CN116813786A - anti-CD 73 antibody and application thereof - Google Patents

anti-CD 73 antibody and application thereof Download PDF

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Publication number
CN116813786A
CN116813786A CN202310968592.5A CN202310968592A CN116813786A CN 116813786 A CN116813786 A CN 116813786A CN 202310968592 A CN202310968592 A CN 202310968592A CN 116813786 A CN116813786 A CN 116813786A
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seq
chain variable
variable region
antibody
sequences shown
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徐汶新
徐德雨
胥阶英
丁列明
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Betta Pharmaceuticals Co Ltd
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Betta Pharmaceuticals Co Ltd
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Priority to CN202310968592.5A priority Critical patent/CN116813786A/en
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Abstract

The invention provides anti-CD 73 antibodies and uses thereof. The antibodies provided are capable of specifically binding CD73. The provided antibody has high affinity and specificity with antigen, has the effect of killing tumor, and can be applied to treating tumor.

Description

anti-CD 73 antibody and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to an anti-CD 73 antibody and application thereof.
Background
CD73, also known as extracellular-5 '-nucleotidase (ecto-5' NT, EC 3.1.3.5), has a protein molecular weight of about 70kDa and is an enzyme with Glucosyl Phosphatidylinositol (GPI) anchored on the cell surface. CD73 is present in most tissues, especially in the endothelial and hematopoietic cell subsets (Resta et al, immunol Rev 1998;161:95-109 and Colgan et al, prinergic Signal 2006; 2:351-60). CD39 upstream of CD73 can catalyze ATP to produce Adenosine Monophosphate (AMP), which is catalytically hydrolyzed by CD73 to adenosine. Adenosine is a widely studied signaling molecule whose biological effects are mediated by several receptors, including A1, A2A, A B and A3. In the tumor microenvironment, adenosine may not only enhance proliferation, angiogenesis and metastasis of tumors (Zhang et al, cancer Res 2010; 70:6407-11), but also significantly inhibit anti-tumor immune responses (f.ghirinhelleli et al, biomed. Res, int.2012). CD73 is therefore considered a powerful cancer therapeutic target.
CD73 is expressed on many different cancers, including colorectal Cancer, lung Cancer, pancreatic Cancer, ovarian Cancer, bladder Cancer, leukemia, glioma, glioblastoma, melanoma, thyroid Cancer, esophageal Cancer, prostate Cancer, and breast Cancer (Jin et al, cancer Res 2010;70:2245-55 and Stagg et al, PNAS2010; 107:1547-52). CD73 also affects various aspects of tumorigenesis, such as proliferation, differentiation, adhesion/migration, angiogenesis, and metastasis. It promotes proliferation of tumor cells by modulating cell cycle, apoptosis and signaling pathways such as EGFR, beta-catenin/cyclin D1, VEGF and AKT/ERK (Zhi X et al, clin Exp Metastasis 2007,24 (6): 439-448;Turcotte M etal, cancer Res2015,75 (21): 4494-4503; wu R et al, oncol Rep2016,35 (3): 1750-1756; gao ZW et al, BMC Cancer2017,17 (1): 135). Moreover, the high expression of CD73 is used as a biomarker and is closely related to the poor prognosis of various types of tumors, including breast cancer, lung cancer, ovarian cancer, gastric cancer, renal cancer, head and neck cancer and the like. In addition to cancer cells, various immune cells such as B cells, T cells, th cells, cd8+ T cells, and the like also express CD73 molecules (Ghasem G et al, 2019,23:2, 127-142). Given the critical role of CD73 in tumor growth and metastasis, it has become one of the hot targets in the field of tumor immunotherapy, and many companies have been in clinical phase 2 or clinical phase 1 studies, and no drugs have been marketed for this target.
Disclosure of Invention
The present invention provides anti-CD 73 antibodies and their use in the treatment of disease. The provided antibodies are capable of specifically binding CD73, exhibiting anti-tumor effects.
In a first aspect the invention provides an anti-CD 73 antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region: comprises the HCDR sequences shown in SEQ ID NO. 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 1, 2 and 3; or comprises the HCDR sequences shown in SEQ ID NOs 4, 5 and 6, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs 4, 5 and 6; or comprises the HCDR sequences shown in SEQ ID NO. 7, 8 and 9, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 7, 8 and 9; or comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS 10, 11 and 12; or comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO 13, 14 and 15; or comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS.16, 17 and 18; or comprises the HCDR sequences shown in SEQ ID NO. 19, 20 and 21, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 19, 20 and 21; the light chain variable region: comprises the LCDR sequences shown in SEQ ID NOs 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs 22, 23 and 24; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 25, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 25; or comprises the LCDR sequences shown in SEQ ID NOS.26, 27 and 28, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.26, 27 and 28; or comprises the LCDR sequences shown in SEQ ID NOS.29, 30 and 31, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.29, 30 and 31; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 32, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 32; or comprises the LCDR sequences shown in SEQ ID NOS.33, 34 and 35, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.33, 34 and 35; or comprises the LCDR sequences shown in SEQ ID NOS.36, 37 and 38, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.36, 37 and 38.
In a second aspect the invention provides an antibody or antigen binding fragment against CD73 comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3 wherein HCDR1 is selected from the sequences shown as SEQ ID No. 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequences shown as SEQ ID No. 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequences shown as SEQ ID No. 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequences shown as SEQ ID No. 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequences shown as SEQ ID No. 23 or 27 or 30 or 34 or 37, LCDR3 is selected from the sequences shown as SEQ ID No. 24 or 25 or 28 or 31 or 32 or 35 or 38.
In a third aspect the invention provides an anti-CD 73 antibody or antigen-binding fragment comprising: the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45 and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO 46 or 47 or 48 or 49 or 50 or 51 or 52.
In a fourth aspect the invention provides a bispecific antibody comprising a first antigen binding portion and a second antigen binding portion; a first antigen binding portion that specifically binds a first epitope, the first antigen binding portion comprising a heavy chain variable region comprising the HCDR sequences of SEQ ID NOs 1, 2 and 3 or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NOs 1, 2 and 3; or comprises the HCDR sequences shown in SEQ ID NOs 4, 5 and 6, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs 4, 5 and 6; or comprises the HCDR sequences shown in SEQ ID NO. 7, 8 and 9, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 7, 8 and 9; or comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS 10, 11 and 12; or comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO 13, 14 and 15; or comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS.16, 17 and 18; or comprises the HCDR sequences shown in SEQ ID NO. 19, 20 and 21, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 19, 20 and 21; the light chain variable region comprises the LCDR sequences shown in SEQ ID NOs 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs 22, 23 and 24; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 25, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 25; or comprises the LCDR sequences shown in SEQ ID NOS.26, 27 and 28, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.26, 27 and 28; or comprises the LCDR sequences shown in SEQ ID NOS.29, 30 and 31, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.29, 30 and 31; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 32, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 32; or comprises the LCDR sequences shown in SEQ ID NOS.33, 34 and 35, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.33, 34 and 35; or comprises the LCDR sequences shown in SEQ ID NOS.36, 37 and 38, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.36, 37 and 38;
The second antigen binding portion specifically binds to a second epitope, and the second antigen binding portion is fused to the first antigen binding portion.
In a fifth aspect of the invention there is provided a bispecific antibody comprising a first antigen binding portion and a second antigen binding portion; the first antigen binding portion specifically binds a first epitope, the first antigen binding portion comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein HCDR1 is selected from the sequences shown in SEQ ID NO 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequences shown in SEQ ID NO 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequences shown in SEQ ID NO 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequences shown in SEQ ID NO 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequences shown in SEQ ID NO 23 or 27 or 30 or 34 or 37, LCDR3 is selected from the sequences shown in SEQ ID NO 24 or 25 or 28 or 31 or 32 or 35 or 38; the second antigen binding portion specifically binds to a second epitope, and the second antigen binding portion is fused to the first antigen binding portion.
In a sixth aspect the invention provides a polynucleotide encoding an antibody or antigen-binding fragment according to any one of the first to third aspects above, or encoding a bispecific antibody according to any one of the fourth to fifth aspects.
In a seventh aspect the invention provides a construct comprising a polynucleotide according to the sixth aspect above.
In an eighth aspect, the invention provides a host cell comprising a polynucleotide as described in the sixth aspect or a construct as described in the seventh aspect.
A ninth aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment of any one of the first to third aspects above, or the bispecific antibody of the fourth to fifth aspects above; and a pharmaceutically acceptable carrier.
According to a tenth aspect of the present invention there is provided an antibody conjugate comprising an antibody or antigen-binding fragment as described in any one of the first to third aspects above, or a bispecific antibody as described in the fourth to fifth aspects; and a functional small molecule linked to the antibody or antigen-binding fragment or to the bispecific antibody.
An eleventh aspect of the present invention provides a kit comprising an antibody or antigen-binding fragment according to any one of the first to third aspects, or a bispecific antibody according to any one of the fourth to fifth aspects described above.
In a twelfth aspect the present invention provides a method of producing an antibody or antigen binding fragment as described above or producing a bispecific antibody as described above, comprising: culturing the host cell of the eighth aspect, and collecting the antibody or antigen-binding fragment or the bispecific antibody from the culture.
A thirteenth aspect of the invention provides a method of preventing and/or treating a disease comprising: administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment of any one of the first to third aspects, or a bispecific antibody of any one of the fourth to fifth aspects, or a pharmaceutical composition of the ninth aspect, or an antibody conjugate of the tenth aspect.
A fourteenth aspect of the present invention provides the use of an antibody or antigen-binding fragment according to any one of the first to third aspects, or a bispecific antibody according to any one of the fourth to fifth aspects, for the preparation of a medicament or kit or for the preparation of an antibody conjugate.
Drawings
Fig. 1 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.
Fig. 2 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.
Fig. 3 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.
Fig. 4 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.
Fig. 5 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.
FIG. 6 is a graph of endocytosis results of different anti-CD 73 antibodies provided according to an embodiment of the present invention.
FIG. 7 is a graph showing the anti-tumor effect of different anti-CD 73 antibodies combined with anti-PD-L1 antibodies according to an embodiment of the present invention.
FIG. 8 is a graph showing the results of blocking the enzymatic activity of different bispecific antibodies provided in accordance with an embodiment of the present invention.
Detailed Description
The following describes the technical scheme of the present invention in detail with reference to specific examples. Meanwhile, in order to facilitate understanding by those skilled in the art, it is to be noted that some terms of the present invention are explained and illustrated only for convenience of understanding by those skilled in the art, and should not be construed as limiting the scope of the present invention.
As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
The term "antibody" is used herein in its broadest sense to refer to a protein or polypeptide comprising an antigen binding site or antigen binding fragment or antigen binding portion, and encompasses natural and artificial antibodies of various structures, including but not limited to whole antibody forms or antigen binding fragments of antibodies. According to a specific embodiment, the mentioned antibodies are monoclonal antibodies.
Herein, the term "bispecific antibody" comprises both an antigen binding moiety that specifically binds to different epitopes of at least two different biomolecules, and an antigen binding moiety that specifically binds to at least two epitopes of the same biomolecule. For the sake of distinction, the portions of bispecific antibodies that bind to different epitopes of the same antigen or different antigens are described as "first antigen binding portion" and "second antigen binding portion", respectively, etc. The first antigen binding portion and the second antigen binding portion are not indicative of importance, nor of the order of binding to the antigen, only for distinguishing between different epitopes of the antigen or for distinguishing between binding to different antigens. The "first antigen binding portion" and "second antigen binding portion" will also be explained and described below. The order of binding to the antigen in the listed bispecific antibodies is arbitrary unless otherwise indicated. Of course, bispecific antibodies, when specifically binding to different biomolecules, are likely to bind to more than one epitope of that particular biomolecule. According to a preferred embodiment of the invention, the bispecific antibody comprises an antigen binding moiety that specifically binds to different epitopes of two different target antigens.
Herein, "whole antibody" or "whole antibody" and the like are used to represent the same meaning, indicating a protein comprising at least two heavy chains (H) and two light chains (L) disulfide-linked to each other. Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region includes a heavy chain constant domain CH1, a heavy chain constant domain CH2, and a heavy chain constant domain CH3. Each light chain consists of a light chain variable region (abbreviated VL) and a light chain constant domain CL. The VH and VL regions may be further divided into complementarity determining regions (also known as hypervariable or hypervariable regions, abbreviated CDRs) separated by conserved Framework Regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus (N-terminus) to carboxy-terminus (C-terminus) according to the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, from the amino terminus, and CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, from the amino terminus.
An "antigen binding fragment" or "antigen binding portion" (e.g., first antigen binding portion, second antigen binding portion) or "antigen binding site" is meant to encompass a portion of a full-length antibody that exhibits antigen binding properties or functions. The term "variable region" or "variable domain" refers to a domain that involves binding of an antigen to an antibody heavy or light chain. As mentioned above, hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen binding fragments or antigen binding portions include, but are not limited to, fab ', F (ab ') 2, bispecific Fab ' and Fv fragments (variable regions), linear antibodies, single chain antibodies, single domain antibodies, and the like. The complete antibody is digested with papain to produce two identical antigen binding fragments, called Fab fragments, each of which contains heavy and light chain variable regions and light and heavy chain constant domains CH1.Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. The complete antibody was digested with pepsin to obtain F (ab') 2 fragments. The F (ab ') 2 fragment has two antigen binding F (ab) moieties linked together by disulfide bonds, and the F (ab') 2 fragment is a diabody. Single chain antibodies are fusion proteins formed by joining the heavy and light chain variable regions of an antibody via a flexible short peptide of about 10-25 amino acids. Single domain antibodies are antibody fragments consisting of variable domains of a single monomer. Single domain antibodies are also commonly referred to as nanobodies, since they are typically derived from the variable region of the heavy chain of camelid antibodies or shark antibodies. Nanobodies contain only heavy chain CDR regions and are the smallest antigen-binding fragments with complete function. Nanobodies include three CDR regions (CDR 1-CDR 3) and four framework regions FR (FR 1-FR 4). The framework regions FR1, FR2, FR3 and FR4 are separated by complementarity determining regions CDR1, CDR2 and CDR3, respectively. Antigen binding fragments may be obtained recombinantly. For example, a nucleic acid encoding the antigen-binding portion of interest may be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen-binding fragment.
The term "and/or" when used in connection with two or more selectable items is understood to mean any one of the selectable items or any two or more of the selectable items.
The term "comprising" or "comprises" is intended to include the stated elements or steps but does not exclude other elements or steps. Of course, unless otherwise indicated, inclusion or inclusion also encompasses a situation consisting of the recited elements or steps. For example, when referring to an antibody variable region comprising a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
Reference herein to "affinity" or "binding affinity" is understood in accordance with the ordinary meaning in the art to reflect the strength and/or stability between an antigen and a binding site on an antibody or antigen binding fragment.
"specific binding" or "specifically binding to", "binding", "specifically targeting" a particular antigen or epitope, or "specific for" a particular antigen or epitope, or "capable of binding" means being distinguished from non-specific interactions, such specific binding can be measured by some method commonly used in the art. The ability of an antibody to bind to an antigen may be measured by an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, the competitive binding of the antibody to be detected and the labeled antibody can be detected by detecting the cells carrying the antigen by flow cytometry and measuring the positive rate index of the cells. The antigen space structure on the cell surface is closer to the form existing in the body, so that the method can reflect the real situation more. In connection with particular embodiments of the present invention, anti-CD 73 antibodies are provided having EC50 values of 10nM or less, 5nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.09nM or less, 0.08nM or less, 0.07nM or less, 0.06nM or less, 0.05nM or less, 0.04nM or less, 0.03nM or less, 0.02nM or less, and 0.01nM or less. In combination with embodiments of the invention, bispecific antibodies are provided having EC50 values of 10nM or less, 5nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.09nM or less, 0.08nM or less, 0.07nM or less, 0.06nM or less, 0.05nM or less, 0.04nM or less, 0.03nM or less, 0.02nM or less, 0.01nM or less, and EC50 values of 10nM or less, 5nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.09nM or less, 0.08nM or less, 0.07nM or less, 0.06nM or less, 0.05nM or less, 0.04nM or less, 0.03nM or less, 0.02nM or less, 0.01nM or less with PD-L1 antigen. In addition, the binding activity of antibodies to antigens can be determined by Surface Plasmon Resonance (SPR) or biological thin film interference (BLI).
Provided herein are antibodies or antigen binding fragments, bispecific antibodies, or polynucleotides, generally, that are isolatable or recombinant. "isolatable" means capable of being identified and isolated and/or recovered from a cell or cell culture expressing a polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. By "isolated antibody" is meant that it is substantially free of other antibodies or antigen-binding fragments thereof having different antigen specificities. "recombinant" means that antibodies can be produced in a foreign host cell using genetic recombination techniques.
"humanized" antibodies generally refer to antibodies based on antigen binding portions derived from non-human species, and based on the partial structure and sequence composition of human immunoglobulin molecules. For example, in humanized antibodies, the entire antibody, except the CDRs, is encoded by polynucleotides of human origin that retain antigen binding activity while reducing immunogenicity.
The CDR sequences of the antibodies or bispecific antibodies shown herein can be obtained in conjunction with existing database analysis. The CDR sequences of the first antigen binding portion and the CDR sequences of the second antigen binding portion of the provided anti-CD 73 antibodies or bispecific antibodies are obtained in combination with IMGT definition schemes (Ehrenmann f., kaas q.and Lefranc m. -p.nucleic Acids res.,38:d301-D307 (2010); ehrenmann, f., lefranc, m. -p.cold Spring Harbor protoc.,6:737-749 (2011)). It is also obtainable by the definition scheme Kabat (see, for example, U.S. Dept. Of Health and Human Servies, "Sequences of Proteins of Immunological Interest" (1983)), chothia (see, for example, J.Mol. Biol.196:901-917 (1987)), and the like. It will be appreciated by those skilled in the art that differences in the CDRs due to differences in the definition of the methods are also within the scope of the present invention.
Antibodies or antigen binding fragments
The present invention provides an anti-CD 73 antibody or antigen binding fragment comprising the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO 46 or 47 or 48 or 49 or 50 or 51 or 52. Antibodies or antigen binding fragments are provided which contain the HCDR1, HCDR2 and HCDR3 sequences, as well as the LCDR1, LCDR2 and LCDR3 sequences, which are obtainable via the variable heavy chain regions of SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45 in combination with the variable light chain regions of SEQ ID NO 46 or 47 or 48 or 49 or 50 or 51 or 52 in combination with the variable heavy chain regions of the different definition schemes. CDR sequences obtained via these heavy chain variable region and light chain variable region may also form different heavy chain variable region or light chain variable region sequences with framework region sequences of different origins as part of an anti-CD 73 antibody, for example, when forming heavy chain variable region or light chain variable region sequences. Such variations are intended to be included within the scope of the present invention.
According to an embodiment of the present invention there is provided an anti-CD 73 antibody or antigen binding fragment comprising a heavy chain variable region comprising the HCDR sequences of SEQ ID NOs 1, 2 and 3 or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NOs 1, 2 and 3; or comprises the HCDR sequences shown in SEQ ID NOs 4, 5 and 6, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs 4, 5 and 6; or comprises the HCDR sequences shown in SEQ ID NO. 7, 8 and 9, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 7, 8 and 9; or comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS 10, 11 and 12; or comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO 13, 14 and 15; or comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS.16, 17 and 18; or comprises the HCDR sequences shown in SEQ ID NO. 19, 20 and 21, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 19, 20 and 21; the light chain variable region comprises the LCDR sequences shown in SEQ ID NOs 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs 22, 23 and 24; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 25, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 25; or comprises the LCDR sequences shown in SEQ ID NOS.26, 27 and 28, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.26, 27 and 28; or comprises the LCDR sequences shown in SEQ ID NOS.29, 30 and 31, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.29, 30 and 31; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 32, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 32; or comprises the LCDR sequences shown in SEQ ID NOS.33, 34 and 35, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.33, 34 and 35; or comprises the LCDR sequences shown in SEQ ID NOS.36, 37 and 38, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.36, 37 and 38.
As used herein, a "conservative amino acid mutation" refers to an amino acid mutation that does not cause a significant change in the conformational structure of a protein or polypeptide, while retaining its biological activity. The conservative amino acid mutations mentioned may be mutations between different amino acids having similar physiological and biochemical properties. For example, through conservative amino acid mutations, the provided antibodies still retain specific binding activity for CD 73. According to embodiments, the conservative amino acid mutations mentioned may be substitutions, deletions or additions of conservative amino acids. According to specific embodiments, the deletion of a conserved amino acid is one amino acid, two amino acids or three amino acids based on the listed CDR sequences. According to specific embodiments, the conservative amino acid increase is one amino acid, two amino acids or three amino acids increase based on the CDR sequences listed. According to a specific embodiment, "conservative amino acid substitution" means the replacement of another amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative amino acid substitutions may occur between amino acid residues with hydrophobic side chains (e.g., met, ala, val, leu and Ile), between residues with neutral hydrophilic side chains (e.g., cys, ser, thr, asn and gin), between residues with acidic side chains (e.g., asp, glu), between amino acids with basic side chains (e.g., his, lys, and Arg), or between residues with aromatic side chains (e.g., trp, tyr, and Phe). As is known in the art, conservative amino acid substitutions typically do not cause a significant change in the conformational structure of the protein, and thus may preserve the biological activity of the protein. The conservative amino acid substitutions mentioned may be 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, etc. The names of amino acids used herein are indicated by single-letter or three-letter codes that are standard in the art. In some embodiments, the provided HCDR sequences have one conservative amino acid substitution with the HCDR sequences shown. In some embodiments, the provided HCDR sequences have a conserved amino acid deletion from the illustrated HCDR sequences. In some embodiments, the HCDR sequences provided have one conservative amino acid increase from the HCDR sequences shown. In some embodiments, the provided LCDR sequences have one conservative amino acid substitution with the illustrated LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid deletion from the illustrated LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid increase from the LCDR sequences shown.
The invention also provides an anti-CD 73 antibody or antigen binding fragment comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 is selected from the sequences shown by SEQ ID NOs 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequences shown by SEQ ID NOs 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequences shown by SEQ ID NOs 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequences shown by SEQ ID NOs 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequences shown by SEQ ID NOs 23 or 27 or 30 or 34 or 37, LCDR3 is selected from the sequences shown by SEQ ID NOs 24 or 25 or 28 or 31 or 32 or 35 or 38.
According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences set forth in SEQ ID NOS: 1, 2 and 3, and the LCDR sequences set forth in SEQ ID NOS: 22, 23 and 24. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS: 4, 5 and 6, and the LCDR sequences shown in SEQ ID NOS: 22, 23 and 25. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS: 7, 8 and 9, and the LCDR sequences shown in SEQ ID NOS: 26, 27 and 28. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, and the LCDR sequences shown in SEQ ID NOS 29, 30 and 31. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS 13, 14 and 15, and the LCDR sequences shown in SEQ ID NOS 22, 23 and 32. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS: 16, 17 and 18, and the LCDR sequences shown in SEQ ID NOS: 33, 34 and 35. According to a specific embodiment, an antibody or antigen binding fragment thereof is provided comprising the HCDR sequences shown in SEQ ID NOS: 19, 20 and 21, and the LCDR sequences shown in SEQ ID NOS: 36, 37 and 38.
In some embodiments, provided antibodies or antigen binding fragments comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO:39 or 40 or 41 or 42 or 43 or 44 or 45, and the light chain variable region is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO:46 or 47 or 48 or 49 or 50 or 51 or 52.
As used herein, "sequence identity", "sequence homology", "sequence similarity" refers to the degree to which two polypeptides or proteins or nucleotide sequences are compared and the sequences are identical. To determine the ratio of sequence identity, this can be accomplished in a variety of ways known in the art. Such as may be obtained using published software such as BLAST, ALIGN, BLAST-2. In some embodiments, sequence identity is due to conservative amino acid mutations. According to specific embodiments, the sequence having sequence identity to the heavy chain variable region or the light chain variable region described above may be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations, or 10 conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity to the heavy chain variable region or the light chain variable region described above differs in the framework regions.
According to a specific embodiment, the antibody provided is mab309 comprising a heavy chain variable region shown in SEQ ID NO. 39 and a light chain variable region shown in SEQ ID NO. 46. According to a specific embodiment, the antibody provided is mab307, which comprises a heavy chain variable region shown in SEQ ID NO. 40 and a light chain variable region shown in SEQ ID NO. 47. According to a specific embodiment, the antibody provided is mab144 comprising a heavy chain variable region shown in SEQ ID NO. 41 and a light chain variable region shown in SEQ ID NO. 48. According to a specific embodiment, the antibody provided is mab235 comprising a heavy chain variable region shown in SEQ ID NO. 42 and a light chain variable region shown in SEQ ID NO. 49. According to a specific embodiment, an antibody is provided that is mab451 comprising a heavy chain variable region shown in SEQ ID NO. 43 and a light chain variable region shown in SEQ ID NO. 50. According to a specific embodiment, the antibody provided is mab540 comprising a heavy chain variable region shown as SEQ ID NO. 44 and a light chain variable region shown as SEQ ID NO. 51. According to a specific embodiment, the antibody provided is mab449, which comprises a heavy chain variable region as set forth in SEQ ID NO. 45 and a light chain variable region as set forth in SEQ ID NO. 52. The antibodies shown exhibit specific binding to CD73, e.g., exhibit strong specific binding activity to mammalian CD 73. The heavy chain constant domain of these antibodies is shown as SEQ ID NO. 100 and the light chain constant domain is shown as SEQ ID NO. 101.
The provided antibodies or antigen binding fragments may also be humanized. By humanization, the immunogenicity of an antibody or antigen-binding fragment can be reduced while maintaining binding activity to the target antigen. In some embodiments, antibodies or antigen binding fragments provided include a heavy chain variable region comprising a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO 66 or 67 or 68 or 69 or 70 or 71 or 73 or 74 or 75 or 76 or 77 or 78 or 79. According to a specific embodiment, the antibody provided is mab309hu1 comprising a heavy chain variable region shown in SEQ ID NO. 53 and a light chain variable region shown in SEQ ID NO. 66. According to a specific embodiment, the antibody provided is mab309hu3 comprising a heavy chain variable region shown in SEQ ID NO. 54 and a light chain variable region shown in SEQ ID NO. 67. According to a specific embodiment, the antibody provided is mab144hu1 comprising a heavy chain variable region shown in SEQ ID NO. 55 and a light chain variable region shown in SEQ ID NO. 68. According to a specific embodiment, the antibody provided is mab144hu2 comprising a heavy chain variable region shown in SEQ ID NO. 56 and a light chain variable region shown in SEQ ID NO. 69. According to a specific embodiment, the antibody provided is mab451hu1 comprising a heavy chain variable region shown in SEQ ID NO. 57 and a light chain variable region shown in SEQ ID NO. 70. According to a specific embodiment, the antibody provided is mab451hu3 comprising a heavy chain variable region shown in SEQ ID NO. 58 and a light chain variable region shown in SEQ ID NO. 71. According to a specific embodiment, the antibody provided is mab451hu4 comprising the heavy chain variable region shown in SEQ ID NO. 59 and the light chain variable region shown in SEQ ID NO. 72. According to a specific embodiment, the antibody provided is mab451hu5 comprising a heavy chain variable region shown in SEQ ID NO. 60 and a light chain variable region shown in SEQ ID NO. 73. According to a specific embodiment, the antibody provided is mab451hu6 comprising the heavy chain variable region shown in SEQ ID NO. 60 and the light chain variable region shown in SEQ ID NO. 74. According to a specific embodiment, the antibody provided is mab540hu1 comprising a heavy chain variable region as set forth in SEQ ID NO. 61 and a light chain variable region as set forth in SEQ ID NO. 75. According to a specific embodiment, the antibody provided is mab540hu3 comprising a heavy chain variable region shown in SEQ ID NO. 62 and a light chain variable region shown in SEQ ID NO. 76. According to a specific embodiment, the antibody provided is mab449hu1 comprising a heavy chain variable region as set forth in SEQ ID NO. 63 and a light chain variable region as set forth in SEQ ID NO. 77. According to a specific embodiment, the antibody provided is mab449hu3 comprising a heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 78. According to a specific embodiment, the antibody provided is mab449hu4 comprising a heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 79. The heavy chain constant domain of these antibodies is shown as SEQ ID NO. 100 and the light chain constant domain is shown as SEQ ID NO. 101.
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According to specific embodiments, antibodies are provided that bind human CD73 or monkey (e.g., cynomolgus) CD73 with an EC50 of 10nM or less, e.g., an affinity EC50 value with CD73 of less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 0.8nM, less than 0.5nM, less than 0.3nM, or less than 0.1nM, less than 0.05nM. The provided antibodies can also promote CD73 endocytosis and exhibit anti-tumor activity.
The anti-CD 73 antibody provided may be from any of the commonly known isotypes, including but not limited to IgA, igG, igM and secretory IgA. IgG isotypes can be divided into different subclasses in different species, for example, igG1, igG2, igG3 and IgG4 in humans and IgG1, igG2a, igG2b and IgG3 in mice. According to embodiments, the provided antibodies further comprise an Fc region, which may be derived from a native Fc region or an engineered Fc region. The native Fc region may be of any origin, including but not limited to murine, primate, human, preferably human. The mentioned Fc region of human origin can be obtained in a public database.
Bispecific antibodies
Bispecific antibodies (Bispecific antibody, bsAb) are antibodies that are capable of specifically binding two antigens or epitopes simultaneously. Since Nisonoff and its co-workers proposed the concept of BsAb for the first time in 1960, bispecific antibodies developed rapidly as the technology of genetically engineered antibodies became mature. Because BsAb can target multiple antigens or epitopes, it exhibits more advantages than monoclonal antibodies, and also gradually exhibits higher efficacy than monoclonal antibody combination therapies. For example, specific immune effector cells can be redirected to adjacent tumor cells to enhance tumor killing; but also the specificity of binding can be increased by the interaction of two different cell surface antigens; meanwhile, compared with the combination of single antibodies, the development cost can be reduced, and the balance of different half-lives of the two antibodies is not needed, so that the administration time is different, and the toxicity is low. Develop the bispecific antibody related to CD73 target spot, have extensive application in fields such as tumor treatment and autoimmune disease. Bispecific antibodies are used to treat diseases and exhibit excellent disease-treating effects.
The present invention provides a bispecific antibody comprising a first antigen binding moiety and a second antigen binding moiety, each of which specifically binds a different epitope. The different epitopes mentioned may be derived from different epitopes of the same antigen or may be derived from different epitopes of different antigens. According to a specific embodiment of the present invention, bispecific antibodies are provided comprising a first antigen binding moiety that specifically binds a first epitope and a second antigen binding moiety that specifically binds a second epitope, the first antigen binding moiety and the second antigen binding moiety being fused. The first epitope and the second epitope may be derived from the same antigen or may be derived from different antigens. According to a preferred embodiment of the invention, the first antigen binding portion specifically binds a first epitope and the second antigen binding portion specifically binds a second epitope from a different antigen. As used herein, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or as indicating a sequential order. The terms "first antigen binding portion", "second antigen binding portion" mean being capable of binding to a different antigen or the same, respectively The portions of the antigen to which different epitopes bind may be whole antibodies or may be part of whole antibodies, e.g.heavy chain variable regions, light chain variable regions, fab ', F (ab') 2 Single chain antibodies (ScFv), or single domain antibodies (sdabs), and the like.
The present invention provides a bispecific antibody comprising a first antigen binding portion and a second antigen binding portion; the first antigen binding portion specifically binds to a first epitope and the second antigen binding portion specifically binds to a second epitope; the first antigen binding portion comprises the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown as SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45 and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown as SEQ ID NO 46 or 47 or 48 or 49 or 50 or 51 or 52. It is noted that each of the CDR sequences mentioned may be obtained via the illustrated heavy chain variable region and light chain variable region sequences in combination with different definition schemes. Those skilled in the art will also appreciate that the CDR sequences of the heavy and light chain variable region sequences shown will also differ somewhat due to differences in the definition of antibody sequences, e.g., the classical Kabat amino acid sequence numbering system, IMGT system, chothia system, etc., as mentioned above. The different CDR sequences analyzed are all included in the scope of the present invention. These CDR sequences may form different heavy chain variable region sequences or light chain variable region sequences with framework region sequences of different origins, and these variations and differences are included within the scope of the present invention.
According to an embodiment of the present invention, there is also provided a bispecific antibody comprising a first antigen binding portion and a second antigen binding portion; the first antigen binding portion specifically binds to a first epitope, the second antigen binding portion specifically binds to a second epitope, and the second antigen binding portion is fused to the first antigen binding portion; the first antigen binding portion comprises a heavy chain variable region comprising the HCDR sequences of SEQ ID NOs 1, 2 and 3 or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NOs 1, 2 and 3; or comprises the HCDR sequences shown in SEQ ID NOs 4, 5 and 6, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs 4, 5 and 6; or comprises the HCDR sequences shown in SEQ ID NO. 7, 8 and 9, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 7, 8 and 9; or comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS 10, 11 and 12; or comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO 13, 14 and 15; or comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS.16, 17 and 18; or comprises the HCDR sequences shown in SEQ ID NO. 19, 20 and 21, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 19, 20 and 21; the light chain variable region comprises the LCDR sequences shown in SEQ ID NOs 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs 22, 23 and 24; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 25, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 25; or comprises the LCDR sequences shown in SEQ ID NOS.26, 27 and 28, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.26, 27 and 28; or comprises the LCDR sequences shown in SEQ ID NOS.29, 30 and 31, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.29, 30 and 31; or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 32, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 32; or comprises the LCDR sequences shown in SEQ ID NOS.33, 34 and 35, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.33, 34 and 35; or comprises the LCDR sequences shown in SEQ ID NOS.36, 37 and 38, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.36, 37 and 38.
The first antigen-binding portion or the second antigen-binding portion of the bispecific antibody referred to may be selected as desired for the whole antibody or only a part of the fragments thereof, provided that it is capable of targeting a different target antigen. The first antigen binding portion or the second antigen binding portion may be only the heavy chain variable region, fab ', F (ab') 2 Single chain antibodies (ScFv), trap, or single domain antibodies (sdAb), and the like.
According to a specific embodiment, bispecific antibodies are provided comprising the HCDR sequences shown in SEQ ID NOS: 1, 2, and 3, and the LCDR sequences shown in SEQ ID NOS: 22, 23, and 24. According to a specific embodiment, bispecific antibodies are provided having an HCDR sequence comprising SEQ ID NOs 4, 5 and 6 and having an LCDR sequence as shown in SEQ ID NOs 22, 23 and 25. According to a specific embodiment, bispecific antibodies are provided having a sequence comprising the HCDR sequences shown in SEQ ID NOS.7, 8, and 9, and the LCDR sequences shown in SEQ ID NOS.26, 27, and 28. According to a specific embodiment, bispecific antibodies are provided comprising the HCDR sequences shown in SEQ ID NOS 10, 11, and 12, and the LCDR sequences shown in SEQ ID NOS 29, 30, and 31. According to a specific embodiment, bispecific antibodies are provided comprising the HCDR sequences shown in SEQ ID NOS 13, 14, and 15, and the LCDR sequences shown in SEQ ID NOS 22, 23, and 32. According to a specific embodiment, bispecific antibodies are provided comprising the HCDR sequences shown in SEQ ID NOS: 16, 17, and 18, and the LCDR sequences shown in SEQ ID NOS: 33, 34, and 35. According to a specific embodiment of the present invention, bispecific antibodies are provided comprising the HCDR sequences shown in SEQ ID NOS.19, 20, and 21, and the LCDR sequences shown in SEQ ID NOS.36, 37, and 38. The HCDR sequences and LCDR sequences shown are shown as the first antigen-binding portion of a bispecific antibody, specifically binding to a target antigen.
In some embodiments, bispecific antibodies are provided that include the following heavy chain variable regions and light chain variable regions: a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence shown in SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45 or 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65 and a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence shown in SEQ ID NO 46 or 47 or 48 or 50 or 51 or 52 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 78 or 79. Wherein SEQ ID NOs 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 are each a humanized heavy chain variable region sequence. 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 and 79, respectively, are humanized light chain variable region sequences. The heavy and light chain variable region sequences are shown as the first antigen binding portion of a bispecific antibody, specifically binding to a target antigen. The sequences of the heavy and light chain variable regions of the first antigen binding portion are less immunogenic after humanization and retain binding activity to the target antigen (e.g., CD 73). Humanized antibody sequences can be obtained by methods commonly used in the art (e.g., complementarity determining region grafting methods). In some embodiments, the provided humanized antibody sequences retain CDR region sequences, replacing framework region sequences with framework region sequences of human origin. The framework region sequences of human origin can be obtained from published sequences of human origin. For example, by database alignment and computer homology modeling, searching for a human FR region template with maximum homology, comprehensively considering key residues for determining whether the FR region needs to be subjected to back mutation and restoring mutation, and obtaining the high-affinity humanized antibody. These sequences are usually described in commonly used databases, such as PDB protein structure database, IMGT, genebank, and the like.
In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 39 and a light chain variable region as set forth in SEQ ID NO. 46. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 40 and a light chain variable region shown as SEQ ID NO. 47. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 41 and a light chain variable region as set forth in SEQ ID NO. 48. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 42 and a light chain variable region as set forth in SEQ ID NO. 49. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 43 and a light chain variable region as set forth in SEQ ID NO. 50. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 44 and a light chain variable region as set forth in SEQ ID NO. 51. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 45 and a light chain variable region as set forth in SEQ ID NO. 52. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 53 and a light chain variable region as set forth in SEQ ID NO. 66. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 54 and a light chain variable region shown as SEQ ID NO. 67. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 55 and a light chain variable region as set forth in SEQ ID NO. 68. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 56 and a light chain variable region as set forth in SEQ ID NO. 69. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 57 and a light chain variable region as set forth in SEQ ID NO. 70. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 58 and a light chain variable region as set forth in SEQ ID NO. 71. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 59 and a light chain variable region as set forth in SEQ ID NO. 72. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 60 and a light chain variable region as set forth in SEQ ID NO. 73. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 60 and a light chain variable region as set forth in SEQ ID NO. 74. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 61 and a light chain variable region as set forth in SEQ ID NO. 75. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 62 and a light chain variable region as set forth in SEQ ID NO. 76. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 63 and a light chain variable region as set forth in SEQ ID NO. 77. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 78. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 79.
According to an embodiment of the invention, the heavy chain variable region and the heavy chain constant domain of the first antigen binding portion are linked, and the light chain variable region and the light chain constant domain of the first antigen binding portion are linked, forming an IgG type structure; the second antigen binding portion is a single domain antibody. According to a specific embodiment of the invention, the second antigen binding portion is linked directly to the carboxy terminus (C-terminus) of the Fc region or via a linker. According to a specific embodiment of the invention, the second antigen binding portion is directly linked to the amino acid (N-terminal) of the heavy chain variable region or the light chain variable region of the first antigen binding portion or is linked via a linker. According to a specific embodiment of the invention, the second antigen binding portion is directly linked to the carboxy-terminus of the light chain constant domain CL of the first antigen binding portion or is linked via a linker. Of course, when the second antigen-binding portion and the first antigen-binding portion are fused, the methods may be not limited to the above-mentioned methods, and these fusion methods may be combined or may be connected by other methods.
According to a preferred embodiment of the invention, as shown in fig. 1, the heavy chain variable region and the heavy chain constant domain of the first antigen binding portion of the bispecific antibody are linked, the light chain variable region and the light chain constant domain of the first antigen binding portion are linked to form an IgG type structure, the second antigen binding portion is a single domain antibody, the second antigen binding portion is linked to the carboxy-terminus of the Fc region through a linker, and the two single domain antibodies are linked to the two ends of the carboxy-terminus of the Fc region through linkers, respectively. The two single domain antibodies comprised by the second antigen binding portion may be the same or different. Of course, one single domain antibody or more single domain antibodies may be linked to only one end of the Fc region at the carboxy terminus, as desired. As shown in fig. 2, the second antigen-binding portion may also comprise four single-domain antibodies, as desired, with two single-domain antibodies linked to each of the two ends of the Fc region at the carboxy terminus. The single domain antibodies that are linked may be the same or different. The single domain antibodies may be linked by a linker.
According to a preferred embodiment of the invention, the heavy chain variable region and the heavy chain constant domain of the first antigen binding portion of the bispecific antibody are linked, the light chain variable region and the light chain constant domain of the first antigen binding portion are linked, forming an IgG type structure, the second antigen binding portion is a single domain antibody, and the second antigen binding portion is linked to the carboxy terminus of the light chain constant domain via a linker. In at least some embodiments, the second antigen binding portion comprises four single domain antibodies, two single domain antibodies each linked to the carboxy terminus of the light chain constant domain by a linker, as shown in fig. 3. The four single domain antibodies comprised by the second antigen binding portion may be the same or different. The second antigen binding portion may also, if desired, comprise only two single domain antibodies, one single domain antibody linked directly or via a linker to the carboxy terminus of the light chain constant domain, as shown in figure 5. The second antigen-binding portion may also comprise four single domain antibodies, one single domain antibody linked to each of the two ends of the Fc region at the carboxy terminus via a linker, and one single domain antibody linked to each of the two ends of the light chain constant domain at the carboxy terminus via a linker, as shown in FIG. 4. The single domain antibodies that are linked may be the same or different. The single domain antibodies may be linked by a linker. Fig. 1 to 5 are only schematic drawings, and for example, the interchain disulfide bond between the light chain and the heavy chain is not shown in the drawings.
According to a preferred embodiment of the invention, the first antigen binding portion is in addition to the heavy chain variable region and the light chain variable region mentioned aboveFurther comprising a heavy chain constant domain CH1 and a light chain constant domain, forming (Fab') 2 The structure, the bispecific antibody further comprises an Fc region, and the second antigen-binding portion comprises a single domain antibody, the single domain antibody being linked to the carboxy terminus of the light chain constant domain and/or to the carboxy terminus of the Fc region. The number of the single domain antibodies is not particularly required, and the single domain antibodies connected with the single domain antibodies can be the same or different.
Herein, "linker" and "linker fragment" have the same meaning. The linker may be some oligopeptide or polypeptide and may be any amino acid sequence capable of providing flexibility. Useful linkers are those commonly used in the art, including but not limited to the group consisting of: GS, SG, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGSGGGS, etc.
The second antigen binding portion referred to specifically binds to an antigen different from the first epitope, including but not limited to: PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, 4-1BB, OX40L, VEGF, VEGF trap, VEGFR, EGFR, GARP, TGF-. Beta., CD47, c-Met, CD3, CD33, TIGIT, PVRIG, CD69, CD96, LEFA1, CD226, FAP2, GITR, CEACAM-1, CD48, TIM-1, VISTA, CD39, CD70, CD27, CD40, claudin18.2, ICOS, BLTA, B7-H3, BCMA, MUC1/16, and the like. For example, a commercially available or in-flight antibody molecule sequence can be combined with the first antigen-binding portion described herein to form a bispecific antibody that can exert a therapeutic effect on a disease. Taking PD-1 antibody as an example, partial or complete sequences of antibody molecules such as Nivolumab, terlipressimab Li Shan, caprilizumab (camrelizumab), tirelizumab (tisllizumab) and the like can be fused with the first antigen-binding portion mentioned herein to form a bispecific antibody. Taking the PD-L1 antibody as an example, part or all of the sequences of the antibody molecules such as the Durvalumab (Durvalumab), the Ab (atezolizumab), the Ab (avelumab) and the like can be fused with the first antigen binding portion mentioned herein to form the bispecific antibody to play a role. For another example, the second antigen binding portion may be a VEGF trap structure. Apacetirizine (aflibercept or VEGF-trap) is able to specifically target VEGFA, VEGFB and PIGF, which by specifically binding and capturing these proteins, prevents angiogenesis. The VEGF-trap molecule and the first antigen binding portion may be combined into a bispecific antibody that exhibits therapeutic effects. Bevacizumab is a recombinant humanized immunoglobulin G1 (IgG 1) monoclonal antibody, which can bind VEGFA and inhibit the binding of VEGFR2, thereby inhibiting the biological action of VEGF. The bevacizumab or a biological analogue thereof may be fused in part or in whole to the first antigen binding portion mentioned herein, and constitutes a bispecific antibody.
According to a specific embodiment, the present invention provides a bispecific antibody comprising a first antigen binding moiety that specifically binds CD73 and a second antigen binding moiety that specifically binds PD-L1. As mentioned above, the expression form of the second antigen binding portion is not limited as long as it can specifically target PD-L1. For example, may be an intact antibody, or a portion thereof, including but not limited to heavy chain variable regions, fab ', F (ab') 2 Single chain antibodies or nanobodies, and the like. The second antigen binding portion may be obtained by screening an antibody library as desired, or may be derived from a commercially available or existing (e.g., published in the literature or clinically developed) antibody sequence. In some embodiments, the second antigen binding portion is a single domain antibody. According to a preferred embodiment of the invention, the bispecific antibody further comprises an Fc region fused to a first antigen binding moiety to an IgG type structure, and a second antigen binding moiety is linked directly to the carboxy-terminus (also known as the C-terminus) of the Fc region or via a linker.
In some embodiments, a second antigen binding portion is provided comprising CDR1, CDR2, and CDR3, wherein the CDR1 is selected from the sequences set forth in SEQ ID NOs 80, 83, or 86, or a sequence having one or two conservative amino acid mutations compared to SEQ ID NOs 80, 83, and 86; CDR2 is selected from the sequences shown in SEQ ID NOS: 81, 84 or 87, or a sequence having one or two conservative amino acid mutations compared to SEQ ID NOS: 81, 84 and 87; and CDR3 is selected from the sequences shown in SEQ ID NO 82, 85, 88 or 89, or a sequence having one, two or three conservative amino acid mutations compared to SEQ ID NO 82, 85, 88 and 89. As mentioned above, it will be appreciated by those skilled in the art that the CDR regions can be characterized using different databases, and that the sequence of the characterized CDR regions can vary and such variations are intended to be within the scope of the present invention.
According to a specific embodiment, the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO. 80, a CDR2 sequence as shown in SEQ ID NO. 81 and a CDR3 sequence as shown in SEQ ID NO. 82. According to a specific embodiment, the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO. 83, a CDR2 sequence as shown in SEQ ID NO. 84 and a CDR3 sequence as shown in SEQ ID NO. 85. According to a specific embodiment, the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO. 86, a CDR2 sequence as shown in SEQ ID NO. 87 and a CDR3 sequence as shown in SEQ ID NO. 88. According to a specific embodiment, the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO. 80, a CDR2 sequence as shown in SEQ ID NO. 81 and a CDR3 sequence as shown in SEQ ID NO. 89. The CDR1, CDR2 and CDR3 sequences shown may form single domain antibodies with framework region FR of different origin. The framework region FR sequence is not particularly limited and may be of murine or human origin, or may be of partially murine or partially human origin.
SEQ ID NO: Functional area Amino acid sequence
80 CDR1 GRSFSSSG
81 CDR2 INSSGGDT
82 CDR3 AAKEGGGPSSIPAIYDY
83 CDR1 GFTFSSYA
84 CDR2 INTGSEIV
85 CDR3 ATGLVSAEHDGI
86 CDR1 GRDFLTYG
87 CDR2 INWSGSMT
88 CDR3 AARRGAVTYASSNEYEH
89 CDR3 AAKEGGGPSSIPAIFDY
In some embodiments, the second antigen binding portion has a sequence as set forth in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the sequence of the second antigen binding portion is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID No. 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. The sequences shown in SEQ ID NOs 95, 96, 97, 98 and 99 are humanized antibody sequences. The provided humanized sequences have low immunogenicity and high affinity for the target protein.
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The sequence of the second antigen binding portion is described in PCT patent application No. PCT/CN2022/110423 and International publication No. WO 2023/01614A, and the patent text describes that an anti-PD-L1 nanobody library is obtained by a alpaca immunization method, and screening and identification are carried out, so that nanobodies with good affinity and specific binding and tumor killing effects are obtained. The disclosures of PCT patent application Ser. No. PCT/CN2022/110423 and International publication No. WO 2023/01614A are also incorporated herein in their entirety or in part by reference thereto, as appropriate.
The present invention also provides a bispecific antibody comprising: a first antigen binding portion capable of binding CD73 and a second antigen binding portion capable of binding PD-L1.
According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS.1, 2 and 3 and the LCDR sequences shown in SEQ ID NOS.22, 23 and 24; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS.4, 5 and 6, and the LCDR sequences shown in SEQ ID NOS.22, 23 and 25; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS.7, 8 and 9, and the LCDR sequences shown in SEQ ID NOS.26, 27 and 28; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, and the LCDR sequences shown in SEQ ID NOS 29, 30 and 31; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS 13, 14 and 15, and the LCDR sequences shown in SEQ ID NOS 22, 23 and 32; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS: 16, 17 and 18, and the LCDR sequences shown in SEQ ID NOS: 33, 34 and 35; the second antigen binding portion comprises the sequence shown in SEQ ID NO 80 or 83 or 86 CDR1 sequence shown in SEQ ID No. 81 or 84 or 87, and CDR3 sequence shown in SEQ ID No. 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequences shown in SEQ ID NOS.19, 20 and 21, and the LCDR sequences shown in SEQ ID NOS.36, 37 and 38; the second antigen binding portion comprises a CDR1 sequence as shown in SEQ ID NO 80 or 83 or 86, a CDR2 sequence as shown in SEQ ID NO 81 or 84 or 87, and a CDR3 sequence as shown in SEQ ID NO 82 or 85 or 88 or 89. According to a specific embodiment, the first antigen binding portion and the second antigen binding portion are bound by (G 4 S) 3-5 The connectors are connected.
In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 39, and a light chain variable region as set forth in SEQ ID NO. 46; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 40, and a light chain variable region shown as SEQ ID NO. 47; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 41 and a light chain variable region shown as SEQ ID NO. 48; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 42 and a light chain variable region as set forth in SEQ ID NO. 49; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 43 and a light chain variable region shown as SEQ ID NO. 50; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 44 and a light chain variable region shown as SEQ ID NO. 51; the second antigen binding portion comprises S EQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 45 and a light chain variable region shown as SEQ ID NO. 52; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 53 and a light chain variable region shown as SEQ ID NO. 66; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 54 and a light chain variable region shown as SEQ ID NO. 67; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 55 and a light chain variable region shown as SEQ ID NO. 68; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 56 and a light chain variable region shown as SEQ ID NO. 69; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 57 and a light chain variable region shown as SEQ ID NO. 70; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 58 and a light chain variable region shown as SEQ ID NO. 71; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 59 and a light chain variable region shown as SEQ ID NO. 72; the second antigen binding portion comprises SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 60 and a light chain variable region shown as SEQ ID NO. 73; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 60 and a light chain variable region shown as SEQ ID NO. 74; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 61 and a light chain variable region shown as SEQ ID NO. 75; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 62 and a light chain variable region as set forth in SEQ ID NO. 76; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO. 63 and a light chain variable region as set forth in SEQ ID NO. 77; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 64 and a light chain variable region shown as SEQ ID NO. 78; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen binding portion comprises a heavy chain variable region shown as SEQ ID NO. 65 and a light chain variable region shown as SEQ ID NO. 79; the second antigen binding portion comprises the sequence shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. According to a specific embodiment, the first antigen binding portion and the second antigen binding portion are bound by (G 4 S) 3-5 The connectors are connected.
According to a specific embodiment, the heavy chain variable region and the heavy chain constant domain of the first antigen binding portion are linked, and the light chain variable region and the light chain constant domain of the first antigen binding portion are linked, forming an IgG type structure. The IgG type structure formed may employ a natural sequence of mammalian origin (e.g., may be a natural sequence of human origin) in addition to the heavy chain variable region and light chain variable region sequences mentioned. According to a specific embodiment, the second antigen binding portion is linked to the carboxy terminus (C-terminus) of the Fc region; or the second antigen binding portion is linked to the amino terminus (N-terminus) of the heavy chain variable region or the light chain variable region of the first antigen binding portion; or the second antigen binding portion is linked to the carboxy terminus (C-terminus) of the light chain constant domain CL.
The Fc region referred to may be a native Fc region sequence or may be a modified Fc region sequence, for example, the sequence of the Fc region may be mutated. ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis) or CDC (complement-mediated cytotoxicity) of antibodies can be modulated by Fc region mutations, and the like. ADCC refers to the binding of Fab fragments of antibodies to epitopes of virus-infected or tumor cells, the binding of their Fc fragments to Fc receptors (FcR) on the surface of killer cells (e.g., NK cells, macrophages, etc.), mediating the direct killing of the target cells by the killer cells. ADCP is primarily mediated by monocytes, macrophages, neutrophils and dendritic cells to phagocytose target cells. CDC is the activation of the classical pathway of complement by binding antibodies to the corresponding antigens on the cell membrane surface, forming complexes that exert lytic effects on target cells. The Fc region sequence mutation may be a mutation of one, two, three, four or five amino acids. Of course, in addition to amino acid mutations in the Fc region, mutations can also occur in other regions, such as the heavy chain constant domain CH1, the hinge region, etc. The amino acid mutations mentioned are known to those skilled in the art and include, but are not limited to, L234F, L234A, L235E, G236R, G237A, D A, K A, P329A, P331S, P331A, N297A, N297G, A330S, V309L, P238S, G236R, L328R and the like. The amino acid mutations mentioned may also be L235V, R292P, K326W, E35333S, S267E, H268F, S T, F243L, R292P, Y L, V L, V305I, P396L, S239D, I332E, A L, S298A, E333A, K334A, etc. The amino acid mutations mentioned may be selected from these mutation sites, or a combination of several mutation sites. In some embodiments, bispecific antibodies provided are substituted with a330S and P331S. In some embodiments, the bispecific antibodies provided are substituted with D270A, K322A, P329A and P331A. In some embodiments, the bispecific antibodies provided are substituted with L234F, L235E, P S and N297A. In some embodiments, the bispecific antibodies provided have the S267E, H268F and S324T mutations. In some embodiments, bispecific antibodies are provided that have undergone K214R, L234F, L235E, P331S, D356E and L358M mutations. Of course, modifications to the Fc region sequence can also be used to improve the half-life of the antibody drug, and the mutation sites for available amino acids are also known to those skilled in the art. The numbering of the mutation sites herein is according to the EU index as proposed by Kabat (Kabat, E.A. et al Sequences of Proteins of Immunological Interest.5) th Edition-USDepartment of Health and human severices,NIH publication n 91-3242,pp662,680,689(1991))。
Polynucleotide
The invention also provides a polynucleotide encoding an antibody or antigen binding fragment or a bispecific antibody. The polynucleotides mentioned are isolatable and include, but are not limited to, DNA, RNA or cDNA, and the like. Isolated polynucleotide sequences encoding bispecific antibodies or antigen-binding fragments can be obtained using methods conventional in the art.
Constructs
To prepare the antibodies of the invention, the polynucleotide sequences encoding the antibodies may be inserted into replicable expression vectors and expressed in host cells or cell-free expression systems. The invention also provides a construct comprising the polynucleotide. A variety of methods commonly used in the art can be used to obtain constructs, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like, for example, polynucleotides can be inserted into expression vectors to form constructs at multiple cloning sites. The construct may contain, as desired, various operators such as promoters, terminators, marker genes, etc., which are operably linked to the polynucleotide. Promoters are commonly used to provide signals to initiate transcription, and the promoters may be selected from lactose promoter (Lac), trp promoter, tac promoter, PL and PR promoters of phage, as desired; terminators provide a signal for transcription termination during transcription, and marker genes on constructs are commonly used for screening. Of course, enhancers may also be used as desired to enhance protein expression. The expression vector is not particularly limited, and may be some commercially available expression vectors, or may be an expression vector artificially modified as required, for example, a plasmid, phage, virus, or the like. The virus may be plant cell virus, mammalian cell virus, etc. The construct may express the antibody or protein in vitro, or may be transferred into cells to express the antibody or protein.
Host cells
The invention also provides a host cell containing the polynucleotide or the construct. Any cell suitable for expression of an antibody or protein by a polynucleotide or construct may be used as a host cell. The host cell may be a prokaryotic cell, such as a bacterial cell; eukaryotic cells, such as yeast cells, mammalian cells, and the like, are also contemplated. Typical host cells may be yeast cells, CHO, HEK-293 cells, COS cells, insect cells of Drosophila S2 or Sf 9. Host cells containing the polynucleotide or construct can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, and the like.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising: the above antibody or antigen binding fragment, or the above bispecific antibody, and a pharmaceutically acceptable carrier.
The pharmaceutical compositions also include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (e.g., as described in Berge, S.M. et al (1977) J.Pharma.Sci.66:1-19). The pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dosage or concentration employed. Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as chlorhexidine, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations may be obtained by methods commonly used in the art, for example by filtration through sterile filtration membranes. The pharmaceutical composition can be prepared into various dosage forms, such as freeze-dried dosage forms, injection and the like by selecting different pharmaceutically acceptable carriers according to the requirements of the person skilled in the art. The different pharmaceutical dosage forms prepared may be formulated for administration to a subject by any suitable route of administration including, but not limited to, intravenous, dermal, intramuscular, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, and the like.
The pharmaceutical compositions mentioned may further comprise other therapeutic agents in addition to the above. The therapeutic agent mentioned is a substance capable of exerting a therapeutic effect. Such as anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-VEGF antibodies, anti-EGFR antibodies, anti-GARP antibodies, anti-CD 3 antibodies, anti-TIGIT antibodies, anti-OX 40 antibodies, anti-TGF-beta antibodies, and the like. By combining these therapeutic antibodies, the therapeutic effect of the disease can be further improved, and thus various diseases can be effectively treated.
Antibody conjugates
The invention also provides an antibody conjugate comprising the bispecific antibody or antigen binding fragment, and a functional small molecule or protease linked to the bispecific antibody or antigen binding fragment. The functional small molecules can be developed or undeveloped small molecule drugs, and the small molecule drugs are chemically coupled with bispecific antibodies or monoclonal antibodies or antigen binding fragments to enhance the treatment effect of the antitumor drugs and reduce adverse reactions. Small molecules such as toxins, chemotherapeutics, photosensitizers, etc. can also be coupled to the mentioned antibodies via linkers. Of course, antibody Protac conjugates can also be obtained by coupling the antibody to a Protac molecule or the like, such as by linking the antibody to a protease targeting ligand (e.g., an E3 ligase ligand) via a linker, bringing the distance between the target protein and the intracellular E3 ubiquitin ligase, and utilizing ubiquitin-proteasome pathway specificity to degrade the target protein.
Kit or medicine box
The invention also provides a kit comprising the bispecific antibody or the monoclonal antibody or the antigen binding fragment. The kit may further include containers, buffer reagents, controls such as positive controls and negative controls, as needed. The person skilled in the art can make the corresponding selections as desired. Accordingly, instructions for use may also be included in the kit to facilitate handling and use by those skilled in the art. In some embodiments, the invention also provides a kit comprising an anti-CD 73 antibody or antigen-binding fragment as described above, or comprising a bispecific antibody as described above. The kit may further comprise an immunotherapeutic agent, wherein the immunotherapeutic agent is one or more of a plurality of therapeutic antibodies, such as an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-CTLA-4 antibody. The anti-PD-L1 antibody, anti-PD-1 antibody or CTLA-4 antibody mentioned is a drug which has been marketed or disclosed in the literature or marketed in the future.
Method for producing antibodies
The present invention also provides a method for producing the bispecific antibody, comprising: culturing the host cell and collecting the bispecific antibody from the culture.
The invention also provides a method of producing an antibody or antigen binding fragment comprising: culturing the host cell and collecting the antibody or antigen binding fragment from the culture.
Bispecific antibodies or monoclonal antibodies or antigen-binding fragments collected from the culture may be purified to obtain substantially pure products. By "substantially pure" is meant that the bispecific antibody or monoclonal antibody or antigen binding fragment is more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and even more than 99.5%, 99.6%, 99.7%, 99.8% pure.
Method for preventing and/or treating diseases
In addition, the present invention also provides a method for preventing and/or treating a disease, comprising: administering to a subject in need thereof an effective amount of the bispecific antibody described above, or the antibody or antigen binding fragment mentioned above.
By "treatment" is meant that can result in a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of the disease asymptomatic phase, or a decrease in pain caused by the disease. The effective amount of antibody used to prevent the disease will generally be less than the effective amount of antibody used to treat the disease. An "effective amount" is an amount sufficient to achieve, or at least partially achieve, the desired effect. After treatment with the bispecific antibody or monoclonal antibody or antigen binding fragment, the inhibition rate of cells in the subject is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, even 90% or more, or 95% or more, as compared to the subject not treated with the antibody. The subject may be an animal or a human. For example, mammals, including cattle, sheep, mice, horses, etc.
The provided bispecific antibodies or monoclonal antibodies or antigen binding fragments thereof can be used for treating diseases including, but not limited to, cancer, inflammatory diseases, chronic infections, cardiovascular and cerebrovascular diseases, fibrosis, neurodegenerative diseases, immunoregulatory diseases, central nervous system diseases, diabetes, and the like. In some embodiments, the methods provided herein can be used to treat diseases associated with the CD73 target, as well as PD-1/PD-L1. The cancers mentioned include, but are not limited to, non-small cell lung cancer, squamous cell carcinoma, glioma, colon cancer, renal cancer, urothelial cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastrointestinal cancer, esophageal cancer, prostate cancer, cervical cancer, uterine cancer, head and neck cancer, adrenal cancer, urinary tract cancer, vaginal cancer, bladder cancer, melanoma, hematological cancer, and the like. In some embodiments, the disease mentioned is non-small cell lung cancer, melanoma.
The mentioned antibodies or antigen binding fragments or bispecific antibodies can also be used in combination with other therapeutic means for the treatment of diseases. Other treatments mentioned may be chemotherapy or radiotherapy, etc. The treatment effect of the medicine can be further improved and the treatment effect of a patient can be improved by combining treatment means such as chemotherapy or radiotherapy clinically.
The application also provides the use of a bispecific antibody or monoclonal antibody or antigen binding fragment in the preparation of a medicament or kit. The medicament is for the treatment of cancer. The bispecific antibody or monoclonal antibody or antigen binding fragment can be used for preparing medicines for treating various diseases. Can also be used for preparing a kit and used as an immunodiagnosis reagent.
The technical scheme of the present application will be described in detail with reference to examples. It should be noted that these examples are only for facilitating understanding of those skilled in the art and should not be construed as limiting the scope of the present application. The following detailed description of embodiments of the application is exemplary and intended to be illustrative of the application and not to be construed as limiting the application. The PD-L1 binding moiety derived from the second antigen binding moiety is a nanobody obtained by alpaca immunization, the sequence preparation and the manner of obtaining of which are described in PCT international patent application text with application number PCT/CN2022/110423, international publication number WO 2023/01614A, the contents of which are incorporated herein in whole or in part as required. Firstly, an anti-PD-L1 nano library is obtained by an alpaca immunization method, and candidate nano antibodies are obtained by screening and identifying. The method comprises the following steps: mixing fusion protein consisting of human PD-L1 protein extracellular domain sequence and human immunoglobulin Fc region sequence with Freund's adjuvant, emulsifying, immunizing healthy alpaca, and stimulating B cell to express antigen-specific nano antibody. Then, alpaca blood is collected, lymphocytes are separated, total RNA is extracted by adopting a Trizol method, cDNA is obtained by reverse transcription, and VHH antibody gene fragments are obtained by PCR amplification from the reverse transcribed cDNA. In the competent yeast of the common electrotransformation of VHH gene fragment and yeast display carrier, the yeast homologous recombinase connects the fragment and carrier to form complete plasmid, establish library of yeast transformant with high library insertion rate and excellent diversity, and keep stable passage in the auxotroph medium. And incubating yeast cells expressing the antibodies and magnetic beads enriched with target protein antigens, and after multiple enrichment culture, identifying the magnetic bead enriched products by using a flow cytometer and sorting. After multiple rounds of screening, 5 positive clones with high expression are obtained through screening and are respectively named as antibodies A to E. The amino acid sequence of the antibody A is shown as SEQ ID NO. 90, the amino acid sequence of the antibody B is shown as SEQ ID NO. 91, the amino acid sequence of the antibody C is shown as SEQ ID NO. 92, the amino acid sequence of the antibody D is shown as SEQ ID NO. 93, and the amino acid sequence of the antibody E is shown as SEQ ID NO. 94. The humanized antibody is an antibody Ahu, and the amino acid sequence is shown as SEQ ID NO. 95; antibody Bhu, the amino acid sequence of which is shown as SEQ ID NO. 96; the amino acid sequence of the antibody Chu is shown as SEQ ID NO. 97; the amino acid sequence of the antibody D1hu is shown as SEQ ID NO. 98; the amino acid sequence of the antibody D2hu is shown as SEQ ID NO. 99.
The antibodies and humanized antibodies have high binding activity to hPD-L1 and cynomolgus monkey PD-L1 proved by FACS and ELISA experiments. But also shows blocking activity against PD-1/PD-L1 and blocking activity against CD 80/PD-L1.
For example, the binding activity of the antibody to human PD-L1 is detected by FACS. The experimental process is as follows: the affinity of different antibodies to the antigen was detected using a flow cytometer. MDA-MB-231 cells endogenously expressing PD-L1 antigen were cultured at 1X 10 5 The individual/well was added to a 96-well plate. Samples of different concentrations were then added and incubated at 4 degrees celsius for 30 minutes. A fluorescently labeled goat anti-human IgG secondary antibody (manufacturer: abcam) was then added to detect antibodies bound to the cell surface. Generating an antibody-antigen binding dose response curve using geometry values and using Graphpad PrismV6.0 software plots the four parameter raw data to determine EC50 results for antibodies binding to the antigen. Experimental results show that the EC50 values of antibody A, antibody B, antibody C, antibody D and antibody E are 0.089nM, 0.031nM, 0.042nM, 0.039nM, 0.053nM, respectively, and similar to positive controls (Abilib, 0.059 nM), showing high binding activity. The binding activity of the humanized antibodies was determined using a similar method, and the EC50 values of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu, antibody D2hu were 0.051nM, 0.18nM, 0.023M, 0.18nM and 0.29nM, respectively.
The blocking activity of the antibodies against PD-1/PD-L1 is determined, for example, by FACS. The experimental process is as follows: the blocking effect of the antibodies on PD-1 and its ligand PD-L1 was detected using a competitive flow cell-based method. Resuscitates the PD-1 overexpressing 293T-PD-1 cell line (manufacturer: kang Yuanbo, biotechnology (Beijing) Co., ltd.) cells were grown at 1X 10 5 Each well was plated in a 96-well plate, and antibody dilutions (initial working concentration 200nM, 4-fold dilution) and PD-L1-Biotin dilutions (2 ug/ml,50 ul/well) were mixed and incubated at room temperature for 30 minutes. The mixture was then added to the cells at 100ul per well, mixed well, incubated at 4℃for 60 min, then washed with FACS-containing buffer (PBS with 2% FBS), centrifuged at 1200rpm for 4 min, and the supernatant discarded. PE-labeled streptavidin was added at 100 ul/well and incubated for 30 min in the dark, after which time the incubation was washed with FACS buffer. Cells were resuspended by adding 120ul of FACS buffer and detected on-press. Live cells were shoot according to FSC/SSC and their mean fluorescence values were measured. Antibody-antigen blocking response curves were generated using geometry values and four parameter plots were plotted using Graphpad Prism V6.0 software to determine IC50 values. The experimental results show that the IC50 values of the antibody A, the antibody B, the antibody C, the antibody D and the antibody E are respectively 1.0nM, 2.1nM, 1.7nM, 2.1nM and 2.5nM, and the antibodies can block the binding of the antigen and the ligand thereof. The blocking activity of the humanized antibodies was determined by a similar method, and the IC50 values of antibodies Ahu, bhu, chu, D1hu, and D2hu were 4.0nM, 3.4nM, 1.0nM, 2.6nM, and 12nM, respectively.
The blocking activity of the antibodies against CD-80/PD-L1 was determined by FACS. The experimental process is as follows: using competition-basedFlow cytometry methods to detect the blocking effect of antibodies on PD-L1 and CD-80. Resuscitates 293T-PD-L1 cell line (manufacturer: kang Yuanbo, biotechnology (Beijing) Co., ltd.) over-expressing PD-L1, 1X 10 cells were isolated 5 Each well was plated in 96-well plates, and antibody dilutions (200 nM at initial working concentration, 4-fold dilution) were added to the cells and incubated at 4℃for 60 min. After the incubation, the cells were washed with FACS-containing buffer, centrifuged at 1200rpm for 4 minutes, and the supernatant was discarded. human-CD80-mFc (2 ug/ml) was then added at 100 ul/well and incubated at 4℃for 30 minutes in the absence of light. After incubation, the cells were washed with FACS buffer. 100ul of a 1:200 dilution of a fluorescence-labeled goat anti-mouse secondary antibody (manufacturer: abcam) was added to each well and incubated at 4℃for 30 minutes in the absence of light. After incubation, the cells were washed with FACS buffer and run on a flow machine. Its average fluorescence value was measured. Antibody-antigen blocking response curves were generated using geometry values and four parameter plots were plotted using Graphpad Prism V6.0 software to determine IC50 values. The experimental results show that the IC50 values of the antibody A, the antibody B, the antibody C, the antibody D and the antibody E are respectively 4.0nM, 3.4nM, 4.1nM, 4.8nM and 3.0nM, and the antibodies can block the binding of the antigen and the ligand thereof. Similar methods determine the blocking activity of the humanized antibodies, antibodies Ahu, bhu, chu and D1hu, with IC50 values of about 1.8nM, 1.7nM, 3.2nM and 1.5nM, respectively.
EXAMPLE 1 preparation of monoclonal antibodies
Mice were immunized with CD73 antigen and screened by hybridoma means to obtain monoclonal antibodies. Mouse myeloma cells and mouse spleen cells were fused and the fused cells plated. Then adding HAT selective medium for screening, myeloma cells can not survive, and spleen cells can survive for a short time. Finally, only the myeloma cells fused with lymphocytes survive, i.e. the hybridoma cells are obtained by screening.
Hybridoma cells capable of producing the specific antibody are then selected by limiting dilution of the cloned cells. The hybridoma cells are diluted multiple times and inoculated on a porous cell culture plate, so that each hole of cells is not more than one, and after clones grow to a certain size, antibodies secreted by cells in supernatant of each hole are detected by ELISA, and culture holes which can be combined with specific antigen (CD 73) are positive holes, and dilution is continued until monoclonal cells are obtained.
By the method, a plurality of positive clones are obtained through screening, and 7 clones with high activity are obtained through differential screening, wherein the clones are respectively numbered as clone309, clone307, clone144, clone235, clone451, clone540 and clone449. All 7 clones obtained showed high affinity to human CD73 antigen, as well as high enzymatic activity blocking activity.
The selected clones were sequenced and the selected cloned variable regions were fused to human derived constant region sequences and constructed into PCDNA3.1 mammalian expression vectors in the form of chimeric antibodies. And then carrying out expression purification on the obtained different antibodies by using a mammalian cell system to obtain different antibodies with the purity of at least more than 90%, wherein the expressed antibodies are respectively numbered as mab309, mab307, mab144, mab235, mab451, mab540 and mab449.
Example 2 identification of antibodies binding Activity to CD73
The ELISA plate was coated with human CD73 (hCD 73, manufacturer: ACRO), 100 ng/well, and incubated overnight at 4 ℃. Then, the wells were washed with PBST, 200ul of 2% BSA was added to each well, and the wells were blocked at 37℃for 1 hour. PBST was used for washing and various concentrations of the antibodies to be tested were added at 100 uL/well (initial concentration of each antibody was 31.25nm, 4-fold gradient dilution) and incubated at 37 ℃ for 2h. PBST was used for washing, and goat anti-human secondary antibody (1% BSA dilution, 1:10000, manufacturer: jackson) was added at 100 uL/well and incubated at 37℃for 1h. After washing with PBST, 100ul of TMB color development solution was added for 10-15min, 50ul of 2M sulfuric acid was added to terminate the reaction. The OD450 values were read using a microplate reader and the EC50 values of the antibodies to be tested were calculated using data processing software Graphpad prism8.0, as shown in table 1 below, wherein the positive control was the anti-CD 73 antibody olecumab (MEDI 9447) from mediune, as Li Kangzi, and the same positive control was used in the following examples.
TABLE 1
The experimental results show that the antibodies can specifically bind to CD 73.
Example 2 antibody binding Activity to MDA-MB-231 cells
The binding activity of the antibody to MDA-MB-231 cells was detected by FACS, with high expression of CD73 antigen on the surface of MDA-MB-231 cells. MDA-MB-231 cells were cultured, and the cell concentration was adjusted to 1-2X 10 6 Each ml was then added to a 96-well plate at 100 ul/well. Centrifugation at 1000rpm for 5min, removal of the culture supernatant, followed by addition of different antibodies to be tested (initial antibody concentration 200nM, 4-fold gradient dilution) at 100 ul/well and incubation at 4℃for 30min. Washing with buffer solution, adding fluorescence labeled goat anti-human secondary antibody (manufacturer: abcam), and incubating at 4deg.C for 30min. The cells were washed 2 times with buffer, resuspended with buffer and the fluorescent signal was detected on-stream. EC50 values of the samples to be tested were calculated using the data processing software Graphpad prism8.0, as shown in table 2 below.
TABLE 2
As can be seen from the results in table 2, all antibodies bind to human CD73 and exhibit properties similar to those of the positive control.
Example 3 Cross-reacting antibodies with monkey CD73
The ELISA plate was coated with monkey CD73 (CynocD 73, manufacturer: ACRO), 100 ng/well, and incubated overnight at 4 ℃. Then, the wells were washed with PBST, 200ul of 2% BSA was added to each well, and the wells were blocked at 37℃for 1 hour. PBST was used for washing, and antibodies to be tested were added at different concentrations (initial concentration of antibody 31.25nM, 4-fold gradient dilution) per well and incubated at 37℃for 2h. PBST was used for washing, and goat anti-human secondary antibody (diluted with 1% BSA, manufacturer: jackson) was added at 100 uL/well and incubated at 37℃for 1h. After washing with PBST, 100ul of TMB color development solution was added for 10-15min, 50ul of 2M sulfuric acid was added to terminate the reaction. The OD450 values were read using a microplate reader and EC50 values of the antibodies to be tested were calculated using data processing software Graphpad prism8.0 as shown in table 3 below.
TABLE 3 Table 3
Antibody numbering EC50(nM)
mab309 0.028
mab307 0.013
mab144 0.027
mab235 0.020
mab451 0.024
mab540 0.041
mab449 0.030
hIgG1 Not combined with
Positive control 0.023
The experimental results show that both the listed antibodies show binding activity to cynoCD 73.
Example 4 antibody enzyme Activity blocking assay
MDA-MB-231 tumor cells were cultured under the conditions of DMEM+10% FBS+1% PS. Cells were digested and resuspended and the cell density was adjusted to 4.5X10 5 Each of the cells was plated at 100 ul/well in 96-well cell culture plates. The antibody to be tested was diluted with medium (initial concentration 200nM, 4-fold gradient dilution). Diluting, mixing, adding into cell culture plate at 50 uL/well, and adding 5% CO at 37deg.C 2 After incubation in the incubator for 1h, 50uL of 0.8mM AMP (manufacturer: sigma) was added to each well. Mixing, and placing at 37deg.C and 5% CO 2 Incubate for 2h. Centrifugation is carried out at 1000rpm for 5min, and 50uL of supernatant is pipetted into a new 96-well plate. 50uL 130uM of ATP (manufacturer: sigma) was added to each well. After mixing, 100ul of Cell titer glo detection reagent (manufacturer: promega) was added to each well and incubated for 5-10 minutes in the dark. Fluorescent signals in 96-well plates were read using an enzyme-labeled instrument. Four parameter curves were drawn with the relative fluorescence values as the y-axis and the concentration of the antibody samples as the x-axis. The curve was analyzed using GraphPad Prism 8.0 software and IC50 values of the antibodies were derived.
TABLE 4 Table 4
Antibody numbering IC50(nM)
mab144 0.30
mab235 0.22
mab451 0.25
mab540 0.18
mab449 0.15
hIgG1 Unblocked
Experimental results show that the mab144 antibody, the mab235 antibody, the mab451 antibody, the mab540 antibody and the mab449 antibody all show enzyme activity blocking activity and are similar to the positive control.
EXAMPLE 5 antibody endocytosis assay
MDA-MB-231 tumor cells were cultured, the cells were digested with 0.25% pancreatin and resuspended for counting, and the cell density was adjusted to 1X 10 6 Each of the cells was plated at 100 ul/well in 96-well plates. Centrifugation was carried out at 1000rpm for 5 minutes, and the supernatant was discarded. The antibody to be tested was diluted to 20nM with FACS buffer, then added to a 96-well plate at 100 uL/well, mixed with the cells and placed in a refrigerator at 4℃for 30 minutes. Then centrifuged at 1000rpm for 10min and washed with FACS buffer. One of the samples is marked as 0h, placed on ice and temporarily stored in a refrigerator at 4 ℃, and the other samples are placed in a constant temperature incubator at 37 ℃ for 4h. Then taking out the sample on ice for 0h and the sample at 37 ℃ for 4h, centrifuging to remove the supernatant, adding 100ul of goat anti-human fluorescent secondary antibody (diluted 1:200, abcam), uniformly mixing, and incubating for 30min at 4 ℃. Then washed with FACS buffer. The cells were resuspended with buffer and the fluorescent signal was detected on-stream. Calculating the endocytosis ratio of the antibody according to the read fluorescence signal, wherein the calculation formula is as follows:
Endocytic ratio (%) = (0 h fluorescent signal-4 h fluorescent signal)/0 h fluorescent signal 100%.
The experimental results are shown in FIG. 6. The results indicate that anti-CD 73 antibodies are able to promote endocytosis of CD 73.
EXAMPLE 6 anti-tumor efficacy of antibodies
CT26-hPDL1-hCD73 cells were cultured to the logarithmic growth phase, the cells were collected, the culture solution was removed and washed with PBS, and the cells were inoculated into BALB/C-hPD1/hPDL1/hCD73 triple transgenic mice (humanized mouse model expressing human PD1, human PDL1 extracellular region protein and human CD73 protein, manufacturer: jiangsu Jiech Kangsu Biotech Co., ltd.) at an inoculum size of 1X 10 6 cells/100. Mu.L/cell. When the average tumor volume reaches 80-120mm 3 When left and right, mice were randomly divided into 10 groups of 6 mice each. The day of the grouping was defined as day D0, and the anti-PD-L1 antibody (antibody number D2hu, administration concentration of 1.5mpk (mg/kg)) and the anti-CD 73 antibody (administration concentration of 10 mpk) were administered in combination at day D0 twice a week for two weeks, and by intraperitoneal injection, the tumor volume variation coefficient CV did not exceed 1/3. Tumor volume and mouse body weight were measured 2-3 times per week following dosing, depending on tumor growth. The tumor volume calculation mode is as follows: tumor volume (mm) 3 ) =0.5×tumor long diameter×tumor short diameter 2 . The experimental results are shown in FIG. 7. Wherein G1 is PBS, G2 is anti-PD-L1 antibody (administration concentration is 1.5 mpk), G3 is anti-PD-L1 antibody and mab307 antibody are administered in combination, G4 is anti-PD-L1 antibody and mab309 antibody are administered in combination, G5 is anti-PD-L1 antibody and mab235 antibody are administered in combination, G6 is anti-PD-L1 antibody and mab540 antibody are administered in combination, G7 is anti-PD-L1 antibody and mab144 antibody are administered in combination, G8 is anti-PD-L1 antibody and mab449 antibody are administered in combination, and G9 is anti-PD-L1 antibody and mab451 antibody are administered in combination.
The results showed that the mouse antitumor efficacy of the mab309 antibody, the mab307 antibody, the mab235 antibody, the mab540 antibody, the mab144 antibody, the mab449 antibody, the mab451 antibody in combination with the PD-L1 antibody was superior to that of the PD-L1 antibody alone.
Example 7
The humanized antibody is obtained by a complementarity determining region Grafting method (CDR-Grafting method), and the humanized antibody is obtained by uniformly dividing the amino acid sequences of the antibody and the template into an FR region and a CDR region, and then Grafting the CDR region onto the FR region of the template, so that the CDR region determining the specificity of the antibody and the framework region of the humanized antibody are mutually combined. Meanwhile, in order to avoid the decrease of the affinity of the antibody, key amino acid residue sites are calculated by computer simulation and other technologies, and the affinity of the humanized antibody is ensured by a back mutation method. Humanized antibodies of the above antibodies were obtained, and the humanized antibodies were named after the numbering of the antibodies by adding the letter "hu" with different numbers to represent different humanized antibodies. In addition, the Fc region of the antibody human source is mutated, and the letter "mu" is added as a suffix to the mutated antibody when it is named. One of the mutations includes the L234F, L235E, P S and N297A mutations, and the mutated antibodies are given the letter "mu1" as suffix in the naming. Another mutation includes the K214R, L234F, L E, P35331S, D E and L358M mutations, and the mutated antibody is added with the letter "mu2" as a suffix when named. For example, mab309hu1 represents a first antibody where the mab309 antibody was humanized. mab144hu2 represents a second antibody to which the mab144 antibody was humanized. mab309hu3mu represents a third antibody where the mab309 antibody was humanized while the Fc region of the antibody was mutated.
Example 8 humanized antibodies binding Activity to MDA-MB-231 cells
MDA-MB-231 cells with high expression of CD73 antigen on their surface are added into 96-well plate at 100 ul/well to obtain cell concentration of 1-2×10 6 And each ml. Washed with FACS buffer (i.e., PBS containing 2% FBS), then added with different antibodies to be tested (initial antibody concentration 200nM, 4-fold gradient dilution) at 100 ul/well and incubated at 4℃for 30min. Washing with buffer solution, adding fluorescence labeled goat anti-human secondary antibody (manufacturer: abcam), and incubating at 4deg.C for 30min. The cells were washed 2 times with buffer, resuspended with buffer and the fluorescent signal was detected on-stream. EC50 values of the samples to be tested were calculated using the data processing software Graphpad prism8.0, as shown in table 5 below.
TABLE 5
Antibody numbering EC50(nM)
mab309hu1mu1 1.1
mab309hu3mu1 3.0
mab144hu2mu1 1.5
mab451hu4mu1 0.68
mab451hu5mu1 0.88
mab451hu6mu1 0.73
mab540hu1mu1 1.3
mab540hu3mu1 0.87
mab449hu4mu1 0.53
Positive control 0.55
hIgG1 Not combined with
The humanized antibodies showed high binding activity to MDA-MB-231 cells, similar to the positive control.
Example 9 Activity of blocking the Activity of humanized antibody
Culturing A375 tumor cells, digesting the cells with 0.25% pancreatin to adjust the cell density to 4.5X10 5 Each of the cells was plated at 100 ul/well in 96-well cell culture plates. Different antibodies to be tested were diluted with medium (initial concentration 200nM, 4-fold gradient dilution). Diluting, mixing, adding into cell culture plate at 50 uL/well, and adding 5% CO at 37deg.C 2 After incubation in the incubator for 1h, 50uL of 0.8mM AMP (manufacturer: sigma) was added to each well. Mixing at 37deg.C with 5% CO 2 Incubate for 2h. Centrifugation at 1000rpm for 5min, 50uL of supernatant was pipetted onto a fresh 96-well cell culture plate, and 50uL 130uM of ATP (manufacturer: sigma) was added per well. After mixing, 100ul of Cell titer glo detection reagent (manufacturer: promega) was added to each well. Incubate for 5-10 min in the dark and read fluorescence signals in 96-well plates with an enzyme-labeled instrument. Four parameter curves were drawn with the relative fluorescence values as the y-axis and the concentration of the antibody samples as the x-axis. The curves were analyzed using GraphPad Prism 8.0 software and IC50 values for the antibodies were obtained as shown in table 6 below.
TABLE 6
Antibody numbering IC50(nM)
mab144hu1mu1 0.087
mab144hu2mu1 0.055
mab449hu4mu1 0.061
mab451hu4mu1 0.072
mab451hu5mu1 0.049
mab451hu6mu1 0.035
hIgG1 Unblocked
The results indicate that the humanized antibody exhibits enzyme activity blocking activity.
EXAMPLE 10 construction of bispecific antibodies
Genetic engineering of an anti-CD 73 antibody as a first antigen-binding portion and an anti-PD-L1 fragment as a second antigen-binding portion to construct a bispecific antibody wherein the anti-PD-L1 fragment is passed through (G 4 S) 4 The joints are connected together. During ligation, the anti-PD-L1 fragment is ligated to the carboxy terminus of the heavy chain constant region domain CH3 of the IgG type structure. There is a carboxyl-terminal linkage of the heavy chain constant region domain CH3 of the anti-PD-L1 single domain antibody (anti-PD-L1 single domain antibody, numbered D2 hu) and the IgG structure, respectively, as shown in FIG. 1. The bispecific antibodies formed are numbered beginning with "b". For example, b144mu-D12hu represents a bispecific antibody formed by a mab144 antibody (with a mutation in the Fc region) as the first antigen-binding moiety and D2hu as the second antigen-binding moiety. For another example, b144hu2mu1-D2hu represents a bispecific antibody formed by humanizing mab144 with a second antibody (with a first mutation in the Fc region) as the first antigen-binding moiety and D2hu as the second antigen-binding moiety. And so on.
Then, the bispecific antibody protein is produced by transiently transfecting CHO-S cells, the supernatant is collected and purified by a protein A affinity column, the purity of the purified antibody is measured by HPLC and SDS-PAGE, and the purity of the bispecific antibody is determined to be more than 90% by detection.
EXAMPLE 11 bispecific antibody binding Activity
CHO-K1-CD73 overexpressing cells were added to 96-well plates at a cell concentration of 1-2X 10 at 100 ul/well 6 And each ml. Washed with FACS buffer (i.e., PBS containing 2% FBS), then added with different antibodies to be tested (initial antibody concentration 200nM, 4-fold gradient dilution) at 100 ul/well and incubated at 4℃for 30min. Washing with buffer solution, adding fluorescence labeled goat anti-human secondary antibody (manufacturer: abcam), and incubating at 4deg.C for 30min. Washing with buffer solution, adding buffer solution to re-suspend the cells, and detecting fluorescent signals by a flow type machine. EC50 values of the samples to be tested were calculated using the data processing software Graphpad prism8.0, as shown in table 7 below.
TABLE 7
Antibody numbering EC50(nM)
b144mu1-D2hu 1.5
b144hu2mu1-D2hu 1.8
b449mu1-D2hu 0.28
b309mu1-D2hu 0.50
b451mu1-D2hu 0.67
hIgG Not combined with
Example 12 determination of bispecific antibody Activity blocking Activity
The enzyme activity blocking activity of the bispecific antibody was examined by the method of reference example 9, and the results are shown in tables 8-1 and 8-2 and FIG. 8. The enzyme activity blocking activity of the bispecific antibody is stronger than that of the positive control, and is at least 2-5 times that of the positive control.
TABLE 8-1
TABLE 8-2
Antibody numbering EC50(nM)
b144hu2mu2-D2hu 0.40
mab144hu2mu2 0.30
mab449hu4mu1 0.42
b451hu4mu1-D2hu 0.68
mab451hu4mu2 0.25
hIgG Unblocked
hIgG1mu2 Unblocked
Example 13 bispecific antibody reporter blocking Activity
The blocking effect of bispecific antibodies on the PD-L1/PD-1 pathway was investigated by reporter experiments. The experiment was performed with the aid of two cell lines, jurkat-PD1-CD3zeta-NFAT-Luc2 cell line (manufacturer: kang Yuanbo, biotechnology (Beijing) Co.) and 293T-hPD-L1 cell line (manufacturer: kang Yuanbo, biotechnology (Beijing) Co.). The Jurkat-PD1-CD3zeta-NFAT-Luc2 cell strain stably expresses fusion protein composed of PD-1ECD and CD3zeta in Jurkat cells, and simultaneously inserts a luciferase reporter gene driven by NF-AT; 293T-hPD-L1 cells are 293T cells expressing human PD-L1. When two cells are co-cultured, the PD-1/PD-L1 interaction is used as a first signal, the intracellular CD3zeta chain is used as a second signal to transmit an activation signal inwards, and the NFAT driven luciferase reporter gene is expressed and emits fluorescence; when the PD-L1 antibody is added into a culture system, the combination of the PD-1/PD-L1 is blocked, the transmission of an activation signal is inhibited, and the luciferase reporter gene cannot be expressed. The experimental procedure was as follows:
293T-hPD-L1 cells were cultured at a rate of 2X 10 4 The wells/well were plated in 96-well plates and Jurkat-PD1-CD3zeta-NFAT-Luc2 effector cells were plated in 2X 10 4 Well continue to be added to 96-well plates; then adding the antibodies to be detected with different concentrations (initial concentration is 60ug/ml, 4-time gradient dilution) at 37 DEG CIncubate for 18-24h. 100ul of luciferase substrate ONE-Glo was added per well TM Luciferase Assay system detection reagent, incubating for 5 minutes in the absence of light; the microplate reader reads the fluorescent signal in the 96-well plate. Four parameter curves were drawn with the relative fluorescence values as the y-axis and the concentration of the antibody samples as the x-axis. The curve was analyzed using GraphPad Prism 8.0 software and the IC50 values of the diabodies were obtained as shown in tables 9-1 and 9-2 below.
TABLE 9-1
TABLE 9-2
Antibody numbering IC50(nM)
b144hu2mu2-D2hu 4.6
b451hu4mu2-D2hu 5.1
D2hu 5.0
hIgG1mu2 Unblocked
In the description of the present specification, a description of the terms "embodiment," "some embodiments," "examples," "detailed description," or "implementation," 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 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 (19)

1. An anti-CD 73 antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region:
comprises the HCDR sequences shown in SEQ ID NO. 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 1, 2 and 3;
or comprises the HCDR sequences shown in SEQ ID NOs 4, 5 and 6, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs 4, 5 and 6;
or comprises the HCDR sequences shown in SEQ ID NO. 7, 8 and 9, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 7, 8 and 9;
or comprises the HCDR sequences shown in SEQ ID NOS 10, 11 and 12, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS 10, 11 and 12;
Or comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO 13, 14 and 15;
or comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOS.16, 17 and 18;
or comprises the HCDR sequences shown in SEQ ID NO. 19, 20 and 21, or has one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NO. 19, 20 and 21;
the light chain variable region:
comprises the LCDR sequences shown in SEQ ID NOs 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOs 22, 23 and 24;
or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 25, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 25;
or comprises the LCDR sequences shown in SEQ ID NOS.26, 27 and 28, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.26, 27 and 28;
Or comprises the LCDR sequences shown in SEQ ID NOS.29, 30 and 31, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.29, 30 and 31;
or comprises the LCDR sequences shown in SEQ ID NOS.22, 23 and 32, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.22, 23 and 32;
or comprises the LCDR sequences shown in SEQ ID NOS.33, 34 and 35, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.33, 34 and 35;
or comprises the LCDR sequences shown in SEQ ID NOS.36, 37 and 38, or has one, two or three conservative amino acid mutations with the LCDR sequences shown in SEQ ID NOS.36, 37 and 38.
2. The antibody or antigen-binding fragment of claim 1, which is selected from at least one of the following:
(1) Comprises the HCDR sequences shown in SEQ ID NO 1, 2 and 3, and the LCDR sequences shown in SEQ ID NO 22, 23 and 24; or alternatively
(2) Comprises the HCDR sequences shown in SEQ ID NO. 4, 5 and 6, and the LCDR sequences shown in SEQ ID NO. 22, 23 and 25; or alternatively
(3) Comprises the HCDR sequences shown in SEQ ID NO 7, 8 and 9, and the LCDR sequences shown in SEQ ID NO 26, 27 and 28; or alternatively
(4) Comprises the HCDR sequences shown in SEQ ID NO 10, 11 and 12, and the LCDR sequences shown in SEQ ID NO 29, 30 and 31; or alternatively
(5) Comprises the HCDR sequences shown in SEQ ID NO 13, 14 and 15, and the LCDR sequences shown in SEQ ID NO 22, 23 and 32; or alternatively
(6) Comprises the HCDR sequences shown in SEQ ID NOS.16, 17 and 18, and the LCDR sequences shown in SEQ ID NOS.33, 34 and 35; or alternatively
(7) Comprises the HCDR sequences shown in SEQ ID NO 19, 20 and 21, and the LCDR sequences shown in SEQ ID NO 36, 37 and 38.
3. An anti-CD 73 antibody or antigen-binding fragment comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein
HCDR1 is selected from the sequences shown in SEQ ID NO 1 or 4 or 7 or 10 or 13 or 16 or 19,
HCDR2 is selected from the sequences shown in SEQ ID NO 2 or 5 or 8 or 11 or 14 or 17 or 20,
HCDR3 is selected from the sequences shown in SEQ ID NO 3 or 6 or 9 or 12 or 15 or 18 or 21,
LCDR1 is selected from the sequences shown in SEQ ID NO. 22 or 26 or 29 or 33 or 36,
LCDR2 is selected from the sequences shown in SEQ ID NO 23 or 27 or 30 or 34 or 37,
LCDR3 is selected from the sequences shown in SEQ ID NO 24 or 25 or 28 or 31 or 32 or 35 or 38.
4. An anti-CD 73 antibody or antigen-binding fragment comprising:
the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO 39 or 40 or 41 or 42 or 43 or 44 or 45 and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO 46 or 47 or 48 or 49 or 50 or 51 or 52.
5. The antibody or antigen-binding fragment of any one of claims 1-4, comprising:
a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence shown in SEQ ID NO 39 or 40 or 41 or 43 or 44 or 45 or 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and
a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO 46 or 47 or 48 or 50 or 52 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79.
6. The antibody or antigen-binding fragment of any one of claims 1 to 5, which is selected from at least one of the following:
has a heavy chain variable region shown in SEQ ID NO. 39 and a light chain variable region shown in SEQ ID NO. 46; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 40 and a light chain variable region shown in SEQ ID NO. 47; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 41 and a light chain variable region shown in SEQ ID NO. 48; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 42 and a light chain variable region shown in SEQ ID NO. 49; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 43 and a light chain variable region shown in SEQ ID NO. 50; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 44 and a light chain variable region shown in SEQ ID NO. 51; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 45 and a light chain variable region shown in SEQ ID NO. 52; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 53 and a light chain variable region shown in SEQ ID NO. 66; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 54 and a light chain variable region shown in SEQ ID NO. 67; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 55 and a light chain variable region shown in SEQ ID NO. 68; or alternatively
Has a heavy chain variable region shown as SEQ ID NO. 56 and a light chain variable region shown as SEQ ID NO. 69; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 57 and a light chain variable region shown in SEQ ID NO. 70; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 58 and a light chain variable region shown in SEQ ID NO. 71; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 59 and a light chain variable region shown in SEQ ID NO. 72; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 60 and a light chain variable region shown in SEQ ID NO. 73; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 60 and a light chain variable region shown in SEQ ID NO. 74; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 61 and a light chain variable region shown in SEQ ID NO. 75; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 62 and a light chain variable region shown in SEQ ID NO. 76; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 63 and a light chain variable region shown in SEQ ID NO. 77; or alternatively
Has a heavy chain variable region shown as SEQ ID NO. 64 and a light chain variable region shown as SEQ ID NO. 78; or alternatively
Has a heavy chain variable region shown in SEQ ID NO. 65 and a light chain variable region shown in SEQ ID NO. 79.
7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody is selected from at least one of the group consisting of: igG1, igG2, igG3, igG4, or variants thereof;
Optionally, the antibody or antigen binding fragment has at least one of the following properties:
(a) Inhibition of CD73 enzymatic activity;
(b) Binding to human CD73 with a Kd of 10nM or less;
(c) Monkey CD73 was bound with a Kd of 10nM or less.
8. A bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion;
the first antigen binding portion specifically binds to a first epitope, the first antigen binding portion comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment of any one of claims 1-7;
the second antigen binding portion specifically binds to a second epitope, the second antigen binding portion being fused to the first antigen binding portion;
optionally, the second antigen binding portion specifically binds to at least one of: PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, 4-1BB, OX40, VEGF trap, VEGFR, EGFR, GARP, TGF-beta, CD47, c-Met, CD3, CD33, TIGIT, PVRIG, CD96, LEFA1, CD226, FAP2, GITR, VISITA, claudin 18.2, ICOS, BTLA, B7-H3, BCMA, MUC1/16;
Optionally, the first antigen binding portion is selected from at least one of:
(1) Has the HCDR sequences shown in SEQ ID NO 1, 2 and 3, and the LCDR sequences shown in SEQ ID NO 22, 23 and 24; or alternatively
(2) Has the HCDR sequences shown in SEQ ID NO. 4, 5 and 6, and the LCDR sequences shown in SEQ ID NO. 22, 23 and 25; or alternatively
(3) Has the HCDR sequences shown in SEQ ID NO 7, 8 and 9, and the LCDR sequences shown in SEQ ID NO 26, 27 and 28; or alternatively
(4) Has the HCDR sequences shown in SEQ ID NO 10, 11 and 12, and the LCDR sequences shown in SEQ ID NO 29, 30 and 31; or alternatively
(5) Has the HCDR sequences shown in SEQ ID NO 13, 14 and 15, and the LCDR sequences shown in SEQ ID NO 22, 23 and 32; or alternatively
(6) Has the HCDR sequences shown in SEQ ID NO 16, 17 and 18, and the LCDR sequences shown in SEQ ID NO 33, 34 and 35; or alternatively
(7) Has the HCDR sequences shown in SEQ ID NO 19, 20 and 21, and the LCDR sequences shown in SEQ ID NO 36, 37 and 38.
9. The bispecific antibody of claim 8, wherein the heavy chain variable region and heavy chain constant domain of the first antigen binding portion are linked, and the light chain variable region and light chain constant domain of the first antigen binding portion are linked to form an IgG-like structure;
The second antigen binding portion is linked to the IgG-like structure, the second antigen binding portion comprising a single domain antibody that specifically binds to a second epitope;
optionally, the second antigen binding portion comprises a single domain antibody that specifically binds a second epitope selected from at least one of PD-1, PD-L1, VEGF trap, TGF- β, GARP, TIGIT, PVRIG, CD 96;
optionally, the second antigen binding portion comprises CDR1, CDR2, and CDR3, the second antigen binding portion being selected from at least one of:
(1) Has a CDR1 sequence shown as SEQ ID NO. 80, a CDR2 sequence shown as SEQ ID NO. 81 and a CDR3 sequence shown as SEQ ID NO. 82; or alternatively
(2) Has a CDR1 sequence shown as SEQ ID NO. 83, a CDR2 sequence shown as SEQ ID NO. 84 and a CDR3 sequence shown as SEQ ID NO. 85; or alternatively
(3) Has a CDR1 sequence shown as SEQ ID NO. 86, a CDR2 sequence shown as SEQ ID NO. 87 and a CDR3 sequence shown as SEQ ID NO. 88; or alternatively
(4) Has a CDR1 sequence shown in SEQ ID NO. 80, a CDR2 sequence shown in SEQ ID NO. 81 and a CDR3 sequence shown in SEQ ID NO. 89;
optionally, the second antigen binding portion is selected from the group consisting of:
(a) Has a sequence as shown in SEQ ID NO 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99;
(b) Sequences having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity than (a).
10. The bispecific antibody of claim 8, wherein the heavy chain variable region and heavy chain constant domain of the first antigen binding portion are linked, and the light chain variable region and light chain constant domain of the first antigen binding portion are linked, forming an IgG type structure;
the second antigen binding portion is linked to the carboxy terminus (C-terminus) of the Fc region; or alternatively
The second antigen binding portion is linked to the amino terminus (N-terminus) of the heavy chain variable region or the light chain variable region of the first antigen binding portion; or alternatively
The second antigen binding portion is linked to the carboxy terminus (C-terminus) of the light chain constant domain CL;
optionally, the bispecific antibody has at least one of the following properties:
(a) Specifically binds to PD-L1 with an EC50 value of 10nM or less;
(b) Specifically binds to CD73 with an EC50 value of 10nM or less;
(c) Has antitumor activity;
(d) Promoting CD73 endocytosis.
11. A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1 to 7 or encoding the bispecific antibody of any one of claims 8 to 10.
12. A construct comprising the polynucleotide of claim 11.
13. A host cell comprising the polynucleotide of claim 11 or the construct of claim 12.
14. A pharmaceutical composition comprising:
the antibody or antigen-binding fragment of any one of claims 1 to 7, or the bispecific antibody of any one of claims 8 to 10; and
a pharmaceutically acceptable carrier.
15. An antibody conjugate, comprising:
the antibody or antigen-binding fragment of any one of claims 1 to 7, or the bispecific antibody of any one of claims 8 to 10; and
a functional small molecule linked to the antibody or antigen-binding fragment or the bispecific antibody.
16. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 or the bispecific antibody of any one of claims 8 to 10.
17. A method of producing the antibody or antigen-binding fragment of any one of claims 1 to 7 or producing the bispecific antibody of any one of claims 8 to 10, comprising:
culturing the host cell of claim 13, and
collecting said antibody or antigen binding fragment or said bispecific antibody from the culture.
18. A method for preventing and/or treating a disease, comprising:
administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1-7, or the bispecific antibody of any one of claims 8-10, or the pharmaceutical composition of claim 14, or the antibody conjugate of claim 15;
optionally, the disease is selected from cancer or autoimmune disease.
19. Use of the antibody or antigen-binding fragment of any one of claims 1 to 7, or the bispecific antibody of any one of claims 8 to 10, in the manufacture of a medicament or kit or antibody conjugate.
CN202310968592.5A 2023-08-03 2023-08-03 anti-CD 73 antibody and application thereof Pending CN116813786A (en)

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