scheme provided by the invention is as follows: a method for immunodetection or determination of PD-L1, said method comprising mixing a sample to be detected with an anti-PD-L1 antibody according to one of the claims, a nucleic acid according to a second of the claims, a recombinant expression vector according to a third of the claims, a transformant according to a fourth of the claims, a multispecific antibody according to a sixth of the claims, or a pharmaceutical composition according to a seventh of the claims.
In a preferred embodiment of the invention, the detection is for non-diagnostic purposes.
The twelve technical schemes provided by the invention are as follows: a method of diagnosing, treating and/or preventing a tumor, the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-PD-L1 antibody according to one of the claims, a multispecific antibody according to claim six, or a pharmaceutical composition according to claim seven; in some embodiments, the medicament is for preventing and/or treating a tumor; preferably, the tumor is, but is not limited to, lymphoma, breast, ovarian, prostate, pancreatic, renal, lung, liver, stomach, colorectal, bladder, rhabdomyosarcoma, esophageal, cervical, multiple myeloma, leukemia, gall bladder, glioblastoma, or melanoma.
In some embodiments, the method further comprises administering to the patient in need thereof a second therapeutic agent, preferably comprising other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
The technical scheme provided by the invention is as follows: the anti-PD-L1 antibody according to one of the claims, the multispecific antibody according to the sixth claim, or the pharmaceutical composition according to the seventh claim as a medicament;
in some embodiments, the medicament is for preventing and/or treating a tumor; preferably, the tumor is, but is not limited to, lymphoma, breast, ovarian, prostate, pancreatic, renal, lung, liver, stomach, colorectal, bladder, rhabdomyosarcoma, esophageal, cervical, multiple myeloma, leukemia, gall bladder, glioblastoma, or melanoma.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The anti-PD-L1 antibody has stronger PD-L1/PD-1 blocking activity, smaller anti-PD-L1 fragments and better tumor infiltration capacity and better drug effect.
Drawings
FIG. 1 shows the PD-L1/PD-1 blocking activity of monoclonal antibodies as measured by the reporter gene method in example 5 of the invention.
FIG. 2 shows the T cell modulating activity of the mab of example 6 of this invention.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the present invention were commercially available according to conventional methods and conditions, or selected according to the commercial specifications, without specifying the specific conditions in the examples below.
The terms used in the present invention are explained specifically as follows:
as used herein, the term "antibody" is used in its broadest sense to encompass monoclonal antibodies, polyclonal antibodies, mono-specific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabody, triabody and tetrabodies, tandem di-scFv, tandem tri-scFv), and also traditional antibodies (tetrapeptide chain structural antibodies made from two identical heavy chains and two identical light chains joined by an interchain disulfide bond), as well as Fab, fab ', F (ab') 2, fv, linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies peptabody, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies) having antigen binding activity. Conventional antibodies (also known as "full length antibodies" or "full antibodies") are typically about 150,000 daltons of iso-tetralin proteins, which are tetrapeptide chain structures made up of two identical light chains (L) and two identical heavy chains (H) linked by interchain disulfide bonds. Full length antibodies each heavy chain consists of a heavy chain variable region (abbreviated VH in the present invention) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are divided into alpha, delta, epsilon, gamma and mu heavy chains. Mammalian light chains are classified as either lambda or kappa light chains. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are immunoglobulin (Ig) A, igD, igE, igG and IgM. The complete antibody forms a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) joined together with disulfide bonds (inter-chain) formed in the hinge. Heavy chains gamma, alpha and delta have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and "CH3 domain", respectively. Each arm of Y includes a variable region and a first constant region (CH 1) of a single heavy chain bound to a single light chain. The "Fc" region is two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domains. Various Fc constant region variants have been disclosed in the art, such as Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (U.S. Pat. No.6,737,056) in 238, 265, 270, 297, 327 and 329 (using EU numbering system), or Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (234, 235, 265, 329 (using EU numbering system), or Fc of the heavy chain constant region of an antibody having one or more amino acid substitutions (see U.S. Pat. No.7,371,826) in 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (using EU numbering system), and the like. These mutations have been shown to confer new properties to antibodies without altering the function of the variable regions of the antibodies.
As used herein, the term "PD-L1" includes any variant or isoform of PD-L1 that is naturally expressed by a cell. The antibodies of the invention are cross-reactive with PD-L1 from a non-human species such as cynomolgus monkey. Alternatively, the antibody may also be specific for human PD-L1 and may not exhibit cross-reactivity with other species. PD-L1 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them or produced by recombinant techniques using techniques common in the art and described herein.
As used herein, the terms "VHH" and "nanobody" have the same meaning and are used interchangeably to refer to nanobodies in which the variable region of the heavy chain of the antibody is cloned, consisting of only one variable region of the heavy chain, which has complete antigen binding function. The VHH specific binding epitope need not be recognized together with other antigen binding domains (unlike conventional tetrapeptide chain structural antibodies, which have epitopes recognized together by pairs of VL and VH). VHH are small stable and efficient antigen recognition units formed from a single heavy chain variable domain. The nano antibody has excellent biological characteristics, has a molecular weight of 12-15kDa, is one tenth of that of an intact antibody, has good tissue penetrability, high specificity and good water solubility. Because of the special structural property, the antibody has the advantages of the traditional antibody and the small molecular medicine, almost perfectly overcomes the defects of long development period, lower stability, harsh preservation condition and the like of the traditional antibody, gradually becomes an emerging force in the treatment of the new generation antibody, and shows wide application prospect in immunodiagnosis and treatment. VHHs include, but are not limited to, natural antibodies produced by camelids, either humanized after antibodies produced by camelids or by phage display technology screening. Methods for obtaining VHH binding to a specific antigen or epitope have been previously disclosed in, for example, the following documents: van der Linden et al Journal of Immunological Methods,240 (2000) 185-195; li et al, J Biol chem, 287 (2012) 13713-13721; deffar et al African Journal of Biotechnology Vol.8 (12), pp.2645-2652, 17June,2009 and WO94/04678.
The term "variable region" or "variable domain" refers to the domain of an antibody that is involved in binding an antigen by an antibody in the heavy or light chain. VHH, VH and VL each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). Wherein the term "complementarity determining region" or "CDR" refers to a region within the variable domain that primarily contributes to binding to an antigen; "framework" or "FR" refers to variable domain residues other than CDR residues. VH or VHH comprise 3 CDR regions: CDR1, CDR2, and CDR3; VL comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.
It is well known to those skilled in the art that CDRs of antibody variable regions can be defined by a variety of methods, such as Chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273, 927-948 (1997)), kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (world Wide Web. Cis. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures. It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. The various numbering systems with their corresponding CDRs are well known to those skilled in the art as shown in table 1:
TABLE 1 antibody CDR definition methods
Kabat AbM Chothia Contact IMGT
LCDR1 L24–L34 L24–L34 L24–L34 L30–L36 L27–L32
LCDR2 L50–L56 L50–L56 L50–L56 L46–L55 L50–L52
LCDR3 L89–L97 L89–L97 L89–L97 L89–L96 L89–L96
HCDR1 H31–H35 H26–H35 H26–H32 H30–H35 H26–H35
HCDR2 H50–H65 H50–H58 H52–H56 H47–H58 H51–H57
HCDR3 H95–H102 H95–H102 H95–H102 H93–H101 H93–H102
Remarks: in Table 1, wherein Laa-Lbb refers to the amino acid sequence from aa to bb according to its corresponding coding rule starting from the N-terminus of the antibody light chain; haa-Hbb refers to the amino acid sequence from aa to bb starting from the N-terminus of the heavy chain of the antibody according to its corresponding coding rules. For example, L24-L34 in the second row and column of Table 1 refers to the amino acid sequence determined from residues 24 to 34 according to the Kabat coding rule starting from the N-terminus of the light chain variable region of the antibody; and so on.
As used herein, the term "nucleic acid" refers to a strand of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analogue thereof, or any substrate capable of incorporation into a strand by a DNA or RNA polymerase.
As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed intracellularly. The vectors of the invention are not generally naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the invention may comprise any type of nucleotide, including, but not limited to, the following DNA and RNA: it may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and it may contain natural, non-natural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In exemplary aspects, the altered nucleotide or non-naturally occurring internucleotide linkages do not hinder transcription or replication of the vector.
The recombinant expression vector of the invention may be any suitable recombinant expression vector that can be used for transformation or transfection to deliver and preferably express one or more genes or sequences of interest into any suitable host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of which include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
As used herein, the term "host cell" refers to any type of cell that may contain a nucleic acid or vector as described herein. The host cell may be a eukaryotic cell, such as a plant, animal, fungus or seaweed; or the host cell may be a prokaryotic cell, such as a bacterium or protozoan. As described herein, a host cell may be a cell derived from or obtained from an individual. The host cell may be derived or obtained from a mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to, mammals of the order Rodentia (order Rodentia), such as mice and hamsters; and mammals of the order Lagomorpha (order Lagomorpha), such as rabbits. Preferably, the mammal is from the order Carnivora (order Carnivora), including felines (cats) and canines (dogs). More preferably, the mammal is from the order artiodactyla (order Artiodactyla), including bovine (bovine) and porcine (porcine), or is of the order singular (order Perssodactyla), including equine (equine). Most preferably, the mammal belongs to the order primates (order primates), the new world monkeys (Ceboids) or the lemois (Simoids) (monkeys) or belongs to the ape subgenera (order Anthropoids) (humans and apes). Particularly preferred mammals are humans.
The expression vector may be transfected or introduced into a suitable host cell. Various techniques may accomplish this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposons (sleep Beauty or PiggyBAC), gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are incubated in medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used, based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to an auxotrophic host, and the like. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, transcriptional promoters, enhancers, and termination signals.
As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies having two or more epitope specificities. Such multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL units have two or more epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies with two or more VHHs, each binding to a different target or a different epitope of the same target.
As used herein, the term "pharmaceutically acceptable carrier" is any of those conventionally used, and is limited only by physical-chemical considerations (such as solubility and lack of reactivity with antibodies targeting PD-L1), and by the route of administration. Pharmaceutically acceptable carriers, such as vehicles, adjuvants, excipients and diluents, described herein are well known to those skilled in the art and are readily available to the public. In one aspect, the pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of the pharmaceutical composition and is a carrier that does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that may be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like; the use thereof is well known in the art.
Therapeutic formulations of compositions, such as polypeptides, polynucleotides or antibodies, suitable for use in practicing the methods disclosed herein can be prepared for storage by mixing a selected composition of the desired purity in the form of a lyophilized cake or aqueous solution with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 18 th edition, ed. A.r. gennaro, mark publication (Mack PublishingCompany) (1990)). The pharmaceutical compositions may be manufactured by blending with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers, suspending agents and the like. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, ointments, salves or in other conventional forms.
The composition used for in vivo administration should be sterile. This is easily achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution. The therapeutic composition is typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some cases, the form should be sterile and should be fluid to the extent that easy injection is possible. It should be stable under the conditions of manufacture and storage and should be preserved from the contaminating action of microorganisms such as bacteria and fungi. Compositions for parenteral administration will generally be stored in lyophilized form or in solution.
The carrier may be a solvent or dispersion medium containing, for example, water or a suitable mixture thereof and vegetable oil. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
The choice of carrier may be determined by the particular type of pharmaceutical composition of the antibody targeting PD-L1 and the route of administration of the pharmaceutical composition. Accordingly, there are a variety of suitable pharmaceutical composition formulations.
The pharmaceutical compositions of the present invention may comprise any pharmaceutically acceptable ingredient, including, for example, acidulants, additives, adsorbents, aerosol propellants (aerosol propellant), air displacement agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antimicrobial agents (anti-aging), matrices, binders, buffers, chelating agents, coating agents, colorants, desiccants, cleaners, diluents, disinfectants (districtants), disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, warming agents, lubricants, mucoadhesives, ointment matrices, ointments, oily vehicles, organic bases, lozenge matrices, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrating agents, solubilizing agents, solvents, stabilizers, suppository matrices, surfactants (surface active agent), surfactants (surfactants), suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxic agents, water-soluble binders, water-softeners, or water-miscible softeners.
The pharmaceutical compositions of antibodies targeting PD-L1 described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal or intraperitoneal administration. The pharmaceutical compositions may be administered by nasal, spray, oral, aerosol, rectal or vaginal administration. The composition may also be administered by infusion, bolus injection, or by implantation of a device.
It will be appreciated by those skilled in the art that in addition to the pharmaceutical compositions described above, the compositions of the present invention may be formulated as inclusion compounds, such as cyclodextrin inclusion compounds, or liposomes.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines.
The term "epitope" refers to a region (area or region) on an antigen that is capable of specifically binding to an antibody. Epitopes can be formed from or comprise a contiguous amino acid string (linear epitope) or comprise a non-contiguous amino acid (conformational epitope), for example becoming spatially close due to folding of the antigen (i.e. tertiary folding of the antigen by the nature of the protein). Conformational epitopes differ from linear epitopes in that: in the presence of denaturing solvents, binding of the antibody to conformational epitopes is lost. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind a particular epitope (i.e., those that bind the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see meth. Mol. Biol.248 (2004) 443-463),
The term "specific binding" refers to an antibody binding to an antigen or epitope within the antigen with a higher affinity than other antigens or epitopes. Typically, the antibodies are present in an amount of about 1X 10 -7 M or less (e.g., about 1X 10) -8 M or less, about 1X 10 - 9 M or less, about 1X 10 -10 M or less, about 1X 10 -11 M or less, or about 1X 10 -12 M or less) binds to an antigen or an epitope within an antigen. In some embodiments, the antibody binds to an antigen with a KD of 10%, or 1% of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using standard procedures, e.g., byAs measured by surface plasmon resonance. However, antibodies that specifically bind to an antigen or an epitope within an antigen mayCross-reactivity to other related antigens, for example cross-reactivity to the same antigen from other species (homologous), such as humans or monkeys, e.g. cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (common marmoset).
As used herein, the term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). As used herein, unless otherwise indicated, "affinity" refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kasloc" or "ka" refers to the rate of association of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein refers to the rate of dissociation of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and is expressed as molar concentration (M). The KD values of antibodies can be determined using well established methods in the art. Methods for determining antibody KD include measuring surface plasmon resonance using a biosensing system such as a system, or measuring affinity in solution by Solution Equilibrium Titration (SET).
The terms "anti-PD-L1 antibody", "an antibody that specifically binds to PD-L1" refer to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting PD-L1. In certain embodiments, the antibodies that bind to PD-L1 have the following dissociation constants (KD)<About 1 mu M,<About 100nM,<About 10nM,<About 1nM,<About 0.1nM,<About 0.01nM or<About 0.001nM (e.g. 10 -8 M or less, e.g. 10 -8 M to 10 -12 M, e.g. 10 -9 M to 10 -10 M). In certain embodiments, the anti-PD-L1 antibodies bind to epitopes conserved in PD-L1 from different species.
Example 1: PD-L1 antibody phage library construction
According to the crystal structure of the human PD-L1 protein, the variable region (variable region) sequence of the human PD-L1 nanobody is designed, VHH sequence is obtained through a gene synthesis technology, and a phage vector (pComb 3XSS, purchased from Beijing allied biosystems) is loaded by using restriction enzymes (XhoI, ncoI, purchased from NEB) to obtain a recombinant plasmid library. Phage plasmids carrying the VHH gene were used to electroporate and transform into SS320 E.coli (SS 320 (MC 1061F') electrocompetent cells, purchased from Lucigen). After proliferation of SS320 to log phase, helper phage (M13K 07, purchased from NEB) was added for infection, cultured overnight, phage were extracted from culture supernatant, and PD-L1 antibody phage library was obtained.
Example 2: screening of PD-L1 antibodies from antibody phage libraries
PD-L1 antibodies were screened from a PD-L1 antibody phage library.
Three rounds of phage display library panning were performed using a protein-based phage antibody panning technique. The first round screening method is as follows: human PD-L1-mFc antigen (purchased from Baipusais) is coated in an ELISA plate, a corresponding phage library is taken, an equal volume of 2% skimmed milk is added for premix incubation, and after the premix is added into a coating hole for reaction, sterile PBST is washed to remove the premix. Phage adsorbed in the well plate were eluted using 75mM sodium citrate buffer; after neutralization of the phage library, the phage library after 100-fold screening was amplified using M13K07 helper phage. The second and third rounds of screening were then performed in a manner similar to the first round of screening. Enriched phage libraries were obtained after three rounds of screening. Enrichment of phage was monitored by the amount of starting phage selected for each round and the titer of phage collected after the selection.
The enriched PD-L1 phage library was used to infect SS320 E.coli (SS 320 (MC 1061F') electrotransduce competent cells, purchased from Lucigen) and then plated on agarose plates for culture. The monoclonal colony is picked, placed into a 96-well deep-hole plate and cultured by 2YT containing ampicillin and kanamycin based on shaking culture at 37 ℃ to obtain a supernatant containing the monoclonal phage. Monoclonal phage supernatants were incubated with the ELISA plates coated with human PD-L1-mFc antigen for 1 hour, then washed with sterile PBST, then Anti-M13-HRP (available from Yinqiao Shenzhou) was added and incubated at 4℃for 30 minutes. And then detecting the combination of phage and antigen by using an enzyme-labeled instrument, and screening the PD-L1 monoclonal phage with strong combination with the antigen.
Example 3: preparation of recombinant PD-L1 monoclonal antibody
The cDNA sequence (VHH) of the variable region of the screened PD-L1 monoclonal phage was cloned into pcDNA3.4 vector (Invitrogen) already containing the antibody constant region, thus obtaining a plurality of anti-PD-L1 monoclonal antibodies, designated 1029, 1031, 1102, 1541, respectively. The Complementarity Determining Region (CDR) sequences of the monoclonal antibodies are shown in table 2 (determined by the Kabat CDR system), the Framework Region (FR) sequences of the monoclonal antibodies are shown in table 3, the amino acid sequences of the variable regions (VHH) are shown in table 4, the amino acid sequences of the linker and heavy chain constant regions of the monoclonal antibodies are shown in table 5, and the full length amino acid sequences of the monoclonal antibodies are shown in table 6. Plasmids were transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant was harvested. Purifying the supernatant by protein A to obtain the purified anti-PD-L1 monoclonal antibody.
TABLE 2 CDR sequences of monoclonal antibodies
Note that: the amino acids are bolded and underlined in the table, which are distinguished from 1029. Wherein X is 1 Is D or E; x is X 2 Is S or T; x is X 3 Is K or Q; x is X 4 Is G or S; x is X 5 Is S or T; x is X 6 Is D or E; x is X 7 Is K or N.
TABLE 3 FR sequences of monoclonal antibodies
Note that: the amino groups are bolded and underlined in the table as distinguished from 1029 And (3) acid. Wherein X is 8 Is L or F; x is X 9 Is A or S; x is X 10 Is K or R; x is X 11 Is A or T.
TABLE 4 amino acid sequences of VHH of monoclonal antibodies
Note that: the CDR sequences described above are bolded and italicized in the table; the amino acids that are distinguishable from 1029 are bolded and underlined.
The amino acid sequence of the general formula of VHH (SEQ ID NO: 30):
EVQLVESGGGLVQPGGSLRLSCAASGFTX 8 X 1 YYX 2 X 3 CWFRQAPGKEREWVSCIX 4 X 5 SX 6 GSTYYADSVKGRFTISRDNX 9 KNTVYLQMNSLX 10 X 11 EDTAVYYCAARX 7 GGPLTIENFFDYWGQGTQVTVSSA
wherein X is 1 Is D or E; x is X 2 Is S or T; x is X 3 Is K or Q; x is X 4 Is G or S; x is X 5 Is S or T; x is X 6 Is D or E; x is X 7 K or N; x is X 8 Is L or F; x is X 9 Is A or S; x is X 10 Is K or R; x is X 11 Is A or T.
TABLE 5 amino acid sequences of the linker and heavy chain constant regions of monoclonal antibodies
TABLE 6 full length amino acid sequence of monoclonal antibodies
Note that: the CDR sequences described above are bolded and italicized in the table; the amino acids that are distinguishable from 1029 are bolded and underlined.
Example 4: binding detection of recombinant PD-L1 monoclonal antibodies to PD-L1
Monoclonal antibodies were tested for their binding capacity to human PD-L1, as well as for species specificity, using enzyme-linked immunosorbent assay (ELISA). The specific method comprises the following steps: antigen human (human) PD-L1, cynomolgus monkey (cynomolgus) PD-L1, rat (rat) PD-L1, mouse (mouse) PD-L1 (all purchased from baipasi), 1 μg/mL, 100 μl per well, overnight incubation at 4 ℃ were coated separately in an elisa plate with carbonate buffer at ph=9.6. PBST was washed 5 times. Blocking with 300. Mu.L/well of PBST containing 1% BSA was performed and incubated for 1 hour at room temperature. PBST was washed 5 times. Monoclonal antibodies were added in a gradient diluted with 1% BSA in PBST, and control PD-L1 mab Durvalumab (IMGT database ID 10010) was added, 100. Mu.L per well, incubated for 1 hour at room temperature. PBST was washed 5 times. HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, cat# 109-035-088) diluted with PBST containing 1% bsa was added and incubated at room temperature for 1 hour at 100 μl per well. PBST was washed 5 times. The colorimetric substrate TMB was added and 100. Mu.L per well was developed for 10 minutes at room temperature, followed by the addition of 1M sulfuric acid to terminate the reaction. Reading OD on a microplate reader 450nm Results were analyzed and EC was calculated using 4-parameter fit to the binding curve 50 。
As shown in table 7, monoclonal antibodies 1029, 1031, 1102, 1541 each bound to human PD-L1 and cynomolgus monkey PD-L1 and were comparable to the control antibodies. The mouse PD-L1 cannot bind with the rat PD-L1.
TABLE 7 binding of monoclonal antibodies to antigens
NB: not combined with
Interactions between monoclonal antibodies and antigens were detected using a non-labeled biomolecular interaction analyzer Gator based on the principle of Biological Layer Interference (BLI). The specific method comprises the following steps: using the PA probe, the mab was diluted to 50nM and added to the probe card, so that the mab was captured by the PA probe. Then adding antigen human PD-L1 which starts from 200nM and performs gradient dilution, wherein the antigen interacts with the bispecific antibody captured by the PA probe, and analyzing the interaction by detecting the signal change of the reflection interference spectrum of the surface of the probe, so as to finally calculate the binding kinetic constant of the antibody.
The results are shown in table 8, with binding kinetics constants between 4.37 and 8.30nM for monoclonal antibodies 1029, 1031, 1102, 1541 to human PD-L1.
TABLE 8 kinetics of binding of monoclonal antibodies to antigens
Example 5: detection of PD-L1/PD-1 blocking Activity of PD-L1 monoclonal antibody by reporter Gene method
A reporter gene detection method for PD-L1/PD-1 blocking activity is established by using Jurkat/PD-1-NFAT-luciferase cells (highly expressed with PD-1 and a luciferase reporter gene under the control of NFAT response elements) and WIL2S/PD-L1 (highly expressed with PD-L1) cells constructed by Ming Ji Biotechnology and anti-CD 20/CD3 bispecific antibodies. The specific method comprises the following steps: WIL2S/PD-L1 cells were collected at a cell density of 4X 10 6 Per mL was resuspended in complete medium (RPMI 1640 containing 10% FBS), 50. Mu.L/well, i.e.2X10 per well 5 Individual cells, spread evenly into 96 well plates; jurrkat-PD-1-NFAT-luciferase cells were collected and assayed at a cell density of 4X 10 6 Per mL was resuspended in complete medium (RPMI 1640 containing 10% FBS), 50. Mu.L/well, i.e.2X10 per well 5 Individual cells were added uniformly to the 96-well plate; complete medium dilution of anti-CD 20/CD3 bispecific antibody, 25. Mu.L/well was added to the 96 well plate described above; complete medium serial dilutions of anti-PD-L1 monoclonal antibody sample and control antibody, 25 μl/well were added to the 96-well plate described above; then incubated at 37℃in a 5% CO2 incubator for 6 hours. After the incubation, 50. Mu.L/well of one-glo reagent (Promega, cat. E6120) was added to the 96-well plate, and the plate was placed on a shaker for 5 minutes with shaking and allowed to stand for 10 minutes. The relative chemiluminescent unit values (RLU) were then read on a microplate reader (MD, spectromax iD 3) using a chemiluminescent module and analyzed.
As shown in FIG. 1, monoclonal antibodies 1029, 1031, 1102, 1541 all blocked the PD-L1 from transmitting a negative signal to PD-1, and the antibodies were similar in activity but slightly more potent than the PD-L1 control monoclonal antibody Durvaumab.
Example 6: t cell regulatory activity of PD-L1 monoclonal antibody
The T cell regulatory activity of the monoclonal antibodies was tested using an MLR assay system with allogeneic DC cells and T cells incubated.
Donated human PBMC cells were resuspended in complete medium (RPMI 1640 with 10% fbs) and inoculated in 10cm cell culture dishes and incubated for 2 hours in a carbon dioxide incubator at 37 ℃. The culture supernatant and the suspension cells are discarded, and the adherent mononuclear cells are obtained. Monocytes were incubated in complete medium containing 100ng/mL GM-CSF (PeproTech, cat. No. 300-03) and 100ng/mL IL-4 (PeproTech, cat. No. 200-04), incubated for 6 days, changed every 2 days, and further incubated with TNF alpha and IL-1β (both purchased from PeproTech) for 2 days to give DC cells.
Allogeneic T cells were isolated from donated human PBMC cells, and specific isolation methods were described in the instructions of the Pan T cell isolation kit (Miltenyi Biotech, cat. No. 130-096-535). Briefly, PBMC were washed once with PBS and then 1X 10 PBMC were washed 7 Cells were resuspended in isolation buffer (2mM EDTA,0.5%BSA,pH =7.2 in PBS) at 40 μl (1×10 for each case 7 Cytometry), 10 μ L Pan T cell Biotin Antibody Cocktail was added and incubated for 5 minutes at 4 ℃. Then, 30. Mu.L of separation buffer and 20. Mu. L Pan T cell MicroBead Cocktail were added thereto, and incubated at 4℃for 10 minutes. And (5) passing through a MACS separation column to obtain the T cells.
Collecting obtained human DC cells and human T cells, re-suspending in complete culture medium (RPMI 1640 contains 10% FBS), inoculating into 96-well plate, and inoculating DC cells and T cells respectively 1×10 4 Well and 1X 10 5 And (3) mixing and culturing. And antibody samples diluted with complete medium sequences were added. The plates were incubated in a carbon dioxide incubator at 37℃for 5 days. After the incubation, the supernatant from the wells was removed and cytokine IFN-. Gamma.was detected using manual protocol in accordance with the kit (Biolegend, cat 430101).
The results are shown in figure 2, where monoclonal antibodies significantly enhance T cell activation.
SEQUENCE LISTING
<110> Mingji biopharmaceutical (Beijing) Co., ltd
<120> anti-PD-L1 antibody, nucleic acid encoding the same, preparation method and use thereof
<130> P22012584C
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR1 of antibody 1029
<400> 1
Asp Tyr Tyr Ser Lys Cys
1 5
<210> 2
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR1 of antibody 1031
<400> 2
Glu Tyr Tyr Thr Lys Cys
1 5
<210> 3
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR1 of antibody 1102 and antibody 1541
<400> 3
Asp Tyr Tyr Thr Gln Cys
1 5
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR2 of antibody 1029
<400> 4
Cys Ile Gly Ser Ser Asp Gly Ser Thr Tyr
1 5 10
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR2 of antibody 1031
<400> 5
Cys Ile Ser Thr Ser Glu Gly Ser Thr Tyr
1 5 10
<210> 6
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR2 of antibody 1102 and antibody 1541
<400> 6
Cys Ile Ser Ser Ser Glu Gly Ser Thr Tyr
1 5 10
<210> 7
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR3 of antibody 1029
<400> 7
Arg Asn Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
1 5 10
<210> 8
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR3 of antibody 1031, antibody 1102, antibody 1541
<400> 8
Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
1 5 10
<210> 9
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR1 of antibody 1029
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu
20 25
<210> 10
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR1 of antibody 1031, antibody 1102 and antibody 1541
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25
<210> 11
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR2 of antibody 1029, antibody 1031, antibody 1102 and antibody 1541
<400> 11
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ser
1 5 10
<210> 12
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> antibody 1029, antibody 1031, and FR3 of antibody 1102
<400> 12
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
1 5 10 15
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
20 25 30
Ala Val Tyr Tyr Cys Ala Ala
35
<210> 13
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR3 of antibody 1541
<400> 13
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
1 5 10 15
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
20 25 30
Ala Val Tyr Tyr Cys Ala Ala
35
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR4 of antibodies 1029, 1031, 1102 and 1541
<400> 14
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
1 5 10
<210> 15
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> VHH of antibody 1029
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ser Lys Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Gly Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asn Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120
<210> 16
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> VHH of antibody 1031
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Tyr Tyr
20 25 30
Thr Lys Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Thr Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120
<210> 17
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> VHH of antibody 1102
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Tyr
20 25 30
Thr Gln Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Ser Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120
<210> 18
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> VHH of antibody 1541
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Tyr
20 25 30
Thr Gln Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Ser Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120
<210> 19
<211> 231
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> heavy chain constant region sequence
<400> 19
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly
225 230
<210> 20
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR1 upper sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa is X1, X1 is D or E
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa is X2, X2 is S or T
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa is X3, X3 is K or Q
<400> 20
Xaa Tyr Tyr Xaa Xaa Cys
1 5
<210> 21
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR2 upper sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa is X4, X4 is G or S
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa is X5, X5 is S or T
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa is X6, X6 is D or E
<400> 21
Cys Ile Xaa Xaa Ser Xaa Gly Ser Thr Tyr
1 5 10
<210> 22
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> CDR3 upper sequence
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is X7, X7 is K or N
<400> 22
Arg Xaa Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
1 5 10
<210> 23
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR1 upper sequence
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa is X8, X8 is L or F
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Xaa
20 25
<210> 24
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> FR3 upper sequence
<220>
<221> misc_feature
<222> (16)..(16)
<223> Xaa is X9, X9 is A or S
<220>
<221> misc_feature
<222> (28)..(28)
<223> Xaa is X10, X10 is K or R
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa is X11, X11 is A or T
<400> 24
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa
1 5 10 15
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Thr
20 25 30
Ala Val Tyr Tyr Cys Ala Ala
35
<210> 25
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> linker sequence
<400> 25
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
<210> 26
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> full Length of antibody 1029
<400> 26
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ser Lys Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Gly Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asn Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Pro Lys Ser
130 135 140
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
145 150 155 160
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly
370
<210> 27
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> antibody 1031 full Length
<400> 27
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Tyr Tyr
20 25 30
Thr Lys Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Thr Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Pro Lys Ser
130 135 140
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
145 150 155 160
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly
370
<210> 28
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> antibody 1102 full Length
<400> 28
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Tyr
20 25 30
Thr Gln Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Ser Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Pro Lys Ser
130 135 140
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
145 150 155 160
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly
370
<210> 29
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> antibody 1541 full Length
<400> 29
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Tyr
20 25 30
Thr Gln Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Ser Ser Ser Glu Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Lys Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Pro Lys Ser
130 135 140
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
145 150 155 160
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly
370
<210> 30
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial sequence)
<220>
<223> VHH Supersequence
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa is X8, X8 is L or F
<220>
<221> misc_feature
<222> (30)..(30)
<223> Xaa is X1, X1 is D or E
<220>
<221> misc_feature
<222> (33)..(33)
<223> Xaa is X2, X2 is S or T
<220>
<221> misc_feature
<222> (34)..(34)
<223> Xaa is X3, X3 is K or Q
<220>
<221> misc_feature
<222> (52)..(52)
<223> Xaa is X4, X4 is G or S
<220>
<221> misc_feature
<222> (53)..(53)
<223> Xaa is X5, X5 is S or T
<220>
<221> misc_feature
<222> (55)..(55)
<223> Xaa is X6, X6 is D or E
<220>
<221> misc_feature
<222> (75)..(75)
<223> Xaa is X9, X9 is A or S
<220>
<221> misc_feature
<222> (87)..(87)
<223> Xaa is X10, X10 is K or R
<220>
<221> misc_feature
<222> (88)..(88)
<223> Xaa is X11, X11 is A or T
<220>
<221> misc_feature
<222> (100)..(100)
<223> Xaa is X7, X7 is K or N
<400> 30
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Xaa Xaa Tyr Tyr
20 25 30
Xaa Xaa Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Cys Ile Xaa Xaa Ser Xaa Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Xaa Gly Gly Pro Leu Thr Ile Glu Asn Phe Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120

Claims (15)

1. An anti-PD-L1 antibody comprising at least one VHH comprising complementarity determining regions CDR1, CDR2 and CDR3 in the amino acid sequence depicted as SEQ ID No. 30;
preferably, the CDR1 comprises the amino acid sequence shown as SEQ ID NO. 20, the CDR2 comprises the amino acid sequence shown as SEQ ID NO. 21, and the CDR3 comprises the amino acid sequence shown as SEQ ID NO. 22.
2. The antibody of claim 1, wherein the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown as SEQ ID No. 18, SEQ ID No. 17, SEQ ID No. 16 or SEQ ID No. 15;
Preferably, the CDR1 comprises an amino acid sequence as shown in SEQ ID NO. 3, SEQ ID NO. 2 or SEQ ID NO. 1, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO. 6, SEQ ID NO. 5 or SEQ ID NO. 4, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO. 8 or SEQ ID NO. 7;
more preferably, the CDR1 comprises the amino acid sequence shown as SEQ ID NO. 3, the CDR2 comprises the amino acid sequence shown as SEQ ID NO. 6, and the CDR3 comprises the amino acid sequence shown as SEQ ID NO. 8; or, the CDR1 comprises an amino acid sequence shown as SEQ ID NO. 1, the CDR2 comprises an amino acid sequence shown as SEQ ID NO. 4, and the CDR3 comprises an amino acid sequence shown as SEQ ID NO. 7; or, the CDR1 comprises an amino acid sequence shown as SEQ ID NO. 2, the CDR2 comprises an amino acid sequence shown as SEQ ID NO. 5, and the CDR3 comprises an amino acid sequence shown as SEQ ID NO. 8;
even more preferably, the VHH comprises CDR1, CDR2 and CDR3 of the amino acid sequences shown as SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, respectively.
3. The antibody of claim 1 or 2, wherein the VHH is a humanized VHH;
preferably, the VHH comprises the framework regions FR1, FR2, FR3 and FR4 in the amino acid sequence shown as SEQ ID NO. 30;
More preferably, said FR1 comprises the amino acid sequence shown as SEQ ID NO. 23, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 24, and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14;
even more preferably, said FR1 comprises the amino acid sequence shown as SEQ ID NO. 10 or SEQ ID NO. 9, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 13 or SEQ ID NO. 12, and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14;
most preferably, said FR1 comprises the amino acid sequence shown as SEQ ID NO. 10, said FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, said FR3 comprises the amino acid sequence shown as SEQ ID NO. 13, and said FR4 comprises the amino acid sequence shown as SEQ ID NO. 14; or, the FR1 comprises an amino acid sequence as shown in SEQ ID NO. 9, the FR2 comprises an amino acid sequence as shown in SEQ ID NO. 11, the FR3 comprises an amino acid sequence as shown in SEQ ID NO. 12, and the FR4 comprises an amino acid sequence as shown in SEQ ID NO. 14; alternatively, the FR1 comprises the amino acid sequence shown as SEQ ID NO. 10, the FR2 comprises the amino acid sequence shown as SEQ ID NO. 11, the FR3 comprises the amino acid sequence shown as SEQ ID NO. 12, and the FR4 comprises the amino acid sequence shown as SEQ ID NO. 14.
4. The antibody of any one of claims 1 to 3, wherein the amino acid sequence of the VHH is shown as SEQ ID No. 18, SEQ ID No. 17, SEQ ID No. 16 or SEQ ID No. 15 or has at least 90% sequence identity with the amino acid sequence shown as SEQ ID No. 18, SEQ ID No. 17, SEQ ID No. 16 or SEQ ID No. 15;
preferably, the amino acid sequence of the VHH is shown as SEQ ID NO. 18, SEQ ID NO. 17, SEQ ID NO. 16 or SEQ ID NO. 15.
5. The antibody of any one of claims 1 to 4, further comprising a heavy chain constant region;
preferably, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3 or IgG 4; the heavy chain constant region is preferably the Fc region of human IgG 1; more preferably, the heavy chain constant region comprises the amino acid sequence shown as SEQ ID NO. 19;
more preferably, the VHH is linked to a heavy chain constant region by a linker; the linker is preferably a linker having a structure as shown in (G) 4 S) x G y A linker of the amino acid sequence shown, wherein x is independently selected from integers from 1 to 20 and y is independently selected from integers from 0 to 4; more preferably a linker as shown in SEQ ID NO. 25;
even more preferably, the amino acid sequence of the antibody is as shown in SEQ ID NO. 29, SEQ ID NO. 28, SEQ ID NO. 27 or SEQ ID NO. 26, or has at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO. 29, SEQ ID NO. 28, SEQ ID NO. 27 or SEQ ID NO. 26; and/or, the antibody has 2 polypeptide chains;
Optimally, the antibody has 2 polypeptide chains; and the amino acid sequence of the polypeptide chain is shown as SEQ ID NO. 29.
6. An isolated nucleic acid encoding the antibody of any one of claims 1-5.
7. A recombinant expression vector comprising the isolated nucleic acid of claim 6; preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; for example, the backbone of the plasmid is pcDNA3.4.
8. A transformant comprising the recombinant expression vector of claim 7;
preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell;
more preferably, the eukaryotic cell is a yeast cell or a mammalian cell;
even more preferably, the mammalian cell is a HEK293 cell.
9. A method of making an anti-PD-L1 antibody, comprising the steps of:
culturing the transformant according to claim 8, and obtaining an anti-PD-L1 antibody from the culture.
10. A multispecific antibody comprising an anti-PD-L1 antibody of any one of claims 1-5, and one or more additional antigen-binding properties of the antibody linked to the anti-PD-L1 antibody.
11. A pharmaceutical composition comprising the anti-PD-L1 antibody of any one of claims 1-5, and a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition further comprises other agents; the other agent is preferably one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
12. Use of an anti-PD-L1 antibody according to any one of claims 1 to 5, a nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, a multispecific antibody according to claim 10, or a pharmaceutical composition according to claim 11 for the preparation of a medicament for the prevention and/or treatment of a tumor.
13. A kit comprising an anti-PD-L1 antibody according to any one of claims 1 to 5, a nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, a multispecific antibody according to claim 10, or a pharmaceutical composition according to claim 11;
Preferably, the kit further comprises (i) a device for administering an antibody or pharmaceutical composition; and/or (ii) instructions for use.
14. A kit of parts, characterized in that it comprises a kit A and a kit B, wherein,
the kit a contains an anti-PD-L1 antibody according to any one of claims 1 to 5, a multispecific antibody according to claim 10 or a pharmaceutical composition according to claim 11;
the kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
15. A method for immunodetection or determination of PD-L1, characterized in that said method comprises mixing a sample to be detected with an anti-PD-L1 antibody according to any one of claims 1 to 5, a nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, a multispecific antibody according to claim 10 or a pharmaceutical composition according to claim 11; preferably, the detection is for non-diagnostic purposes.
CN202210616273.3A 2022-05-31 2022-05-31 anti-PD-L1 antibody, nucleic acid encoding same, preparation method and application Pending CN117186223A (en)

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CN108997500A (en) * 2018-09-12 2018-12-14 首都医科大学附属北京胸科医院 A kind of anti human PD-L 1 antibody and its application
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