Anti-garp antibodies or antigen-binding fragments thereof and uses thereof

Abstract

This invention relates to an anti-GARP antibody or its antigen-binding fragment and its uses. The anti-GARP antibody or its antigen-binding fragment provided by this invention can specifically recognize GARP and the GARP / Latent TGF-β1 complex, and does not bind to Latent TGF-β1 or TGF-β1 alone, exhibiting high target activity and ADCC activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to anti-GARP antibodies or their antigen-binding fragments and their uses. This invention also relates to nucleic acid molecules encoding the anti-GARP antibody or its antigen-binding fragment, vectors containing the nucleic acid molecules, host cells; and conjugates containing the anti-GARP antibody or its antigen-binding fragment. This invention further relates to the use of anti-GARP antibodies or their antigen-binding fragments, pharmaceutical compositions containing them, and conjugates. This invention also relates to methods for preventing and / or treating diseases using anti-GARP antibodies or their antigen-binding fragments. This invention further relates to kits containing anti-GARP antibodies or their antigen-binding fragments. Background Technology

[0002] Glycoprotein-A repetitions predominant (GARP) can form a complex with latent transforming growth factor β1 (Latent TGF-β1) on the cell surface, releasing active TGF-β1 under the action of integrins. The GARP / Latent TGF-β1 complex is overexpressed in activated regulatory T cells (Tregs), various tumor cells, and platelets. Furthermore, the role of soluble GARP has been reported; soluble GARP lacking a transmembrane region can be directly added to CD4+. + In T cells, soluble GARP can inhibit their proliferation; therefore, the possibility of an immunosuppressive mechanism involving the release of TGF-β1 by soluble GARP cannot be ruled out (Hahn SA, Stahl HF, Becker C, Correll A, Schneider FJ, Tuettenberg A, Jonuleit H. Soluble GARP has potent antiinflammatory and immunomodulatory impact on human CD4). + T cells. Blood. 2013 Aug 15;122(7):1182-91. doi: 10.1182 / blood-2012-12-474478. Epub 2013 Jul1. PMID: 23818544).

[0003] Therefore, eliminating Treg cells and tumor cells that highly express GARP / Latent TGF-β1 in the tumor microenvironment, while neutralizing soluble GARP, is seen as a promising approach to cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to provide an antibody or antigen-binding fragment thereof that targets GARP, which kills Treg cells and tumor cells expressing GARP in the tumor microenvironment through antibody-mediated ADCC effect, thereby achieving better tumor treatment results.

[0005] The anti-GARP antibody or its antigen-binding fragment provided by this invention can specifically recognize GARP and the GARP / LatentTGF-β1 complex (in this document, "GARP / Latent TGF-β1" is sometimes also referred to as "GARP / TGF-β1" or "GARP / LTGF-β1"), and does not bind to Latent TGF-β1 or TGF-β1 alone. The anti-GARP antibody or its antigen-binding fragment provided by this invention can eliminate Treg cells and tumor cells in the tumor microenvironment, and has good therapeutic safety. Compared with existing anti-GARP antibodies, it has higher target binding activity and ADCC activity.

[0006] Specifically, in a first aspect, the present invention provides an anti-GARP antibody or an antigen-binding fragment thereof.

[0007] In some embodiments, the anti-GARP antibody or its antigen-binding fragment comprises: The three heavy chain CDRs (VH CDR1, VHCDR2, VH CDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 20 or 22 and / or the three light chain CDRs (VLCDR1, VL CDR2, VL CDR3) contained in the light chain variable region as shown in SEQ ID NO: 21 or 23.

[0008] Based on the variable region amino acid sequence contained in the given antibody or its fragment according to the present invention, those skilled in the art can routinely determine the CDR contained therein. For example, the CDR in the variable region amino acid sequence can be determined using methods known in the art, such as the Kabat numbering scheme, the Chothia numbering scheme, or the IMGT numbering scheme.

[0009] In some embodiments, the present invention provides an anti-GARP antibody or an antigen-binding fragment thereof comprising one or more amino acid sequences (according to the Kabat numbering scheme) from (a) to (f): (a) VH CDR1, comprising an amino acid sequence as shown in SEQ ID NO: 1 or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 or 7, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO: 1 or 7; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO: 2; (c) VH CDR3, comprising an amino acid sequence as shown in SEQ ID NO: 3 or 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 3 or 8, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO: 3 or 8; (d) VL CDR1, comprising an amino acid sequence as shown in SEQ ID NO: 4 or 9, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 4 or 9, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO: 4 or 9; (e) VL CDR2, comprising the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO: 5; (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 6.

[0010] In some preferred embodiments, the antibody or its antigen-binding fragment comprises VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 3, VL CDR1 as shown in SEQ ID NO: 4, VL CDR2 as shown in SEQ ID NO: 5, and VL CDR3 as shown in SEQ ID NO: 6.

[0011] In some preferred embodiments, the antibody or its antigen-binding fragment comprises VH CDR1 as shown in SEQ ID NO: 7, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 8, VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 5, and VL CDR3 as shown in SEQ ID NO: 6.

[0012] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises at least a heavy chain variable region and / or a light chain variable region, both of which include the aforementioned CDRs and spaced frame regions (FRs), with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the maximum 20% difference in amino acid sequence resulting from the "at least 80% sequence identity" may exist in any frame region within the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or its fragment of the present invention. This difference may be caused by amino acid substitution, deletion, or insertion at any position.

[0013] In some embodiments, the frame region comprises any one or more of the amino acid sequences shown in SEQ ID NO: 10-19, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one or more of the amino acid sequences shown in SEQ ID NO: 10-19, or an amino acid sequence having one or more conserved amino acid substitutions with any one or more of the amino acid sequences shown in SEQ ID NO: 10-19.

[0014] In some preferred embodiments, the frame region comprises an amino acid sequence as shown in any one of SEQ ID NO: 10-17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 10-17, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NO: 10-17.

[0015] In some preferred embodiments, the frame region comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 13-19, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 11, 13-19, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NO: 11, 13-19.

[0016] In some preferred embodiments, the frame region includes heavy chain FR1, heavy chain FR2, heavy chain FR3, heavy chain FR4, light chain FR1, light chain FR2, light chain FR3, and light chain FR4; Preferably: The heavy chain FR1 comprises an amino acid sequence as shown in SEQ ID NO: 10 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 10 or 18, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10 or 18. The heavy chain FR2 comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 11. The heavy chain FR3 comprises an amino acid sequence as shown in SEQ ID NO: 12 or 19, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 12 or 19, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 12 or 19. The heavy chain FR4 comprises the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 13. The light chain FR1 comprises the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 14. The light chain FR2 comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 15. The light chain FR3 comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 16. The light chain FR4 comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 17.

[0017] In some preferred embodiments, the frame region includes heavy chain FR1 as shown in SEQ ID NO: 10, heavy chain FR2 as shown in SEQ ID NO: 11, heavy chain FR3 as shown in SEQ ID NO: 12, heavy chain FR4 as shown in SEQ ID NO: 13, light chain FR1 as shown in SEQ ID NO: 14, light chain FR2 as shown in SEQ ID NO: 15, light chain FR3 as shown in SEQ ID NO: 16, and light chain FR4 as shown in SEQ ID NO: 17.

[0018] In some preferred embodiments, the frame region includes heavy chain FR1 as shown in SEQ ID NO: 18, heavy chain FR2 as shown in SEQ ID NO: 11, heavy chain FR3 as shown in SEQ ID NO: 19, heavy chain FR4 as shown in SEQ ID NO: 13, light chain FR1 as shown in SEQ ID NO: 14, light chain FR2 as shown in SEQ ID NO: 15, light chain FR3 as shown in SEQ ID NO: 16, and light chain FR4 as shown in SEQ ID NO: 17.

[0019] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:20, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO:20.

[0020] In some preferred embodiments, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 26.

[0021] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:22, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO:22.

[0022] In some preferred embodiments, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 28.

[0023] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO:21.

[0024] In some preferred embodiments, the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 27.

[0025] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence having one or more conserved amino acid substitutions with respect to the amino acid sequence shown in SEQ ID NO:23.

[0026] In some preferred embodiments, the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 29.

[0027] In some embodiments, the present invention provides an anti-GARP antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 20, and the light chain variable region comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 21.

[0028] In some embodiments, the present invention provides an anti-GARP antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 22, and the light chain variable region comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 23.

[0029] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region or a functional variant thereof.

[0030] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, and the heavy chain constant region of the monoclonal antibody is an IgG1, IgG2, IgG3 or IgG4 subtype or a functional variant thereof; preferably IgG1 or a defucosylated IgG1 variant.

[0031] In some embodiments, the heavy chain constant region of the antibody or its antigen-binding fragment comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 24, and the light chain constant region of the antibody or its antigen-binding fragment comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 25.

[0032] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and / or a light chain, wherein: The heavy chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 32; The heavy chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 34; The light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 33; or The light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 35.

[0033] In some preferred embodiments, the coding sequence of the heavy chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 36 or 38; and the coding sequence of the light chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 37 or 39.

[0034] In some preferred embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 32, and the light chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 33.

[0035] In some preferred embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 34, and the light chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 35.

[0036] In some preferred embodiments, the coding sequence of the heavy chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 36, and the coding sequence of the light chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 37.

[0037] In some preferred embodiments, the coding sequence of the heavy chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 38, and the coding sequence of the light chain comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 39.

[0038] In most cases, the C-terminal lysine (K) of the antibody heavy chain is lost to varying degrees. This is usually caused by carboxypeptidase produced during cell culture, resulting in each heavy chain of the generated antibody having 0 or 1 lysine at the C-terminus, thus making the antibody exhibit C-terminal heterogeneity. However, this does not affect the activity of the antibody itself. The antibodies described in this disclosure cover various antibodies with C-terminal heterogeneity.

[0039] In some embodiments of the antibody described in any of the foregoing embodiments, the antibody is any form of monoclonal antibody, single-chain antibody, bispecific antibody, multispecific antibody, murine antibody, fully or partially humanized antibody, or chimeric antibody. In one specific embodiment, the antibody is a monoclonal antibody. In a preferred embodiment, the antibody is a chimeric antibody. In a more preferred embodiment, the antibody is a human-mouse chimeric antibody.

[0040] In some embodiments of the antibody mentioned above, the antibody is a full-length antibody.

[0041] In some embodiments, the anti-GARP antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the anti-GARP antibody of the present invention covers only its antigen-binding portion, such as: Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv, or (dsFv)2 fragment.

[0042] In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody, such as Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv, or (dsFv)2 fragments; preferably, the antigen-binding fragment is an antigen-binding fragment of a human-mouse chimeric antibody.

[0043] In some embodiments of the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof may be used in the form of antibody-drug conjugates or CAR molecules.

[0044] In certain embodiments of the antibody or antigen-binding fragment thereof of any of the foregoing claims, the antibody or antigen-binding fragment thereof has at least one of the following properties (1)-(5): (1) It has inhibitory activity against the immunosuppressive function of regulatory T cells; (2) It has antibody-dependent cytotoxic activity; (3) It has in vivo and / or in vitro antitumor activity; (4) Specifically binds to GARP, the GARP / Latent TGF-β1 complex, and cells expressing the above proteins; preferably, the GARP includes human GARP, monkey GARP, and mouse GARP; preferably, the GARP / Latent TGF-β1 complex includes human GARP / Latent TGF-β1 complex, monkey GARP / Latent TGF-β1 complex, and mouse GARP / Latent TGF-β1 complex; preferably, the cells include tumor cells, engineered cells, immune cells, and platelets; (5) It does not bind to either Latent TGF-β1 or TGF-β1 alone.

[0045] In a second aspect, the present invention provides a biomaterial comprising: (i) A nucleic acid molecule encoding the anti-GARP antibody or its antigen-binding fragment as described in this invention; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

[0046] The nucleic acid molecule comprises a coding nucleic acid molecule that, due to codon degeneracy, can be translated to yield the antibody or its antigen-binding fragment. The nucleic acid molecule includes, but is not limited to, DNA and RNA. In some embodiments, the nucleic acid molecule is DNA, including cDNA, genomic DNA, or synthetically produced DNA. In some embodiments, the DNA is double-stranded. In some embodiments, the DNA is a coding strand or a non-coding strand.

[0047] In some implementations, the nucleic acid molecule may be an isolated nucleic acid molecule.

[0048] The nucleic acid molecules of the present invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, the present invention also provides a vector comprising the nucleic acid molecule described in (i). In some embodiments, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, and a bacteriophage vector. In some embodiments, the expression vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, and eukaryotic cell expression plasmids; preferably, eukaryotic cell expression plasmids.

[0049] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, the present invention also provides host cells comprising the vector described in (ii) or the nucleic acid molecule described in (i), or said host cells transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell includes plant cells, microbial cells, and mammalian cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In some embodiments, the host cell is eukaryotic, such as a mammalian cell or an engineered variant thereof. In some embodiments, the host cell is another cell suitable for preparing antibodies or antigen-binding fragments thereof.

[0050] The antibodies or antigen-binding fragments thereof provided by this invention can be obtained using any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody can be obtained first from the nucleic acid molecule provided by this invention, or the heavy chain and / or light chain of the antibody can be obtained, and then assembled with optional other structural domains of the antibody to form an antibody.

[0051] Thirdly, the present invention provides a method for preparing the anti-GARP antibody or antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0052] Fourthly, the present invention provides a conjugate comprising the anti-GARP antibody or its antigen-binding fragment as described in the present invention and a conjugated portion, wherein the conjugated portion is another molecule; preferably, the conjugated portion is a cytotoxin, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an oligonucleotide, or an enzyme, etc.

[0053] The antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells and / or conjugates provided by the present invention can be included in pharmaceutical compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed.

[0054] Fifthly, the present invention provides a pharmaceutical composition comprising the anti-GARP antibody of the first aspect of the present invention or its antigen-binding fragment, or the biological material (including nucleic acid molecules, carriers, host cells), conjugate, and one or more pharmaceutically acceptable carriers, sustained-release agents, or excipients of the present invention. In some embodiments of the present invention, the pharmaceutical composition further comprises a second therapeutic agent and / or optionally a pharmaceutical excipient, the second therapeutic agent being selected from cytokines, antibodies, chemotherapeutic agents, and small molecule drugs.

[0055] In a sixth aspect, the present invention provides the use of the anti-GARP antibody or its antigen-binding fragment, the biological material, the conjugate, or the pharmaceutical composition in the preparation of any of the following products: (I) Products for the prevention and / or treatment of diseases suffered by the subjects; (II) Products that stimulate or enhance the immune response; (III) Products tested by GARP.

[0056] In some implementations, the disease includes cancer; preferably, the cancer includes solid tumors and hematologic malignancies. Preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, soft tissue sarcoma, and other solid tumors. Preferably, the hematologic malignancies include lymphoma, etc.

[0057] In a seventh aspect, the present invention provides a method for eliminating Treg cells or tumor cells and / or soluble GARP protein that highly express GARP / Latent TGF-β1 complex or GARP in the tumor microenvironment, comprising administering to a subject in need an effective amount of the anti-GARP antibody or its antigen-binding fragment thereof, the biological material, the conjugate, or the pharmaceutical composition of the present invention. In some embodiments, the GARP / Latent TGF-β1 complex includes a human GARP / Latent TGF-β1 complex, a monkey GARP / Latent TGF-β1 complex, and a mouse GARP / Latent TGF-β1 complex.

[0058] Eighthly, the present invention provides a kit comprising the antibody or its antigen-binding fragment, conjugate, or pharmaceutical composition of the present invention. The kit can be used to detect the presence or expression level of GARP and / or the GARP / Latent TGF-β1 complex in a sample, and can also be used for the diagnosis or prognosis of diseases. In some embodiments, the diseases include benign tumors and cancers; the cancers include solid tumors and hematological malignancies. Preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, soft tissue sarcoma, and other solid tumors. Preferably, the hematological malignancies include lymphoma, etc.

[0059] In a ninth aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need an effective amount of the anti-GARP antibody or its antigen-binding fragment, the biological material, the conjugate, or the pharmaceutical composition. In some embodiments, the disease includes cancer; the cancer includes solid tumors and hematologic malignancies. Preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, soft tissue sarcoma, and other solid tumors. Preferably, the hematologic malignancies include lymphoma, etc.

[0060] In a tenth aspect, the present invention provides the anti-GARP antibody or its antigen-binding fragment thereof, the biological material, the conjugate, or the pharmaceutical composition thereof, which can be used as a medicament or for treatment.

[0061] Eleventhly, the present invention provides a method for detecting the presence or level of GARP and / or GARP / Latent TGF-β1 complex in a sample, comprising contacting the anti-GARP antibody or its antigen-binding fragment or conjugate of the present invention with the sample, and detecting whether the antibody or its antigen-binding fragment or conjugate forms a complex with the GARP and / or GARP / Latent TGF-β1 complex. Attached Figure Description

[0062] Figure 1A and Figure 1B The results show the antibody titers detected by FACS in mice #1-4 after immunization.

[0063] Figure 2 The results of the chimeric antibody purity test are displayed.

[0064] Figure 3 The activity of the chimeric antibody in binding to 293T-hGARP-LTGFβ1 was demonstrated by FACS detection.

[0065] Figure 4 The activity of the chimeric antibody in binding to 293T-hGARP was demonstrated by FACS detection.

[0066] Figure 5A and Figure 5B The activity of the chimeric antibody in binding to human Latent TGF-β1 and human TGF-β1 was demonstrated by ELISA.

[0067] Figure 6 The activity of the chimeric antibody in binding to 293T-cGARP-LTGFβ1 was demonstrated by FACS detection.

[0068] Figure 7 The activity of the chimeric antibody in binding to mouse GARP / Latent TGF-β1 was demonstrated by ELISA.

[0069] Figure 8 The activity of the chimeric antibody in binding to Hs 578T cells was demonstrated by FACS detection.

[0070] Figure 9 The activity of the chimeric antibody in binding to L-428 cells was demonstrated by FACS detection.

[0071] Figure 10 The results of the ADCC effect detection of chimeric antibodies are shown. Detailed Implementation

[0072] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0073] definition Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0074] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0075] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

[0076] In this document, the term "GARP" has the same meaning as the GARP protein. The GARP described herein can be directly purified and used from GARP-expressing cells of humans or non-human mammals (e.g., rats, mice, etc.), or components isolated from the aforementioned cells can be prepared and used as the GARP. Alternatively, GARP can also be synthesized in vitro or obtained by genetically manipulating host cells to produce GARP using methods known in the art.

[0077] The term "antibody" is used in its broadest sense herein to refer to a protein that contains an antigen-binding site and is capable of specifically recognizing and binding to an antigen. An antibody can be a complete antibody or any antigen-binding fragment thereof, or a single chain thereof; therefore, the term "antibody" includes any protein or polypeptide that contains at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen.

[0078] "Amino acid" refers to α-aminocarboxylic acid, which can be encoded by nucleic acids directly or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". As used in this application, the term "amino acid" refers to naturally occurring α-aminocarboxylic acids, including alanine (three-letter code: Ala, or one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0079] The term "antibody" is used in the broadest sense herein to refer to a protein containing an antigen-binding site that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof; therefore, the term "antibody" includes any protein or polypeptide containing at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. The anti-GARP antibody described in this application can be a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (such as a bispecific antibody), and preferably a monoclonal antibody.

[0080] An "antibody fragment" comprises a portion of a full-length antibody, typically containing its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; dimers; linear antibodies; single-chain antibodies; and multispecific antibodies formed from antibody fragments.

[0081] In this article, the term "cytotoxicity" is used to refer to pathological changes induced in cells in any given manner. It refers not only to direct trauma but also to all types of structural or functional damage to cells, such as DNA cleavage, base dimer formation, chromosome cleavage, impairment of cell mitosis, and reduction of the activity of various types of enzymes.

[0082] In this paper, the term "cytotoxic activity" refers to the activity that causes the aforementioned cytotoxicity.

[0083] In this article, the term “antibody-dependent cytotoxicity” means “antibody-dependent cytotoxic (ADCC) activity”, and this activity refers to the effect or activity of damaging target cells, such as tumor cells, via antibody-mediated NK cells.

[0084] The term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies, and is generally used interchangeably with the term "antibody" in this invention. IgG immunoglobulins are heterotetrameric glycoproteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, the VH-CH1 of the heavy chain typically pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules linked by immunoglobulin hinge regions and two dimerized Fc regions. Immunoglobulin heavy chains can be classified into one of five categories based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0085] The "complementarity-determining region" (CDR) or "hypervariant region" is a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0086] Several schemes are known in the art for determining the CDR sequence in a given VH or VL amino acid sequence: the Kabat complementarity-determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the location of the structural loop (Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:877-883), and the IMGT numbering scheme (The Martin (Enhanced Chothia) Numbering Scheme, Lefranc, 2003).

[0087] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different numbering scheme rules or combinations).

[0088] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0089] The terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region in an antibody molecule that actually binds to the antigen. Antigen binding sites include, but are not limited to, Fv, Fab fragments, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv), VHH, etc.

[0090] The term "variable region" or "variable domain" of an antibody refers to the domain of the antibody's heavy or light chain involved in the binding of the antibody to the antigen. The variable region of an antibody can be further divided into hypervariable regions (i.e., complementarity-determining regions (CDRs)) and more conserved regions interspersed between the hypervariable regions (i.e., frame regions (FRs)). In the case of IgG immunoglobulins, the heavy or light chain variable regions, from the N-terminus to the C-terminus, consist of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively. In the case of heavy chain antibodies (also referred to herein as nanobodies), such as those derived from camelid heavy chains, the antigen-binding site consists of a single VH domain (i.e., the "VHH" domain). The VHH of natural heavy chain antibodies has a similar structure to the heavy chain variable region of natural IgG antibodies, containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs).

[0091] "Heavy chain constant region domain" or "heavy chain constant region" refers to a constant region domain derived from, obtained from, or derived from the immunoglobulin heavy chain, including heavy chain constant regions CH1, CH2, and CH3 sequentially and covalently linked from the N-terminus to the C-terminus, and optionally heavy chain constant region CH4. In most cases, heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but where appropriate, they can also be connected by a flexible linker.

[0092] The term "EC" 50 The "half-maximum concentration" (WMC), also known as the "half-maximum effective concentration," refers to the concentration of a drug, antibody, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time.

[0093] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0094] "Affinity" or "binding affinity" refers to the intrinsic binding capacity that reflects the interaction between members of a binding pair. Binding affinity can be measured by common methods known in the art.

[0095] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. These variants may contain conserved modifications.

[0096] Monoclonal antibodies (mAbs) are antibodies prepared from the same immune cells, which are all clones of a single parent cell. Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the part of the antigen that the antibody recognizes). In contrast, polyclonal antibodies bind to multiple epitopes and are typically secreted by several different plasma cells. Monoclonal antibodies can also be modified into bispecific monoclonal antibodies by adding a therapeutic target to two epitopes. Monoclonal antibodies can be prepared using hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0097] The terms "patient" and "subject" are used interchangeably to refer to mammals, including humans and non-human mammals. In some embodiments, the patient is a human. In some embodiments, the patient is a non-human mammal, such as a wild, domesticated, or farm animal. In other embodiments, the patient is a dog, cat, mouse, rat, rabbit, guinea pig, or primate such as a cynomolgus monkey.

[0098] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of this invention are used to delay disease development or to slow disease progression.

[0099] The term "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0100] The term "effective amount" refers to such an amount or dose of the antibody or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health condition; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration method; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0101] "Administration" and "dosage" are used interchangeably and can be accomplished by injection, infusion, parenteral administration, intravenous administration, mucosal administration, sublingual administration, intramuscular administration, intradermal administration, intranasal administration, intraperitoneal administration, intra-arterial administration, subcutaneous absorption, or by any method in combination with other known techniques. "Administration" can also refer to the prescription of a composition with instructions for administration using one or more of the aforementioned techniques. In some embodiments, administration is systemic.

[0102] As used herein, the phrase "need" means that a patient has been identified as requiring a specific method or treatment. In some implementations, this identification can be made by any diagnostic means. In any of the methods and treatments described herein, the patient may require these methods and treatments.

[0103] The term "pharmaceutical composition" refers to a composition that exists in a form that allows for the biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition. Such pharmaceutical compositions include, but are not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0104] Biomaterials (nucleic acid molecules, carriers, and host cells) This invention provides nucleic acid molecules encoding any of the above-described antibody molecules or their antigen-binding fragments. The polynucleotide sequences encoding the antibody molecules or their antigen-binding fragments can be generated using methods well-known in the art, either through de novo solid-phase DNA synthesis or through genetic engineering methods. Furthermore, the polynucleotides and nucleic acids of this invention may include segments encoding secretion signal peptides and be operatively linked to segments encoding the antibody molecules or their antigen-binding fragments, thereby guiding the secretory expression of the antibody molecules or their antigen-binding fragments.

[0105] This invention also provides vectors comprising the nucleic acid molecules of this invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector and a prokaryotic expression vector. "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, including viscera, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0106] This invention also provides prokaryotic and eukaryotic host cells comprising the nucleic acid molecule or the vector. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. Suitable host cells for replicating and supporting the expression of the antibody molecules of this invention or their antigen-binding fragments are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antibody molecules.

[0107] Conjugates, compositions and pharmaceutical formulations This invention provides conjugates comprising the antibody molecule of this invention or its antigen-binding fragment or biological material. The conjugate includes a coupling portion, which is another molecule. The coupling portion includes, but is not limited to, cytotoxins, radioisotopes, fluorescent substances, luminescent substances, colored substances, oligonucleotides, and enzymes. Preferably, the oligonucleotide includes: antisense oligonucleotides (ASO), small interfering RNA (siRNA), aptamers, microRNAs (miRNA), small activating RNAs (saRNA), and CpG oligonucleotides, etc.

[0108] The present invention also provides compositions comprising the antibody molecule of the present invention or its antigen-binding fragment, biological material or conjugate. Preferably, the composition is a pharmaceutical composition.

[0109] The compositions of the present invention also include pharmaceutical excipients, such as pharmaceutically acceptable carriers, sustained-release agents, pharmaceutical excipients, etc., known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises the anti-GARP antibody of the present invention or its antigen-binding fragment, and a combination of one or more other therapeutic agents.

[0110] As used in this article, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0111] For information on the use and applications of pharmaceutical excipients, see also “Handbook of Pharmaceutical Excipients”, 8th edition, RCRowe, PJSeskey and SC Owen, Pharmaceutical Press, London, Chicago.

[0112] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as powders or suspensions, liquid solutions (e.g., injectable and infusionable solutions), liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0113] The compositions of the present invention can be administered by known methods, such as orally, intravenously, intraperitoneally, intracerebrally, intramuscularly, intraocularly, intra-arterially, or intralesionally; via a continuous release system or via an implantable device. In some embodiments, the compositions can be administered by bolus injection, by continuous infusion, or via an implantable device.

[0114] Subjects may be mammals, such as primates, or humans (e.g., individuals with or at risk of having the diseases described herein). In one embodiment, the subject has or is at risk of having the diseases described herein (e.g., cancer or an autoimmune disease). In some embodiments, the subject has received or has received other treatments.

[0115] A pharmaceutical preparation comprising the antibody described herein can be prepared by mixing the anti-GARP antibody of the present invention, or its antigen-binding fragment, having the desired purity, with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0116] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents. The active ingredients are suitably combined in amounts effective for the intended use.

[0117] The compositions or formulations of the present invention can be used to prepare sustained-release formulations. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0118] Preparation of the antibody of the present invention In one embodiment, the present invention provides a method for preparing an anti-GARP antibody, wherein the method includes culturing a host cell containing a nucleic acid encoding an anti-GARP antibody or an expression vector containing said nucleic acid under conditions suitable for expressing a nucleic acid encoding said anti-GARP antibody, and optionally isolating said anti-GARP antibody. In one embodiment, the method further includes recovering the anti-GARP antibody from said host cell (or host cell culture medium).

[0119] To recombinantly generate the anti-GARP antibody of the present invention, the nucleic acid encoding the anti-GARP antibody of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the anti-GARP antibody of the present invention.

[0120] The anti-GARP antibody of the present invention, prepared as described herein, can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the anti-GARP antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0121] Treatment methods and uses This invention provides a method for preventing and / or treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a biomaterial of the present invention, a conjugate of the present invention, or a therapeutically effective amount of a pharmaceutical composition of the present invention in the preparation of any of the following products: (I) Products for the prevention and / or treatment of diseases suffered by the subjects; (II) Products that stimulate or enhance the immune response; (III) Products tested by GARP.

[0122] In some embodiments, the disease includes cancer; the cancer includes solid tumors and hematologic malignancies; preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, soft tissue sarcoma, etc.; the hematologic malignancies include lymphoma, etc. In one embodiment, the disease includes cancer. In some embodiments, the cancer includes solid tumors and hematologic malignancies. Preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, soft tissue sarcoma, etc. Preferably, the hematologic malignancies include lymphoma, etc.

[0123] In one embodiment, the present invention provides Treg cells or tumor cells that highly express GARP / Latent TGF-β1 complex or GARP in the tumor microenvironment, as well as soluble GARP protein, comprising administering to the subject an effective amount of the antibody of the present invention or its antigen-binding fragment, or an effective amount of the pharmaceutical composition of the present invention.

[0124] The antibody molecules or antigen-binding fragments thereof of the present invention (and pharmaceutical compositions comprising them) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0125] For the prevention or treatment of disease, the appropriate dose of the antibody molecule or its antigen-binding fragment of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether it is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history, and the judgment of the attending physician.

[0126] Example The following embodiments further illustrate the present invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0127] Unless otherwise expressly stated, the present invention will be practiced using conventional chemical, biochemical, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology methods in the art. Unless otherwise specified, the experimental methods described herein are conventional methods using default parameters and procedures; the experimental materials used are commercially available products. Where specific techniques or conditions are not specified in the examples, they shall be performed according to the techniques or conditions described in the literature in the art, or according to the corresponding product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0128] Example 1 In this embodiment, animals were immunized, and the antibody levels in the animals were detected after immunization.

[0129] Female Balb / c mice aged 6-8 weeks were selected, with four mice per group. Recombinant human GARP / Latent TGF-β1 protein (Bepsys, catalog number GA1-H52W9) or synthetic DNA using human GARP sequence (SEQ IN NO: 30) and human Latent TGF-β1 sequence (SEQ IN NO: 31) as references were used as immunogens. The recombinant protein was mixed with TiterMax® Gold Adjuvant (Sigma-Aldrich, catalog number T2684) in a specific ratio for immunization. The initial immunization dose was 20 μg / mouse, followed by 10 μg / mouse for subsequent immunizations. The immunization cycle was 14 days, for a total of four immunizations. For DNA immunization, the initial dose was 200 μg / mouse, followed by 100 μg / mouse for subsequent immunizations, with an immunization cycle of 7 days. A total of 12 immunizations were administered. For mice with sufficient antibody titers, a booster immunization was given 10 days after the final immunization, with 20 μg of recombinant protein and 200 μg of DNA per mouse.

[0130] FACS method for detecting antibody titer A lentiviral vector containing the human GARP gene and the human Latent TGF-β1 gene was transduced into 293F cells to obtain cells overexpressing the human GARP / Latent TGF-β1 protein complex, named 293F-hGARP / TGF-β1 (constructed by WuXi Biologics; the amino acid sequence of human GARP is shown in SEQ IN NO: 30, and the amino acid sequence of human Latent TGF-β1 is shown in SEQ IN NO: 31). A lentiviral vector containing the monkey GARP gene and the monkey Latent TGF-β1 gene was transduced into 293F cells to obtain cells overexpressing the monkey GARP / Latent TGF-β1 protein complex, named 293F-cGARP / TGF-β1 (constructed by WuXi Biologics; the monkey GARP amino acid sequence accession number is A0A2K5X2X9-1, and the monkey Latent TGF-β1 amino acid sequence accession number is A0A2K5TJB2). 293F-hGARP / TGF-β1 cells and 293F-cGARP / TGF-β1 cells (3E4 cells / well) were added to 384-well microplates, respectively. Then, 30 μL of mouse serum sample diluted with 1% BSA (starting at 1 / 100, 3-fold dilution) was added to each well, and the plates were incubated at 4°C for 1 h. After washing twice, 30 μL of Goat anti-Mouse IgG Fc Alexa647 (500-fold dilution) was added to each well, and the plates were incubated at 4°C in the dark for 30 min. The cells were washed twice, and then resuspended in 30 μL of 1% BSA / 1×PBS solution in each well. The median fluorescence intensity of the cells was measured by flow cytometry (iQue3, Sartorius), and the data were analyzed. Results are shown below. Figure 1A and Figure 1B .pass Figure 1A and Figure 1B It can be seen that the antibody levels in the serum of all mice reached a high level.

[0131] Example 2 This embodiment demonstrates cell fusion and hybridoma screening.

[0132] 2.1 Cell Fusion Spleens and lymph nodes of mice meeting the titer requirements were harvested, ground, and lymphocytes were released. After filtration through a 40 μm sterile cell sieve, lymphocytes were collected. Plasma cells were enriched using the EasySep Mouse CD138 Positive Selection Kit (STEMCELL, catalog number 18957). These plasma cells were then electrofused with mouse myeloma SP2 / 0 cells at a 1:2 ratio. Cells were seeded at 1E4 cells per well and cultured at 37°C in a 5% CO2 incubator for 7–10 days.

[0133] 2.2 Hybridoma Screening On day 10 post-fusion, cell culture supernatant was collected for preliminary FACS screening of 293F-hGARP / TGF-β1. FACS-positive clones were then subjected to FACS binding assays for 293F-cGARP / TGF-β1, ELISA binding assays for recombinant human GARP / Latent TGF-β1 protein, ELISA binding assays for recombinant human GARP protein (Sinochem, catalog number 15508-H08H), and ELISA binding assays for recombinant human Latent TGF-β1 protein (Bestsys, catalog number TG1-H524x). Target clones were then subjected to subcloning. After 10 days, subclone supernatant was collected for FACS binding assays of 293F-hGARP / TGF-β1 and 293F-cGARP / TGF-β1, as well as ELISA binding assays for recombinant human GARP and recombinant human Latent TGF-β1 proteins.

[0134] (1) FACS detection of the binding activity of hybridoma antibody to 293F-hGARP / TGF-β1 293F-hGARP / TGF-β1 cells (3E4 cells / well) were added to 384-well microplates, followed by 30 μL of hybridoma culture supernatant per well. The plates were incubated at 4°C for 1 h, washed twice, and then 30 μL of Goat anti-mouse IgG Fc Alexa647 (500-fold dilution) was added to each well. The plates were incubated at 4°C in the dark for 30 min. Cells were washed twice, and then resuspended in 30 μL of 1% BSA / 1×PBS solution per well. The median fluorescence intensity of the cells was measured by flow cytometry (iQue3, Sartorius), and the data were analyzed. Immunohistochemical serum was used as a positive control, and HAT medium was used as a negative control. A total of 360 clones were positive.

[0135] (2) FACS detection of the binding activity of hybridoma antibody to 293F-cGARP / TGF-β1 293F-cGARP / TGF-β1 cells (3E4 cells / well) were added to 384-well microplates, followed by 30 μL of hybridoma culture supernatant per well. The plates were incubated at 4°C for 1 h. After washing twice, 30 μL of Goat anti-mouse IgG Fc Alexa647 (500-fold dilution) was added to each well, and the plates were incubated at 4°C in the dark for 30 min. The cells were washed twice, and then resuspended in 30 μL of 1% BSA / 1×PBS solution in each well. The median fluorescence intensity of the cells was measured by flow cytometry (iQue3, Sartorius), and the data were analyzed. Control antibody 1 (Livmoniplimab analog, the heavy chain of which is shown in SEQ ID NO: 16 of CN110945027B and the light chain of which is shown in SEQ ID NO: 17 of CN110945027B) and control antibody 2 (DS-1055 analog, the heavy chain of which is shown in SEQ ID NO: 33 of CN108026521B and the light chain of which is shown in SEQ ID NO: 37 of CN108026521B) were added as positive controls at a concentration of 10 nM. The secondary antibody was Goat anti-human IgG Fc Alexa647 (500-fold dilution). HAT medium was used as a negative control.

[0136] (3) ELISA detection of the binding activity between hybridoma antibody and recombinant protein MonoRab rabbit anti-His antibody (GenScript, catalog number A01857) was diluted to the working concentration (1 μg / mL) with coating buffer, and 30 µL was added to each well of a 384-well microplate. The plate was sealed with a sealing membrane and incubated overnight at 4°C. On the second day, after washing three times with PBST, 80 μL of 2% BSA blocking buffer was added, the plate was sealed with a sealing membrane, and incubated at 25°C for 1 h. After washing, recombinant human GARP / Latent TGF-β1 (Bepsys, catalog number GA1-H52W9), recombinant human GARP (Sinosure, catalog number 15508-H08H) were diluted to 0.125 μg / mL, and recombinant human Latent TGF-β1 (Bepsys, catalog number TG1-H524x) was diluted to 0.25 μg / mL, and 30 µL was added to each well. The plate was incubated at 25°C for 1 h. After washing, add 30 μL of hybridoma culture supernatant to each well, seal with a membrane, and incubate at 25°C for 1.5 h. After washing, add 30 μL of HRP-labeled goat anti-mouse IgG Fc antibody (1:5000 dilution) to each well, seal with a membrane, and incubate at 25°C for 1 h. After washing, add 30 μL of TMB substrate solution to each well, seal with a membrane, incubate at room temperature for 4 min, and add 30 μL of stop solution to each well to terminate the reaction. Read the OD at 450 nm using a microplate reader. Control antibodies 1-2 are positive controls, and HAT medium is used as a negative control.

[0137] Through the screening methods described above, a total of 54 clones were obtained that can simultaneously bind human GARP / Latent TGF-β1, monkey GARP / Latent TGF-β1, and human GARP without binding human Latent TGF-β1.

[0138] Example 3 This embodiment describes the sequencing of the variable region gene of hybridoma antibodies and the expression and purification of chimeric antibodies.

[0139] RNA lysis buffer (74136, QIAGEN) was added to hybridoma cells (1E6 cells), and RNA was extracted using an RNA extraction kit (74136, QIAGEN). After extraction, RNA concentration was measured using Nanodrop, and RNA was reverse transcribed into cDNA using 5'-RACE. After reverse transcription, PCR amplification was performed on the variable regions of the antibody light and heavy chains using specific primers. After the reaction, the PCR stock solution was recovered by agarose gel electrophoresis (CW2302M, Kangwei Century), and the recovered product concentration was measured using Nanodrop. After the assay, the fragment was ligated to the vector using a seamless cloning enzyme (C116-02, Novozymes) and transformed into DH5α competent cells. The cells were then plated onto LB plates containing ampicillin and sent to a sequencing company for single-clone sequencing the following day. The sequence data were analyzed using SnapGene software.

[0140] The sequenced antibody gene was cloned into a pcDNA3.4 vector containing the human IgG1 constant region using a seamless cloning enzyme. A mammalian cell expression plasmid was constructed, and HEK293F cells were transfected using liposomes. After 72 hours, the supernatant was collected, and the expressed antibody was purified using Protein A. The purified antibody was then analyzed by SDS-PAGE and SEC-HPLC, showing a purity greater than 95%. (See attached results). Figure 2 .

[0141] Example 4 In this embodiment, the in vitro activity of the chimeric antibody was tested.

[0142] 4.1 FACS detection of the binding activity of chimeric antibody to 293T-hGARP-LTGFβ1 A lentiviral vector containing the human GARP gene and the human Latent TGF-β1 gene was transduced into 293T cells to obtain cells overexpressing the human GARP / Latent TGF-β1 protein complex, named 293T-hGARP-LTGFβ1 (constructed by Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., with the human GARP amino acid sequence shown in SEQ IN NO: 30 and the human Latent TGF-β1 amino acid sequence shown in SEQ IN NO: 31). 293T-hGARP-LTGFβ1 cells (1E5 cells / well) were added to 96-well polypropylene microplates (Thermo, 267350) and centrifuged at 1500 rpm for 5 min at 4°C, removing the supernatant. 50 μL of chimeric antibody at different concentrations (starting from 20 μg / mL, serially diluted 3.16-fold in 12 steps) was added to each well, mixed thoroughly, and incubated at 4°C in the dark for 1 hour. After the reaction, cells were washed with 400 μL of 2% FBS / 1×PBS solution. Goat F(ab')2 anti-human IgG-Fc(PE) (Southern Biotech, catalog number 2010-09) was diluted 1000-fold with 2% FBS / 1×PBS solution, added to the cells, and mixed thoroughly by pipetting. The cells were incubated at 4°C in the dark for 40 min. Cells were washed twice, then resuspended in 200 μL of 2% FBS / 1×PBS solution. The median fluorescence intensity of the cells was measured by flow cytometry (Attune NxT AcousticFocusing Cytometer, Invitrogen), and the data were analyzed. The experimental data were calculated using a four-parameter nonlinear fitting method in GraphPad Prism7 software to calculate EC50. 50 Values. Control antibodies 1 and 2 served as positive controls, and the IgG1 isotype control antibody served as a negative control. Results are shown below. Figure 3 See Table 1. The results show that the two chimeric antibodies, B1354-hIgG1 and B1362-hIgG1, exhibited superior 293T-GARP-LTGFβ1 binding activity compared to control antibodies 1 and 2 (B1362-hIgG1 showed better EC50 binding activity). 50 B1354-hIgG1, on the other hand, exhibited a stronger maximum binding signal. The chimeric antibodies B1354-hIgG1 and B1362-hIgG1 respectively contain the following CDR combinations and the following framework regions:

[0143]

[0144] 4.2 FACS detection of the activity of chimeric antibody binding to 293T-GARP The lentiviral vector of the human GARP gene was transduced into 293T cells to obtain cells overexpressing human GARP protein, named 293T-hGARP (constructed by Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., the amino acid sequence of human GARP is shown in SEQ INNO: 30). 293T-hGARP cells (1E5 cells / well) were added to 96-well polypropylene microplates (Thermo, 267350) and centrifuged at 1500 rpm for 5 min at 4°C, removing the supernatant. 50 μL of chimeric antibodies of different concentrations (starting from 20 μg / mL, serially diluted 3.16-fold, 12 gradients) were added to the wells, mixed thoroughly by pipetting, and incubated at 4°C in the dark for 1 hour. After the reaction, the cells were washed with 400 μL of 2% FBS / 1×PBS solution. Goat F(ab')2 anti-human IgG-Fc(PE) (Southern Biotech, catalog number 2010-09) was diluted 1000-fold with 2% FBS / 1×PBS solution, then added to cells and mixed by pipetting. The cells were incubated at 4°C in the dark for 40 min. Cells were washed twice and resuspended in 200 μL of 2% FBS / 1×PBS solution. The median fluorescence intensity of the cells was measured by flow cytometry (Attune NxT Acoustic Focusing Cytometer, Invitrogen), and the data were analyzed. EC50 was calculated using four-parameter nonlinear fitting in GraphPad Prism7 software. 50 Values. Control antibodies 1 and 2 served as positive controls, and the IgG1 isotype control antibody served as a negative control. Results are shown below. Figure 4 See Table 1. The results show that the two chimeric antibodies, B1354-hIgG1 and B1362-hIgG1, exhibited superior 293T-hGARP binding activity compared to control antibody 2 (both candidate antibodies showed EC50% activity comparable to control antibody 2). 50 (and can generate a stronger maximum binding signal), control antibody 1 does not bind 293T-hGARP, a result consistent with the description in the patent for control antibody 1.

[0145] Table 1

[0146] N / A indicates that it cannot be obtained.

[0147] 4.3 ELISA detection of the binding activity of chimeric antibody to human Latent TGF-β1 Recombinant human Latent TGF-β1 protein (Bepsys, catalog number TG1-H524x) or recombinant human TGF-β1 (Bepsys, catalog number TG1-H4212) was diluted to the working concentration (2 μg / mL) with coating buffer, and 100 µL was added to each well of the ELISA plate. The plate was sealed with a sealing membrane and incubated overnight at 4°C. On the second day, after washing three times with PBST, 300 μL of 2% BSA blocking buffer was added, and the plate was sealed with a sealing membrane and incubated at 37°C for 1.5 h. After washing, the chimeric antibody was serially diluted (starting from 10 μg / mL, 5-fold serial dilutions, 8 dilutions), and 100 μL was added to each well. The plate was sealed with a sealing membrane and incubated at 37°C for 1 h. After washing, add 100 μL of HRP-labeled goat anti-human IgG-Fc antibody (BETHYL, catalog number: A80-304P, 10000-fold dilution) to each well, seal the plate with a sealing membrane, and incubate at 37°C for 1 h. After washing, add 100 μL of Pierce 1-Step Ultra TMB-ELISA substrate solution to each well, seal the plate with a sealing membrane, incubate at room temperature for 3 min, and add 100 μL of stop solution to each well to stop the reaction. Read the OD at 450 nm using a microplate reader. Results are shown in the figure. Figure 5A and Figure 5B The results show that neither the B1354-hIgG1 nor the B1362-hIgG1 chimeric antibodies, nor the control antibodies 1 and 2, bind to human Latent TGF-β1 protein or human TGF-β1 protein. TGF-β1 has a wide range of complex physiological functions in the human body, and direct binding to it may lead to unpredictable side effects. This characteristic of the candidate antibodies suggests that they have good safety profiles.

[0148] 4.4 FACS detection of the activity of chimeric antibody binding to 293T-cGARP-LTGFβ1 cells Lentiviral vectors containing the monkey GARP gene and the monkey Latent TGF-β1 gene were transduced into 293T cells to obtain cells overexpressing monkey GARP / Latent TGF-β1 protein, named 293T-cGARP-LTGFβ1 (constructed by Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., monkey GARP amino acid sequence accession number A0A2K5X2X9-1, monkey Latent TGF-β1 amino acid sequence accession number A0A2K5TJB2). 293T-cGARP-LTGFβ1 cells (1E5 cells / well) were added to 96-well polypropylene microplates (Thermo, 267350) and centrifuged at 1500 rpm for 5 min at 4°C, removing the supernatant. 50 μL of chimeric antibody at different concentrations (starting from 20 μg / mL, serially diluted 3.16-fold in 12 steps) was added to each well, mixed thoroughly, and incubated at 4°C in the dark for 1 hour. After the reaction, cells were washed with 400 μL of 2% FBS / 1×PBS solution. Goat F(ab')2 anti-human IgG-Fc(PE) (Southern Biotech, catalog number 2010-09) was diluted 1000-fold with 2% FBS / 1×PBS solution, added to the cells, and mixed thoroughly by pipetting. The cells were incubated at 4°C in the dark for 40 min. Cells were washed twice, then resuspended in 200 μL of 2% FBS / 1×PBS solution. The median fluorescence intensity of the cells was measured using a flow cytometer (Attune NxT Acoustic Focusing Cytometer, Invitrogen), and the data were analyzed. The experimental data were calculated using a four-parameter nonlinear fitting method in GraphPad Prism7 software to calculate EC50. 50 Values. Control antibody 2 served as a positive control, and IgG1 isotype control antibody (20 μg / mL) served as a negative control. Results are shown below. Figure 6 See Table 2. The results show that the binding levels of both antibodies to monkey GARP / Latent TGF-β1 at lower concentrations are basically the same as those of control antibody 2, but the binding levels at higher concentrations are significantly higher than those of control antibody 2, indicating their potential for in vivo experiments in monkeys.

[0149] Table 2

[0150] N / A indicates that it cannot be obtained.

[0151] 4.5 ELISA detection of the binding activity of chimeric antibody to mouse GARP / Latent TGF-β1 MonoRab rabbit anti-His antibody (GenScript, catalog number A01857) was diluted to the working concentration (1 μg / mL) with coating buffer, and 100 µL was added to each well of a 96-well microplate. The plates were sealed with a membrane and incubated overnight at 4°C. On the second day, after washing three times with PBST, 300 μL of 2% BSA blocking buffer was added, and the plates were sealed with a membrane and incubated at 37°C for 1.5 h. After washing, recombinant mouse GARP / LatentTGF-β1 (Bepsys, catalog number GA1-M52W2) was diluted to 2 μg / mL, and 100 µL was added to each well. The plates were incubated at 37°C for 1 h. After washing, different concentrations of chimeric antibody (starting from 20 μg / mL, 5-fold serial dilutions, 8 gradients) were added, 100 μL to each well, and the plates were sealed with a membrane and incubated at 37°C for 1 h. After washing, add 100 μL of HRP-labeled goat anti-human IgG-Fc antibody (BETHYL, catalog number: A80-304P, 10000-fold dilution) to each well, seal the plate with a membrane, and incubate at 37°C for 1 h. After washing, add 100 μL of Pierce 1-Step Ultra TMB-ELISA substrate solution to each well, seal the plate with a membrane, incubate at room temperature for 3 min, and add 100 μL of stop solution to each well to stop the reaction. Read the OD at 450 nm using a microplate reader. Results are shown below. Figure 7 See Table 3. The results show that both the B1354-hIgG1 and B1362-hIgG1 chimeric antibodies bind to mouse GARP / Latent TGF-β1. Specifically, B1362-hIgG1 binds more strongly to mouse GARP / Latent TGF-β1, while B1354-hIgG1 binds less strongly, indicating that B1362-hIgG1 has the potential for in vivo mouse assays. Control antibodies 1 and 2 do not bind to mouse GARP / Latent TGF-β1 protein.

[0152] Table 3

[0153] N / A indicates that it cannot be obtained.

[0154] 4.6 FACS detection of the activity of chimeric antibody binding to Hs 578T cells Hs 578T cells (purchased from Peking Union Medical College Cell Bank) are human breast cancer cells expressing endogenous GARP and the GARP / LatentTGF-β1 complex. Hs 578T cells (1E5 cells / well) were added to 96-well polypropylene microplates (Thermo, 267350) and centrifuged at 1500 rpm for 5 min at 4°C, removing the supernatant. 50 μL of chimeric antibodies at different concentrations (starting from 20 μg / mL, serially diluted 3.16-fold in 12 steps) were added to the wells, mixed thoroughly, and incubated at 4°C in the dark for 1 hour. After the reaction, the cells were washed with 400 μL of 2% FBS / 1×PBS solution. Goat F(ab')2 anti-human IgG-Fc(PE) (Southern Biotech, catalog number 2010-09) was diluted 1000-fold with 2% FBS / 1×PBS solution, then added to cells and mixed by pipetting. The cells were incubated at 4°C in the dark for 40 min. Cells were washed twice and resuspended in 200 μL of 2% FBS / 1×PBS solution. The median fluorescence intensity of the cells was measured by flow cytometry (Attune NxT Acoustic Focusing Cytometer, Invitrogen), and the data were analyzed. EC50 was calculated using four-parameter nonlinear fitting in GraphPad Prism7 software. 50 Values. Control antibodies 1-2 served as positive controls, and IgG1 isotype control antibody served as a negative control. Results are shown in […]. Figure 8 See Table 4. The results show that on Hs 578T cells, the two chimeric antibodies, B1354-hIgG1 and B1362-hIgG1, bound stronger than control antibodies 1 and 2 at any concentration. The maximum binding signal (MFI MAX) was increased by approximately 48% and 60% compared to control antibody 1, and by approximately 24% and 34% compared to control antibody 2, respectively.

[0155] Table 4

[0156] N / A indicates that it cannot be obtained.

[0157] 4.7 FACS detection of the binding of chimeric antibody to tumor cells L-428 L-428 cells (purchased from DSMZ) are human Hodgkin's lymphoma cells expressing endogenous GARP and the human GARP / LatentTGF-β1 complex. According to the HUMAN PROTEIN ATLAS database, the GARP mRNA level in L-428 cells is lower than that in Hs 578T. L-428 cells (1E5 cells / well) were added to 96-well polypropylene microplates (Thermo, 267350) and centrifuged at 1500 rpm for 5 min at 4°C, removing the supernatant. 50 μL of chimeric antibodies of different concentrations (starting from 20 μg / mL, serially diluted 3.16-fold in 12 steps) were added to the wells, mixed thoroughly, and incubated at 4°C in the dark for 1 hour. After the reaction, the cells were washed with 400 μL of 2% FBS / 1×PBS solution. Goat F(ab')2 anti-human IgG-Fc(PE) (Southern Biotech, catalog number 2010-09) was diluted 1000-fold with 2% FBS / 1×PBS solution, then added to cells and mixed by pipetting. The cells were incubated at 4°C in the dark for 40 min. Cells were washed twice and resuspended in 200 μL of 2% FBS / 1×PBS solution. The median fluorescence intensity of the cells was measured by flow cytometry (Attune NxT Acoustic Focusing Cytometer, Invitrogen), and the data were analyzed. EC50 was calculated using four-parameter nonlinear fitting in GraphPad Prism7 software. 50 Values. Antibody 2 was used as a positive control, and IgG1 isotype control antibody was used as a negative control. Results are shown below. Figure 9 See Table 5. The results show that the binding activity of the two chimeric antibodies, B1354-hIgG1 and B1362-hIgG1, to L-428 cells is superior to that of the control antibody 2.

[0158] Table 5

[0159] N / A indicates that it cannot be obtained.

[0160] The experiments in sections 4.6 and 4.7 demonstrate that, in two different cell lines (breast cancer and lymphoma), they maintained EC levels comparable to or better than the optimal control antibody. 50 In both cell lines, they can mediate stronger or comparable maximum binding signals (MFI MAX).

[0161] 4.8 Detection of the ADCC effect of chimeric antibodies FcγRⅢA (CD16a) can mediate ADCC (antibody-mediated cell-mediated death) activity of NK cells against tumor cells. In this study, Jurkat-NFAT-Luc2-CD16a-V158 cells were used as alternative effector cells and co-incubated with target cells to activate the NFAT-luc2 luciferase reporter system. The ADCC activity of the antibody was detected by measuring the luciferase content. L-428 cells were used as target cells in this experiment. Cells in the logarithmic growth phase were harvested and counted using a platelet counter. Cell viability was assessed using the trypan blue rejection assay to ensure it was above 90%. 45 µL of effector cells and target cells were added to each well of a 96-well plate at a 1:1 ratio for co-incubation. The cell count was 2E4 cells / well. A 10-fold final concentration antibody solution was prepared, and 10 µL of antibody solution was added to each well of the 96-well plate containing both effector and target cells. The initial working antibody concentration was 20 μg / mL, with nine concentrations and 3.16-fold serial dilutions. The 96-well plates were incubated at 37°C and 5% CO2 for 6 hours. Bright-Glo reagent was melted and the plates were equilibrated to room temperature for 30 minutes. An equal volume of Bright-Glo solution was added to each well, and the plates were shaken on a track-mounted shaker for 15 minutes to induce cell lysis. The bioluminescence readings were then recorded. Data were analyzed using GraphPad Prism 7 software to fit dose-response curves and calculate EC50. 50 Control antibody 2 served as a positive control, IgG1 isotype control antibody served as an isotype control, target cells and effector cells without the test antibody served as negative controls, PBS wells without cells and antibodies served as blank controls, effector cells alone without antibodies and target cells served as effector cell controls, and target cells alone without antibodies and effector cells served as target cell controls. Results are shown below. Figure 10 See Table 6. The results show that both chimeric antibodies, B1354-hIgG1 and B1362-hIgG1, exhibited superior ADCC effects on L-428 cells compared to control antibody 2. Specifically, at low concentrations, the activation signals of the ADCC receptor by B1354-hIgG1 and B1362-hIgG1 were similar to those of control antibody 2, while at higher concentrations, the activation signals were significantly higher. Specifically, the Luminescence Max of B1354-hIgG1 was 5.4 times that of control antibody 2 (21208), and the Luminescence Max of B1362-hIgG1 was 3.2 times that of control antibody 2 (21208).

[0162] Table 6

[0163] In this context, "N / A" means that the information cannot be obtained, and " / " means that the information is not applicable.

[0164] 4.9 Determination of Tm value of chimeric antibody The chimeric antibody was diluted to 0.5 mg / mL with 20 mM citrate buffer (containing 150 mM sodium chloride, pH 5.5). The reaction mixture was prepared using the Protein Thermal Shift Dye Kit™ (Thermo, catalog number 4461146), and 20 μL was added to each well of the reaction plate. Detection was performed using a 7500 Fast Real-time PCR System (Thermo). Step 1: 25℃ for 2 min; Step 2: 99℃ for 2 min. Continuous temperature increases were performed at a rate of 1%. Results are shown in Table 7. The results show that the Tm1 values ​​of the B1354-hIgG1 and B1362-hIgG1 chimeric antibodies are similar to those of the control antibody 2, and both are greater than 65℃, indicating that the chimeric antibodies have good thermal stability comparable to the reference antibody 2. This provides important positive data for subsequent process development, formulation studies, and storage.

[0165] Table 7

[0166] In this context, "N / A" means that the information cannot be obtained.

[0167] Sequence information:

Claims

1. An anti-GARP antibody or an antigen-binding fragment thereof, comprising one or more amino acid sequences from (a) to (f): (a) VH CDR1, comprising an amino acid sequence as shown in SEQ ID NO: 1 or 7, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 7, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 1 or 7. (b) VH CDR2, which contains the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 2; (c) VH CDR3, comprising an amino acid sequence as shown in SEQ ID NO: 3 or 8, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or 8, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 3 or 8. (d) VL CDR1, which contains an amino acid sequence as shown in SEQ ID NO: 4 or 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 9, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 4 or 9. (e) VL CDR2, which contains the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO:

5. (f) VL CDR3, which contains the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 3, VL CDR1 as shown in SEQ ID NO: 4, VL CDR2 as shown in SEQ ID NO: 5, and VL CDR3 as shown in SEQ ID NO: 6; or The antibody or its antigen-binding fragment comprises VH CDR1 as shown in SEQ ID NO: 7, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 8, VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 5, and VL CDR3 as shown in SEQ ID NO:

6.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment further includes a frame region comprising heavy chain FR1, heavy chain FR2, heavy chain FR3, heavy chain FR4, light chain FR1, light chain FR2, light chain FR3, and light chain FR4. Preferably: The heavy chain FR1 comprises an amino acid sequence as shown in SEQ ID NO: 10 or 18, or an amino acid sequence having at least 80% sequence identity with an amino acid sequence as shown in SEQ ID NO: 10 or 18, or an amino acid sequence having one or more conserved amino acid substitutions compared to an amino acid sequence as shown in SEQ ID NO: 10 or 18. The heavy chain FR2 comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

11. The heavy chain FR3 comprises an amino acid sequence as shown in SEQ ID NO: 12 or 19, or an amino acid sequence having at least 80% sequence identity with an amino acid sequence as shown in SEQ ID NO: 12 or 19, or an amino acid sequence having one or more conserved amino acid substitutions compared to an amino acid sequence as shown in SEQ ID NO: 12 or 19. The heavy chain FR4 comprises an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

13. The light chain FR1 comprises an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

14. The light chain FR2 comprises an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

15. The light chain FR3 comprises an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

16. The light chain FR4 comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

17.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 20 or 22, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 or 22, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 20 or 22; and / or The variable region of the light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 21 or 23, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or 23, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 21 or 23.

5. An anti-GARP antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 21; or The heavy chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO: 22, and the light chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:

23.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The antibody is any form of monoclonal antibody, single-chain antibody, bispecific antibody, multispecific antibody, murine antibody, fully or partially humanized antibody, or chimeric antibody; preferably, the antibody is a human-mouse chimeric antibody; and / or The antigen-binding fragment is an antigen-binding fragment of an antibody; preferably, the antigen-binding fragment is a Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv, or (dsFv)2 fragment; preferably, the antigen-binding fragment is an antigen-binding fragment of a human-mouse chimeric antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region or a functional variant thereof.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein, The antibody is a monoclonal antibody, and the heavy chain constant region of the monoclonal antibody is an IgG1, IgG2, IgG3 or IgG4 subtype or a functional variant thereof; preferably IgG1 or a defucosylated IgG1 variant.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein, The heavy chain constant region of the antibody or its antigen-binding fragment comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 24; the light chain constant region of the antibody or its antigen-binding fragment comprises or is composed of an amino acid sequence as shown in SEQ ID NO:

25.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, comprising a heavy chain and / or a light chain, wherein: The heavy chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 32; The heavy chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 34; The light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 33; or The light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO:

35.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein, The application forms of the antibody or its antigen-binding fragment include antibody-drug conjugates and CAR molecules.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein, The antibody or its antigen-binding fragment has at least one of the following properties (1)-(5): (1) It has inhibitory activity against the immunosuppressive function of regulatory T cells; (2) It has antibody-dependent cytotoxic activity; (3) It has in vivo and / or in vitro antitumor activity; (4) Specifically binds to GARP, the GARP / Latent TGF-β1 complex, and cells expressing the above proteins; preferably, the GARP includes human GARP, monkey GARP, and mouse GARP; preferably, the GARP / Latent TGF-β1 complex includes human GARP / Latent TGF-β1 complex, monkey GARP / Latent TGF-β1 complex, and mouse GARP / Latent TGF-β1 complex; preferably, the cells include tumor cells, engineered cells, immune cells, and platelets; (5) It does not bind to either Latent TGF-β1 or TGF-β1 alone.

13. A biomaterial comprising: (i) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-12; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

14. The biomaterial according to claim 13, wherein, The nucleic acid molecule includes DNA or RNA, preferably DNA, and the DNA includes cDNA, genomic DNA or artificially synthesized DNA; the DNA is single-stranded or double-stranded; the DNA is a coding strand or a non-coding strand; The vector includes an expression vector; preferably, the expression vector includes bacterial plasmids, phage plasmids, yeast plasmids, plant cell viruses, mammalian cell viruses, and eukaryotic cell expression plasmids; more preferably, it is a eukaryotic cell expression plasmid; and / or The host cell is prokaryotic or eukaryotic; preferably, the host cell includes plant cells, microbial cells, and mammalian cells; more preferably, it is a mammalian cell or an engineered variant thereof.

15. A conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-12 and a conjugation moiety, wherein, The coupling portion is another molecule; preferably, the coupling portion is a cytotoxin, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an oligonucleotide, or an enzyme.

16. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-12, a biological material as described in claim 13 or 14 or a conjugate as described in claim 15, and one or more pharmaceutically acceptable carriers, sustained-release agents or excipients; optionally, the pharmaceutical composition further comprises a second therapeutic agent and / or optionally a pharmaceutical excipient, the second therapeutic agent being selected from cytokines, antibodies, chemotherapeutic agents and small molecule drugs.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-12, the biomaterial according to claim 13 or 14, the conjugate according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of any of the following products: (I) Products for the prevention and / or treatment of diseases suffered by the subjects; (II) Products that stimulate or enhance the immune response; (III) Products tested by GARP.

18. The use according to claim 17, wherein, The diseases include cancer; the cancers include solid tumors and hematologic cancers; preferably, the solid tumors include lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, breast cancer, nervous system tumors, bladder cancer, urethral cancer, prostate cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, and soft tissue sarcoma; the hematologic cancers include lymphoma.

19. A kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-12, a conjugate as described in claim 15, or a pharmaceutical composition as described in claim 16.

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