CN114907478A - LAG-3 binding antibodies and uses thereof - Google Patents

LAG-3 binding antibodies and uses thereof Download PDF

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CN114907478A
CN114907478A CN202210118342.8A CN202210118342A CN114907478A CN 114907478 A CN114907478 A CN 114907478A CN 202210118342 A CN202210118342 A CN 202210118342A CN 114907478 A CN114907478 A CN 114907478A
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variable region
chain variable
amino acid
antibody
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王荣娟
焦莎莎
张畅
王双
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Maiwei Shanghai Biotechnology Co ltd
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Abstract

The present invention relates to antibodies and antibody fragments that specifically bind LAG-3 and pharmaceutical compositions containing the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding said antibodies and host cells comprising said nucleic acids, as well as to methods of producing said antibodies. The invention also relates to therapeutic and diagnostic uses of these antibodies that bind LAG-3.

Description

LAG-3 binding antibodies and uses thereof
Technical Field
The invention relates to the field of antibody drugs. In particular, the invention relates to novel antibodies and antibody fragments that specifically bind LAG-3 and compositions comprising the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding said antibodies and host cells comprising said nucleic acids, as well as to methods of producing said antibodies. The invention also relates to therapeutic and diagnostic uses of these antibodies that bind LAG-3.
Background
Immune cells are regulated by a variety of inhibitory immune checkpoint molecules that act as "safety brakes" at various stages of the immune response. These regulations are important in preventing destruction of body tissues due to inappropriate immune responses to invading pathogens. However, cancer cells utilize such inhibitory immune checkpoint molecules to evade immune responses in which immune cells specifically destroy tumors.
In the prior art, cancer therapy to restore tumor-specific immune cell function by blocking immune checkpoint molecules using monoclonal antibodies (mabs) against cytotoxic T-lymphocyte-associated protein 4(CTLA-4, CD152), programmed death 1(PD-1, CD279) and its ligand PD-L1(CD274, B7-H1) significantly improved patient survival in a proportion of tumor patients. However, the frequency of patient response to monoclonal antibodies used against the immune checkpoint molecule is not ideal due to Tumor resistance, lack of Tumor Infiltrating Lymphocytes (TILs) and presence of inhibitory Myeloid Cells (Awad, R.M et al, Turn Back the TIME: Targeting Tumor Infiltrating Myeloid Cells to reverse Cancer progression. front. immunological.2018, 9,1977). Furthermore, the occurrence of immune-related adverse events (immune-related adverse events) is also responsible for discontinuing the use of mabs that block the immune checkpoint in patients. In order to increase the response rate of patients and prevent immune-related adverse events, researchers have also investigated other inhibitory immune checkpoint molecules that play a role in cancer cell escape from anti-cancer immunity, where lymphocyte activation gene-3protein (LAG-3) is becoming an important target for cancer treatment and evaluation of prognosis.
LAG-3, also known as CD223, is a member of the immunoglobulin superfamily, and consists of 3 portions of extracellular, transmembrane and cytoplasmic regions. The LAG-3 gene is located on chromosome 12 (12P13), and has similar location and structure to CD4 molecule on chromosome, but the amino acid sequences of the two have only 20% homology.
LAG-3 is expressed predominantly on the surface of T cells (particularly activated T cells), natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 has been shown to be an inhibitory receptor, has functions of maintaining homeostasis and participating in immune regulation, and is closely related to the development of tumors. The major ligand of LAG-3 is the class II histocompatibility complex (MHC-II), and in addition, Galectin 3(Galectin-3), antral endothelial lectin (LSECtin) and fibrinogen-like protein 1(FGL-1) are also ligands thereof. LSECtin, galectin 3 and FGL-1 can interact with LAG-3 in a tumor microenvironment to inhibit the antitumor activity of CD8+ T cells.
In cancer, LAG-3 expressing Regulatory T cells (tregs) have enhanced suppressive antitumor activity, while LAG-3 expressing cytotoxic CD8+ T cells have reduced proliferation rate and effector cytokine production (Scurr m. et al, high potency clinical cancer-encapsulating LAP + Foxp3 - T cells exhibit more potent immunosuppressive activity than Foxp3 + regulatory T cells, Mucosal Immunol.2014,7(2): 428-. LAG-3 is expressed in elevated levels in a number of tumor-infiltrating Treg cells, which are also expressed in peripheral blood and tumor tissues of melanoma and colorectal cancer patients, and the interaction between MHC-II expressed on Antigen Presenting Cells (APC) and LAG-3 on Treg cells can produce immunosuppressionA cytokine, thereby inhibiting CD8+ TILs activity.
Since LAG-3 expression levels and LAG-3+ cell infiltration in tumors are associated with tumor progression, poor prognosis (Maruhashi T, Sugiura D, Okazaki I et al, Journal for ImmunoTherapy of Cancer 2020; 8: e001014), there remains a need in the art to develop new anti-LAG-3 antibodies, particularly humanized antibodies, that modulate LAG-3 activity to activate the immune system, for example for Cancer ImmunoTherapy and the treatment of other diseases such as infectious diseases.
Summary of The Invention
The present inventors have made studies to develop a novel group of anti-LAG-3 antibodies (e.g., humanized antibody molecules) that bind LAG-3 with high affinity and activate the immune system by blocking LAG-3 activity, thereby satisfying the above-mentioned needs. The antibodies or antigen-binding fragments of the invention that specifically bind LAG-3 have one or more of the following properties:
(a) binding LAG-3 with high affinity, e.g., human LAG-3 and cynomolgus monkey LAG-3, e.g., K binding between the anti-LAG-3 antibody or antigen binding fragment thereof and LAG-3 D Is about 10 -7 M to about 10 -12 M, preferably, about 10 -8 M to about 10 -12 M, as measured by ForteBio kinetic binding assay;
(b) specifically block the binding of LAG-3 to FGL-1;
(c) specifically block binding of LAG-3 to cell surface MHC class II molecules (e.g., human HLA);
(d) maintaining the T cells in an activated state;
(e) has antitumor effect in vivo.
Thus, in a first aspect, the invention provides an isolated antibody or antigen-binding fragment that specifically binds LAG-3, comprising
(a) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 1 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 2; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(b) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 4; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(c) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 5 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 6; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(d) 3 CDRs in the amino acid sequence of heavy chain variable region shown in SEQ ID NO. 7 and 3 CDRs in the amino acid sequence of light chain variable region shown in SEQ ID NO. 8; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(e) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 9 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 10; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(f) 11 and 12, 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO; or a variant having a single CDR or multiple CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions.
In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein
(a) The heavy chain variable region comprises HCDR1 as represented by SYGIS (SEQ ID NO:31), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRSDNTYYNGKFKG (SEQ ID NO:32) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYYGSNYYAMDY (SEQ ID NO:33), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown in RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of said LCDR1 with NO more than 2 amino acid changes, or NO more than 1 amino acid change; LCDR2 as shown by KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(b) said heavy chain variable region comprises HCDR1 as indicated by NYAMS (SEQ ID NO:37), or a variant of said HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; TITYGTTYTFYSDNVKG (SEQ ID NO:38) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GEYGSSFAY (SEQ ID NO:39), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASQNVRTAVA (SEQ ID NO:40), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LASNRHT (SEQ ID NO:41), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and LQHWNYPLT (SEQ ID NO:42) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(c) the heavy chain variable region comprises HCDR1 as represented by DYAVS (SEQ ID NO:43), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; VVWGDGSTNYHSALIS (SEQ ID NO:44) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GGGGMDY (SEQ ID NO:45), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RASSSVSYMH (SEQ ID NO:46), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in ATSNLAS (SEQ ID NO:47), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and QHYNTNPPTWT (SEQ ID NO:48) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(d) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; RIDPEDVETKYDPKFQG (SEQ ID NO:50) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes, or does not exceed 1 amino acid change; and SFYSNYVNYFDQ (SEQ ID NO:51), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in GASNRYT (SEQ ID NO:53), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and GQSYSYPYT (SEQ ID NO:54), or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
for example, the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIX 1 PEDVETKYDPKFQG (SEQ ID NO:64), preferably, HCDR2 wherein X is 1 Is D or N; and SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3;
the light chain variable region comprises X 2 ASENVGTYVS (SEQ ID NO:68), preferably wherein X is 2 Is K or R; x 3 ASX 4 RYT (SEQ ID NO:69), preferably LCDR 2; wherein X 3 Is G or A; x 4 Is N or T; and GQSYSYPYT (SEQ ID NO:54) under the conditions of LCDR 3;
for example, the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) is shown as HCDR 2; and SFYSNYVNYFDQ (SEQ ID NO:51) of HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at RASENVGTYVS (SEQ ID NO:65), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54); the heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54); or
The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
(e) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; WIDPENGETEYASKFQG (SEQ ID NO:55) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and FDY (SEQ ID NO:56) or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KSSQSLLDSDGKTYLN (SEQ ID NO:57), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LVSKLDF (SEQ ID NO:58), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and WQGTHFPQT (SEQ ID NO:59), or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; or
(f) The heavy chain variable region comprises HCDR1 as indicated by NYGIS (SEQ ID NO:60), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO:61) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYFGSNYYAMDY (SEQ ID NO:62), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change.
In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein
(a) The heavy chain variable region comprises the sequence of SEQ ID NO 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(b) the heavy chain variable region comprises the sequence of SEQ ID NO 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(c) the heavy chain variable region comprises the sequence of SEQ ID NO 5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(d) the heavy chain variable region comprises the sequence of SEQ ID NO 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
the heavy chain variable region comprises the sequence of SEQ ID NO 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or
The heavy chain variable region comprises the sequence of SEQ ID NO 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 17 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(e) the heavy chain variable region comprises the sequence of SEQ ID NO 9 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(f) the heavy chain variable region comprises the sequence of SEQ ID NO 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, an isolated antibody or antigen-binding fragment of the invention is a humanized antibody.
In some embodiments, the isolated antibody or antigen binding fragment of the invention is an IgG1, IgG2, IgG3, or IgG4 antibody; preferably, is an IgG1 or IgG4 antibody; more preferably, it is a humanized IgG1 or a humanized IgG4 antibody. In some embodiments, the antigen binding fragment of the invention is a Fab, Fab ', F (ab') 2 Fv, single-chain Fab or diabody (diabody).
In a second aspect, the invention provides a nucleic acid encoding the antibody or antigen-binding fragment of the first aspect described above, a vector (preferably, an expression vector) comprising the nucleic acid, a host cell comprising the nucleic acid or the vector. In some embodiments, the host cell is prokaryotic or eukaryotic, e.g., selected from an escherichia coli cell, a yeast cell, a mammalian cell, or other cells suitable for the production of antibodies or antigen-binding fragments, multispecific antibodies. In some embodiments, the host cell is a 293 cell or a CHO cell.
In a third aspect, the invention provides a method of making an antibody or antigen-binding fragment of the invention, the method comprising culturing a host cell of the invention under conditions suitable for expression of a polypeptide encoding the antibody or antigen-binding fragment of the invention, optionally recovering the antibody or antigen-binding fragment of the invention from the host cell or from the culture medium.
In a fourth aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention and a pharmaceutically acceptable carrier.
In some embodiments, the invention provides a pharmaceutical composition comprising an antibody of the invention and an additional therapeutic agent, and optionally a pharmaceutically acceptable carrier; preferably, the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, additional antibodies (e.g., anti-PD-1 antibody or anti-PD-L1 antibody), cytotoxic agents.
In some embodiments, the invention provides a combination product comprising an antibody of the invention, and one or more other therapeutic agents, e.g., a chemotherapeutic agent, a cytotoxic agent, an additional antibody, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody.
In a fifth aspect, the present invention provides a method of preventing or treating a tumor or an infectious disease in a subject or individual, comprising administering to the subject an effective amount of the antibody, the pharmaceutical composition, or the combination product of the present invention.
In some embodiments, the tumor prevented or treated by the antibody, pharmaceutical composition, or combination product of the invention is a cancer, e.g., a cancer with an elevated expression level of LAG-3; or the antibody, pharmaceutical composition, or combination product of the invention prevents or treats an infectious disease, such as a bacterial infection, a viral infection, a fungal infection, or a protozoal infection, preferably the infectious disease is a chronic infection, in which elevated levels of LAG-3 expression result in low immunity in the subject or individual.
In a sixth aspect, the present invention provides a kit for detecting LAG-3 in a sample, said kit comprising an antibody of the invention for performing the steps of:
(a) contacting the sample with an antibody of the invention; and
(b) detecting formation of a complex between the LAG-3 antibody and LAG-3; optionally, the LAG-3 antibody is detectably labeled,
thereby, it is determined whether an elevated expression level of LAG-3 is present in a sample from the subject or individual.
Brief Description of Drawings
The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
FIG. 1: shows the results of detecting the specific binding activity of the anti-human LAG-3 chimeric antibody to 293/rhLAG-3 cell surface LAG-3 by FACS method. The negative control in the figure is an isotype control antibody, purified human IgG4 isotype monoclonal antibody that does not bind to LAG-3 molecule, and MFI represents mean fluorescence intensity.
FIG. 2 is a schematic diagram: shows the results of detecting that anti-human LAG-3 chimeric antibody blocks binding of 293/rhHLA cells to hLAG-3 by FACS method.
FIG. 3: results of detecting binding of the anti-human LAG-3 humanized antibody to recombinant human LAG-3 by ELISA are shown.
FIG. 4: results showing the detection of binding of anti-human LAG-3 humanized antibody to recombinant cynomolgus LAG-3 by ELISA.
FIG. 5 is a schematic view of: results of detection of binding of anti-human LAG-3 humanized antibody to recombinant mouse LAG-3 by ELISA are shown.
FIG. 6: shows a graph of the results of the affinity of the anti-human LAG-3 humanized antibody of the present invention for human LAG-3 extracellular region recombinant protein as measured by ForteBio kinetic binding assay.
FIG. 7: a graph showing the results of BMS-986016 affinity for human LAG-3 extracellular domain recombinant protein as measured by the ForteBio kinetic binding assay. BMS-986016 is a monoclonal antibody targeting LAG-3 developed by BMS corporation.
FIG. 8: shows the results of detecting the specific binding of the anti-human LAG-3 humanized antibody of the present invention to 293/rhLAG-3 cell surface LAG-3 by FACS method.
FIG. 9: shows the results of detecting that the anti-human LAG-3 humanized antibody of the present invention blocks the binding of human FGL-1 to LAG-3 by ELISA.
FIG. 10: shows the results of detecting the binding of anti-human LAG-3 humanized antibody to hLAG-3 by FACS detection in 293/rhHLA cells.
FIG. 11: shows the inhibitory effect of the anti-human LAG-3 humanized antibody of the present invention on tumor growth in a B6-huLAG3 humanized mouse subcutaneous graft tumor model.
FIG. 12: the effect of the anti-human LAG-3 humanized antibody of the present invention on the body weight of mice in the B6-huLAG3 humanized mouse subcutaneous graft tumor model is shown.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, 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 be apparent from the description and drawings, and from the claims.
I. Definition of
For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.
As used herein, the term "and/or" means any one of the options or two or more of the options.
When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments. Preferably, the antibody of the invention is a single domain antibody or a heavy chain antibody.
"antibody fragment" or "antigen-binding fragment" are used interchangeably herein to refer to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, Fv, single chain Fab, or diabodies (diabodies).
An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity. In one embodiment, the reference antibody is a monoclonal antibody targeting LAG-3 developed by BMS: BMS-986016.
"complementarity determining regions" or "CDR regions" or "CDRs" are regions of antibody variable domains that are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or regions that contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given heavy chain variable region amino acid sequence can be determined using any one or combination of a number of well known antibody CDR assignment systems, including, for example: chothia (Chothia et Al (1989) Nature 342:877- & 883, Al-Lazikani et Al, "Standard constraints for the structural characterization of Immunological constructs", Journal of Molecular Biology,273,927-948(1997)), based on antibody sequence variations Kabat (Kabat et Al, Sequences of Proteins of Immunological constructs, 4 th edition, U.S. Department of Health and Human Services, National instruments of Health (1987)), AbM (fundamental of balance), action compatibility, International Cluster of Molecular Biology (IMG), and the use of the structural characterization of biological constructs/homology.
Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.
CDRs can also be determined based on Kabat numbered positions that are the same as reference CDR sequences (e.g., any of the CDRs exemplified herein). In the present invention, when referring to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system.
Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the region of minimum overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM may be replaced by conserved amino acid residues.
The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is altered, replaced or exchanged such that the antigen-binding site is linked to a constant region of a different or altered class and/or species or an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that confers novel properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a murine antibody may be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with human constant regions, the chimeric antibody can retain its specificity in recognizing antigens while having reduced antigenicity in humans as compared to the original murine antibody.
"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in the humanized antibody correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.
"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed.
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three Complementarity Determining Regions (CDRs). (see, e.g., Kindt et al Kuby Immunology, 6 th ed., page 2007, w.h.freeman and co.91). A single VH or VL domain may be sufficient to confer antigen binding specificity.
As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques or other conventional binding assays known in the art.
The term "immune checkpoint molecule" means a class of inhibitory signal molecules present in The immune system, which avoid tissue damage by modulating The persistence and intensity of The immune response in peripheral tissues and are involved in maintaining tolerance to autoantigens (pardol DM., The block of immune responses in Cancer immunological therapy. nat Rev Cancer,2012,12(4): 252-. It has been found that one of the reasons that tumor cells are able to escape the immune system in vivo and proliferate uncontrollably is to utilize the inhibitory signaling pathway of immune checkpoint molecules, thereby inhibiting T lymphocyte activity, rendering T lymphocytes ineffective in exerting a killing effect on tumors (Yao S, Zhu Y and Chen l., Advances in targeting cell surface signaling molecules for tumor modulation. nat Rev Drug Discov,2013,12(2): 130-146). Immune checkpoint molecules include, but are not limited to, LAG-3, programmed death 1(PD-1), PD-L2, CTLA-4, TIM-3.
The term "co-stimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response (e.g., without limitation, proliferation) of the cell. Costimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to the antigen receptor or its ligand. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD 28. In some embodiments, a "co-stimulatory molecule" is CD28, OX40, GITR, 4-1BB (i.e., CD137), and/or CD 27.
An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
The term "inhibit" or "block" refers to a decrease in certain parameters (e.g., activity) of a given molecule (e.g., an immune checkpoint molecule). For example, this term includes substances that cause a given molecule (e.g., LAG-3) to be inhibited by at least 5%, 10%, 20%, 30%, 40%, or more of its activity. Thus, the inhibitory effect need not be 100%.
The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.
The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.
The terms "cancer" and "cancerous" refer to a physiological condition in mammals in which cell growth is unregulated.
The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive as they are referred to herein.
"tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infection, bacterial infection, fungal infection, or protozoan infection such as parasitic infection.
The term "label" as used herein refers to a compound or composition that is conjugated or fused, directly or indirectly, to an agent (such as an antibody) and facilitates detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.
An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the LAG-3 antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b848: 79-87(2007).
An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An "isolated nucleic acid encoding a LAG-3 antibody" refers to one or more nucleic acid molecules encoding a chain of a LAG-3 antibody or fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.
The calculation of sequence identity between sequences is performed as follows.
To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
Sequence comparisons between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48: 444-. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com), using the NWSgapdna. CMP matrix and GAP weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can also be determined using the PAM120 weighted residue table, gap length penalty 12, gap penalty 4), using the e.meyers and w.miller algorithms that have been incorporated into the ALIGN program (version 2.0) ((1989) cabaos, 4: 11-17).
Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.
As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes hybridization and wash conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous methods are described in this reference and either method may be used. In some embodiments, specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions are those that wash twice in 6 Xsodium chloride/sodium citrate (SSC) at about 45 ℃ followed by at least 50 ℃ (for low stringency conditions, the temperature of the wash can be increased to 55 ℃) in 0.2 XSSC, 0.1% SDS; 2) moderate stringency hybridization conditions are one or more washes in 6 XSSC at about 45 ℃ followed by 0.2 XSSC, 0.1% SDS at 60 ℃; 3) high stringency hybridization conditions are one or more washes in 6 XSSC at about 45 ℃ followed by 0.2 XSSC, 0.1% SDS at 65 ℃; and preferably 4) very high stringency hybridization conditions are one or more washes in 0.5M sodium phosphate, 7% SDS at 65 ℃ followed by 0.2 XSSC, 0.1% SDS at 65 ℃. The extremely high stringency condition (4) is the preferred condition and one that should be used unless otherwise specified.
The term "pharmaceutical composition" refers to a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.
The term "pharmaceutically acceptable carrier" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers and the like, which are administered with the active substance.
As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the invention are used to delay the progression of a disease or to slow the progression of a disease.
As used herein, "prevention" includes inhibition of the onset or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly prior to the onset of symptoms of cancer in a subject at risk for cancer.
The term "effective amount" refers to an amount or dose of an antibody or composition of the invention that produces the desired effect in a patient in need of treatment or prevention following administration to the patient in a single or multiple dose. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors: species such as mammals; body weight, age, and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.
"therapeutically effective amount" means an amount effective, at dosages and for periods of time as required, to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or antibody fragment or composition thereof may vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or deleterious effects of the antibody or antibody fragment or composition thereof are less than therapeutically beneficial. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system predictive of efficacy in human tumors.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time as required, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.
The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response. Immune modulators include immune checkpoint molecule inhibitors and co-stimulatory molecule activators.
The term "small molecule drug" refers to low molecular weight organic compounds capable of modulating biological processes.
As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Examples of cytotoxic agents are disclosed in WO2015/153513, WO2016/028672 or WO 2015/138920.
The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of a microorganism, such as a virus, bacterium, fungus, or protozoan, e.g., a parasite, but does not fate the host at the concentrations administered and at the dosing intervals. As used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a particular aspect, the anti-infective active agent is non-toxic to the host at the administration concentration and interval.
The term "combination product" refers to a kit of parts for combined administration or a fixed combination or a non-fixed combination in one dosage unit form, wherein two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially when these time intervals allow the combined therapeutic agents to exhibit a synergistic, e.g. synergistic, effect. The term "fixed combination" means that the antibody of the invention and a combination partner (e.g. other therapeutic agent, such as an anti-PD-1 antibody or an anti-PD-L1 antibody) are administered to a patient simultaneously in the form of a single entity or dose. The term "non-fixed combination" means that the LAG-3 antibody and a combination partner of the invention (e.g. other therapeutic agent, such as an anti-PD-1 antibody or an anti-PD-L1 antibody) are administered to a patient as separate entities simultaneously, concurrently or sequentially, without specific time constraints, wherein such administration provides therapeutically effective levels of both therapeutic agents in the patient. The latter also applies to cocktail therapies, such as the administration of three or more therapeutic agents. In a preferred embodiment, the pharmaceutical combination is a non-fixed combination.
The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents to treat a cancer or infection as described in the present disclosure. Such administration includes co-administering the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration or separate administration or sequential administration in multiple or separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In some embodiments, administering further comprises using each type of therapeutic agent at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.
The term "vector" as used herein when referring to a nucleic acid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell. Host cells are any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E.coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
"subject/patient sample" refers to a collection of cells, tissues or body fluids obtained from a patient or subject. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.
Antibodies of the invention that specifically bind LAG-3
The antibodies of the invention that specifically bind LAG-3 comprise a heavy chain variable region and a light chain variable region, wherein:
(a) the heavy chain variable region comprises HCDR1 as represented by SYGIS (SEQ ID NO:31), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRSDNTYYNGKFKG (SEQ ID NO:32) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYYGSNYYAMDY (SEQ ID NO:33), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown in RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of said LCDR1 with NO more than 2 amino acid changes, or NO more than 1 amino acid change; LCDR2 as shown in KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(b) said heavy chain variable region comprises HCDR1 as indicated by NYAMS (SEQ ID NO:37), or a variant of said HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; TITYGTTYTFYSDNVKG (SEQ ID NO:38) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GEYGSSFAY (SEQ ID NO:39), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASQNVRTAVA (SEQ ID NO:40), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LASNRHT (SEQ ID NO:41), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and LQHWNYPLT (SEQ ID NO:42) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(c) the heavy chain variable region comprises HCDR1 as represented by DYAVS (SEQ ID NO:43), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; VVWGDGSTNYHSALIS (SEQ ID NO:44) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GGGGMDY (SEQ ID NO:45), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RASSSVSYMH (SEQ ID NO:46), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in ATSNLAS (SEQ ID NO:47), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and QHYNTNPPTWT (SEQ ID NO:48) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(d) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; RIDPEDVETKYDPKFQG (SEQ ID NO:50) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and SFYSNYVNYFDQ (SEQ ID NO:51), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in GASNRYT (SEQ ID NO:53), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and GQSYSYPYT (SEQ ID NO:54) of LCDR3, or a variant of said LCDR3 that does not exceed 2 amino acid changes, or does not exceed 1 amino acid change;
(e) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; WIDPENGETEYASKFQG (SEQ ID NO:55) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and FDY (SEQ ID NO:56) or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KSSQSLLDSDGKTYLN (SEQ ID NO:57), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LVSKLDF (SEQ ID NO:58), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and WQGTHFPQT (SEQ ID NO:59) of LCDR3, or a variant of said LCDR3 having NO more than 2 amino acid changes or NO more than 1 amino acid change; or
(f) The heavy chain variable region comprises HCDR1 as indicated by NYGIS (SEQ ID NO:60), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO:61) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYFGSNYYAMDY (SEQ ID NO:62), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown by KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
wherein the amino acid change is an addition, deletion or substitution of an amino acid, e.g., the amino acid change is a conservative amino acid substitution. The CDRs are CDRs according to the Kabat numbering scheme.
In some embodiments, an antibody of the invention binds mammalian LAG-3, e.g., human LAG-3, monkey LAG-3. In some embodiments, the LAG-3 antibodies of the invention bind to one or more extracellular domains of LAG-3.
In some embodiments, the antibodies of the invention have one or more of the following properties:
(1) binding LAG-3 with high affinity, e.g., human LAG-3 and cynomolgus monkey LAG-3, e.g., K binding between the anti-LAG-3 antibody or antigen binding fragment thereof and LAG-3 D Is about 10 -7 M to about 10 -12 M, preferably, about 10 -8 M to about 10 -12 M, as measured by ForteBio kinetic binding assay;
(2) specifically block the binding of LAG-3 to FGL-1;
(3) specifically block binding of LAG-3 to cell surface HLA;
(4) maintaining the T cells in an activated state;
(5) has antitumor effect in vivo.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in SYGIS (SEQ ID NO: 31); EIYPRSDNTYYNGKFKG (SEQ ID NO:32) and HCDR 2; and RAFYYYGSNYYAMDY (SEQ ID NO:33) shown as HCDR 3; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO: 34); LCDR2 as shown by KVSNRFS (SEQ ID NO: 35); and LCDR3 as shown in FQGSHVPWT (SEQ ID NO: 36).
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in NYAMS (SEQ ID NO: 37); TITYGTTYTFYSDNVKG (SEQ ID NO:38) and HCDR 2; and GEYGSSFAY (SEQ ID NO:39) shown as HCDR 3; the light chain variable region comprises LCDR1 as shown at KASQNVRTAVA (SEQ ID NO: 40); LCDR2 as shown in LASNRHT (SEQ ID NO: 41); and LCDR3 as shown in LQHWNYPLT (SEQ ID NO: 42).
In one embodiment, the LAG-3-specifically binding antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DYAVS (SEQ ID NO: 43); VVWGDGSTNYHSALIS (SEQ ID NO:44) shown in HCDR 2; and HCDR3 shown by GGGGMDY (SEQ ID NO: 45); the light chain variable region comprises LCDR1 as shown at RASSSVSYMH (SEQ ID NO: 46); LCDR2 as shown by ATSNLAS (SEQ ID NO: 47); and LCDR3 as shown in QHYNTNPPTWT (SEQ ID NO: 48).
In some embodiments, an antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable regionA variable region wherein the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIX 1 PEDVETKYDPKFQG (SEQ ID NO:64), preferably, HCDR2 wherein X is 1 Is D or N; and SFYSNYVNYFDQ (SEQ ID NO:51) of HCDR 3;
the light chain variable region comprises X 2 ASENVGTYVS (SEQ ID NO:68), preferably wherein X is 2 Is K or R; x 3 ASX 4 RYT (SEQ ID NO:69), preferably LCDR 2; wherein X 3 Is G or A; x 4 Is N or T; and GQSYSYPYT (SEQ ID NO:54) shown in LCDR 3.
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) is shown as HCDR 2; and SFYSNYVNYFDQ (SEQ ID NO:51) of HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at RASENVGTYVS (SEQ ID NO:65), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in DDYMH (SEQ ID NO: 49); WIDPENGETEYASKFQG (SEQ ID NO:55) and HCDR 2; and HCDR3 as shown by FDY (SEQ ID NO: 56); the light chain variable region comprises LCDR1 as shown at KSSQSLLDSDGKTYLN (SEQ ID NO: 57); LCDR2 as shown in LVSKLDF (SEQ ID NO: 58); and LCDR3 as shown at WQGTHFPQT (SEQ ID NO: 59); or
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as set forth in NYSIS (SEQ ID NO: 60); EIYPRGGNTYYNGKFKG (SEQ ID NO:61) and HCDR 2; and RAFYYFGSNYYAMDY (SEQ ID NO:62) HCDR 3; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO: 34); LCDR2 as shown by KVSNRFS (SEQ ID NO: 35); and LCDR3 as shown in FQGSHVPWT (SEQ ID NO: 36).
In some embodiments, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region each N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C from the N-terminus to the C-terminus, wherein 6 CDRs of each antibody are as defined above and FRs are human antibody-derived FRs. For example, in some embodiments, the heavy chain variable region of a humanized LAG-3 antibody or antigen-binding fragment thereof comprises FR1 having amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:80), EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:81), EVQLVQSGAEVKKPGASVKVSCKASGYTIK (SEQ ID NO:82), EVQLVQSGAEVKKPGASVKVSCKASGVNFT (SEQ ID NO:83) or EVQLVQSGAEVKKPGASVKVSCKASGVNIK (SEQ ID NO:84), FR2 having amino acid sequence WVRQAPGQGLEWMG (SEQ ID NO:85) or WVRQAPGQGLEWIG (SEQ ID NO:86), FR3 having amino acid sequence RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:87), RVTITRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:88) or RVTITADTSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:89), and FR4 having amino acid sequence WGQGTLVTVSS (SEQ ID NO: 90); the light chain variable region contains FR1 having amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:70) or EIVMTQSPATLSLSPGERATLSC (SEQ ID NO:71), FR2 having amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO:72), WFQQKPGKAPKLLIY (SEQ ID NO:73), WYQQKPGQAPRLLIY (SEQ ID NO:74) or WFQQKPGQAPRLLIY (SEQ ID NO:75), FR3 having amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:76), GIPARFSGSGSGTDFTLTISSLQPEDFAVYYC (SEQ ID NO:77) or GIPDRFSGSGSGTDFTLTISSLQPEDFAVYYC (SEQ ID NO:78), and FR4 having amino acid sequence FGQGTKLEIK (SEQ ID NO: 79).
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:80, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:80, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence SEQ ID NO 71, FR2 comprising the amino acid sequence SEQ ID NO 74, FR3 comprising the amino acid sequence SEQ ID NO 77 and FR4 comprising the amino acid sequence SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:86, FR3 comprising amino acid sequence SEQ ID NO:87, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:70, FR2 comprising the amino acid sequence of SEQ ID NO:73, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:86, FR3 comprising amino acid sequence SEQ ID NO:87, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:85, FR3 comprising amino acid sequence SEQ ID NO:88, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:70, FR2 comprising the amino acid sequence of SEQ ID NO:73, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:85, FR3 comprising amino acid sequence SEQ ID NO:88, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:86, FR3 comprising amino acid sequence SEQ ID NO:87, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:74, FR3 comprising the amino acid sequence of SEQ ID NO:77 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising amino acid sequence SEQ ID NO:81, FR2 comprising amino acid sequence SEQ ID NO:85, FR3 comprising amino acid sequence SEQ ID NO:88, and FR4 comprising amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:74, FR3 comprising the amino acid sequence of SEQ ID NO:77 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:81, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:70, FR2 comprising the amino acid sequence of SEQ ID NO:73, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:81, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:81, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:74, FR3 comprising the amino acid sequence of SEQ ID NO:77 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the present invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and comprises FR1 having the amino acid sequence SEQ ID NO:81, FR2 having the amino acid sequence SEQ ID NO:85, FR3 having the amino acid sequence SEQ ID NO:87, and FR4 having the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:75, FR3 comprising the amino acid sequence of SEQ ID NO:78 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the present invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and comprises FR1 having the amino acid sequence SEQ ID No. 82, FR2 having the amino acid sequence SEQ ID No. 85, FR3 having the amino acid sequence SEQ ID No. 87, and FR4 having the amino acid sequence SEQ ID No. 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, which are FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 73, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:82, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:82, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:74, FR3 comprising the amino acid sequence of SEQ ID NO:77 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:82, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:75, FR3 comprising the amino acid sequence of SEQ ID NO:78 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:83, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:70, FR2 comprising the amino acid sequence of SEQ ID NO:73, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:83, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:83, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:74, FR3 comprising the amino acid sequence of SEQ ID NO:77 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:83, FR2 comprising the amino acid sequence SEQ ID NO:85, FR3 comprising the amino acid sequence SEQ ID NO:87, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, which are FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 71, FR2 comprising the amino acid sequence of SEQ ID NO 75, FR3 comprising the amino acid sequence of SEQ ID NO 78 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the present invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and comprises FR1 having the amino acid sequence SEQ ID No. 84, FR2 having the amino acid sequence SEQ ID No. 86, FR3 having the amino acid sequence SEQ ID No. 89, and FR4 having the amino acid sequence SEQ ID No. 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:70, FR2 comprising the amino acid sequence of SEQ ID NO:73, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:84, FR2 comprising the amino acid sequence SEQ ID NO:86, FR3 comprising the amino acid sequence SEQ ID NO:89, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO 70, FR2 comprising the amino acid sequence of SEQ ID NO 72, FR3 comprising the amino acid sequence of SEQ ID NO 76 and FR4 comprising the amino acid sequence of SEQ ID NO 79.
In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3, and FR4, respectively, and FR1 comprising the amino acid sequence SEQ ID NO:84, FR2 comprising the amino acid sequence SEQ ID NO:86, FR3 comprising the amino acid sequence SEQ ID NO:89, and FR4 comprising the amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, FR1, FR2, FR3 and FR4 respectively, and FR1 comprising the amino acid sequence of SEQ ID NO:71, FR2 comprising the amino acid sequence of SEQ ID NO:75, FR3 comprising the amino acid sequence of SEQ ID NO:76 and FR4 comprising the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the antibodies or antigen-binding fragments of the invention that specifically bind LAG-3 comprise or consist of the heavy chain variable region and the light chain variable region of:
(i) selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16, and 15/17;
(ii) and a sequence selected from SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16, and 15/17, wherein any one of the sequences has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the heavy chain variable region sequence/light chain variable region sequences; or
(iii) And a sequence selected from SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16, and 15/17, preferably, the amino acid change does not occur in the CDR region, compared to or consisting of an amino acid sequence having 1 or more (preferably, no more than 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably, amino acid substitutions, more preferably, amino acid conservative substitutions) in any one of the heavy chain variable region sequence/light chain variable region sequence.
In some embodiments, the antibodies of the invention have an Fc region, e.g., from an IgG, e.g., IgG1, IgG2, IgG3, or IgG 4. In some embodiments, the Fc region is from IgG 4. In some embodiments, the Fc region is from human IgG 4.
In some embodiments of the invention, the amino acid changes described herein comprise substitutions, insertions or deletions of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
In a preferred embodiment, the amino acid changes described herein occur in a region outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes according to the invention occur in regions outside the heavy chain variable region and the light chain variable region. In some embodiments, the substitution is a conservative substitution. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid. Exemplary substitutions are shown in table 1 below:
TABLE 1
Figure BDA0003497456830000231
Figure BDA0003497456830000241
In certain embodiments, the LAG-3 antibodies provided herein are altered to increase or decrease the degree of glycosylation thereof. The glycosylation sites of LAG-3 antibodies are added or deleted by altering the amino acid sequence to create or remove one or more glycosylation sites. When the LAG-3 antibody comprises an Fc region, the carbohydrate attached to the Fc region may be altered. In some applications, modifications that remove unwanted glycosylation sites are advantageous, such as removal of fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be made to modulate Complement Dependent Cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of LAG-3 antibodies provided herein, thereby generating Fc region variants, in order to enhance, for example, the effectiveness of the antibodies of the invention in treating cancer or infectious diseases.
Nucleic acids of the invention and host cells comprising the same
In one aspect, the invention provides a nucleic acid encoding any of the above LAG-3 antibodies or antigen-binding fragments thereof, or any chain thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell), or other cell suitable for production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.
For example, the nucleic acid of the invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs 1-12, 15-28, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 1-12, 15-28.
The invention also encompasses nucleic acids that hybridize under stringent conditions to or encode polypeptide sequences having one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions as compared to the following nucleic acids: a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of those set forth in any one of SEQ ID NOs 1-12, 15-28; or a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of those set forth in any of SEQ ID NOs 1-12, 15-28.
In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs). In one embodiment, the vector is a pcdna3.1 vector.
Once an expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used based on the present specification and methods known in the art.
Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance, etc., to an auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.
In one embodiment, a host cell comprising a polynucleotide of the invention is provided. In some embodiments, host cells comprising the expression vectors of the invention are provided. In some embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the production of antibodies. Suitable host cells include prokaryotic microorganisms such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells and the like. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), Vero cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Bufaro rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, and the like. For a review of mammalian host cell lines suitable for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, NJ), pp.255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or 293 cell.
IV. production and purification of antibodies of the invention
In one embodiment, the present invention provides a method of making a LAG-3 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the LAG-3 antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the LAG-3 antibody, and optionally isolating the LAG-3 antibody. In a certain embodiment, the method further comprises recovering LAG-3 antibody from the host cell (or host cell culture medium).
For recombinant production of the antibodies of the invention, a nucleic acid encoding the LAG-3 antibody of the invention is first isolated and inserted into a vector for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the LAG-3 antibodies of the invention.
Antibodies of the invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on net charge, hydrophobicity, hydrophilicity, and like factors, and will be apparent to those skilled in the art. The purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
V. Activity assay of antibodies of the invention and Activity of antibodies of the invention
The LAG-3 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like. Binding to LAG-3 can be determined using methods known in the art, exemplary methods being disclosed herein. In some embodiments, binding of an antibody of the invention to LAG-3 is determined using SPR or ForteBio kinetic binding assays.
The invention also provides assays for identifying LAG-3 antibodies having biological activity. The biological activity may include, for example, binding to cell surface LAG-3 (e.g., human LAG-3, monkey LAG-3), inhibition of binding of LAG-3/MHC class II molecules or binding of LAG-3/FGL-1, or the like.
Cells for use in any of the above in vitro assays include cell lines that naturally express LAG-3 or are engineered to express LAG-3. The LAG-3-expressing cell line engineered to express LAG-3 is a cell line that does not normally express LAG-3 and that expresses LAG-3 after transfection of DNA encoding LAG-3 into cells.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention specifically binds human LAG-3 protein with its affinity (K) as measured by ForteBio kinetic binding assay D ) About 4.17E-11M, while the control antibody BMS-986016 binds human LAG-3 protein with an affinity (K) D ) Is about 1.61E-10M. Thus, the LAG-3 antibodies of the invention are capable of specifically binding LAG-3 proteins with high affinity.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention can effectively bind human LAG-3 recombinant protein with a half-effective binding concentration (EC50) value of about 0.034nM, as measured by an ELISA assay.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the present invention can efficiently bind cell-surface human LAG-3 protein with a median effective binding concentration (EC50) value of about 1.6nM, comparable to the level of binding of the control antibody BMS-986016(EC 50: 1.5nM) to cell-surface human LAG-3 protein, as measured by FACS method.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention specifically blocks cell surface HLA binding to recombinant LAG-3 protein with a corresponding median effective inhibitory concentration IC50 value of 4.8nM, superior blocking capacity to the control antibody BMS-986016(IC 50: 9.9nM), as measured by FACS.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention is capable of blocking binding of human LAG-3 to FGL-1 with a corresponding half maximal inhibitory concentration IC50 value of 0.038nM, comparable to the blocking activity of the control antibody BMS-986016(IC 50: 0.038nM), as measured by an ELISA assay.
In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention has good in vivo efficacy in mice as determined by experimental animal models. In a B6-huPD1huLAG3 humanized mouse subcutaneous transplanted tumor model, the anti-tumor effect of the anti-LAG-3 humanized antibody hz7F10 is superior to that of a control antibody BMS-986016, and the weight of animals in each experimental group is steadily increased without obvious toxic effect.
Pharmaceutical compositions and pharmaceutical formulations
In some embodiments, the present invention provides a composition comprising any LAG-3 antibody or immunoconjugate thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, a composition (e.g., a pharmaceutical composition) comprises a combination of an antibody or immunoconjugate thereof of the invention, and one or more additional therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, an additional antibody, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody).
In some embodiments, the composition is for preventing or treating a tumor. In some embodiments, the tumor is a cancer. In some embodiments, the compositions are used to prevent or treat an infection, e.g., a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.
The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a LAG-3 antibody or immunoconjugate thereof and/or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding a LAG-3 antibody. These compositions may also comprise suitable pharmaceutically acceptable carriers, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers, as known in the art.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of Excipients and their use, see also "Handbook of Pharmaceutical Excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, Pharmaceutical Press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
Pharmaceutical formulations comprising the LAG-3 antibody described herein may be prepared by mixing an antibody of the invention having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.
The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-cancer or anti-infective active ingredients, such as chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulators, such as anti-PD-1 antibodies, anti-PD-L1 antibodies and the like. The active ingredients are suitably present in combination in an amount effective for the intended use.
Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
Combination product or kit
In some embodiments, the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof of the invention, or an immunoconjugate thereof, and one or more additional therapeutic agents (e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, anti-infective active agent, small molecule drug, or immunomodulatory agent, etc.). In some embodiments, the other antibody is, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody.
In some embodiments, the combination product is for use in the prevention or treatment of a tumor. In some embodiments, the tumor is a cancer or the like. In some embodiments, the combination product is used to prevent or treat an infection, e.g., a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.
In some embodiments, two or more of the ingredients of the combination product may be administered to a subject in combination, sequentially, separately or simultaneously.
In some embodiments, the invention also provides a kit comprising an antibody, pharmaceutical composition, or combination product of the invention, and optionally a package insert directing administration.
In some embodiments, the invention also provides a pharmaceutical product comprising an antibody, pharmaceutical composition combination of the invention, optionally further comprising a package insert directing administration.
Use of the antibodies of the invention
In one aspect, the invention relates to a method of modulating an immune response in an individual. The method comprises administering to the subject an effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical composition or combination product comprising the LAG-3 antibody, thereby modulating an immune response in the subject. In one embodiment, a therapeutically effective amount of a LAG-3 antibody or pharmaceutical composition or combination disclosed herein restores, enhances, stimulates or increases an immune response in a subject.
In some embodiments, the present invention relates to a method of inhibiting the activity of LAG-3, blocking binding of LAG-3 to MHC class II molecules, blocking binding of LAG-3 to FGL-1 molecules in an individual, comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical composition or combination product comprising the same.
In another aspect, the present invention relates to a method of preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein or a pharmaceutical composition or combination product comprising the same. In some embodiments, the tumor is an immune-escaping tumor.
In another aspect, the present invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein or a pharmaceutical composition or combination product comprising the same.
In another aspect, the present invention relates to a method of eliciting antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical composition or combination product comprising the same.
The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disease as described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., a tumor or infectious disease as described herein). In certain embodiments, the subject receives or has received other treatment, such as chemotherapy treatment and/or radiation therapy. Alternatively or in combination, the subject is or is at risk of being immunocompromised due to the infection.
In some embodiments, a tumor, e.g., a cancer, described herein includes, but is not limited to, a solid tumor, a hematologic cancer, a soft tissue tumor, and a metastatic lesion.
Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas of various organ systems (including adenocarcinomas and squamous cell carcinomas), such as those that affect the liver, lung, breast, lymph, gastrointestinal tract (e.g., colon), genito-urinary tract (e.g., kidney, bladder epithelial cells), prostate, and pharynx. The adenocarcinoma includes malignant tumors such as most of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer among lung cancer, small intestine cancer and esophageal cancer. Squamous cell carcinomas include malignancies such as those in the lung, esophagus, skin, head and neck regions, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, e.g., advanced melanoma. In one embodiment, the cancer is renal cell carcinoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention.
Non-limiting examples of preferred cancers for treatment include lymphoma (e.g., diffuse large B-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma), breast cancer (e.g., metastatic breast cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (e.g., multiple myeloma), leukemia (e.g., chronic myelogenous leukemia), skin cancer (e.g., melanoma (e.g., stage III or IV melanoma), or Merkel cell carcinoma), head and neck cancer (e.g., Head and Neck Squamous Cell Carcinoma (HNSCC)), myelodysplastic syndrome, bladder cancer (e.g., transitional cell carcinoma), kidney cancer (e.g., renal cell cancer, e.g., clear cell renal cell cancer, e.g., advanced or metastatic clear cell renal cell carcinoma) and colon cancer. In addition, refractory or recurrent malignancies can be treated using the LAG-3 antibodies described herein or pharmaceutical compositions or combination products comprising the same.
In one embodiment, the disease is a disease with elevated levels of LAG-3 (nucleic acid or protein). In some embodiments, the tumor is a tumor that is capable of being inhibited by inhibiting the binding of LAG-3 to MHC class II molecules and/or FGL-1 molecules, e.g., a cancer. In some embodiments, the tumor or infection is a disease that would benefit from LAG-3 at the inhibitory nucleic acid or protein level.
In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is a persistent infection, a latent infection, or a slow infection. In some embodiments, the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa.
In some embodiments, an antibody of the invention, or a composition or combination comprising the same, delays the onset of a disorder and/or symptoms associated with the disorder.
In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual a LAG-3 antibody or pharmaceutical composition or combination product disclosed herein in combination with one or more other therapies, e.g., a therapeutic modality and/or other therapeutic agents.
In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which an irradiation region is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ) or focused irradiation), and the like. The focused irradiation may be selected from stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity modulated radiotherapy. The focused irradiation may have a radiation source selected from a particle beam (protons), cobalt-60 (photons) and a linear accelerator (X-rays), for example as described in WO 2012/177624.
Radiation therapy can be administered by one of several methods or a combination of methods including, without limitation, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole-body radiotherapy, and permanent or transient interstitial brachytherapy.
In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, another antibody, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule).
Exemplary additional antibodies include, but are not limited to, inhibitors of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, or anti-CEACAM); an antibody that stimulates an immune cell (e.g., an agonistic GITR antibody or a CD137 antibody), and the like. Preferably, the other antibody is selected from an anti-PD-1 antibody and/or an anti-PD-L1 antibody. More preferably, the anti-PD-1 antibody is Nivolumab (Nivolumab) of Bevacizumab (BMS), Pembrolizumab (Pembrolizumab) of Merck (Merck); the anti-PD-L1 antibody was atezolizumab, developed by Roche (Roche), avelumab, developed by cooperation of Merck KGaA and Merck KGaA in Germany and Pfizer in America, durvalumab, developed by Aslicon.
In some embodiments, the immunomodulatory agent is an agonist of a costimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from an agonist of (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion of) the following molecules: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands.
The combination therapies of the invention encompass both combined administration (wherein the two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, administration of the antibody or immunoconjugate of the invention, etc., may be carried out before, concurrently with, and/or after administration of the other therapy.
In one embodiment, administration of the LAG-3 antibody and administration of the other therapy (e.g., treatment modality or therapeutic agent) occur within about one month, or within about one, two, or three weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.
The antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising the same) can be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of the antibody of the 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 type of LAG-3 antibody, the severity and course of the disease, whether the LAG-3 antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the LAG-3 antibody, and the discretion of the attending physician. The LAG-3 antibody is suitably administered to the patient in one treatment or over a series of treatments. The dosage and treatment regimen for the LAG-3 antibody can be determined by the skilled artisan.
It will be appreciated that any of the prophylaxis or treatment described above can be carried out using a composition or combination of the invention in place of LAG-3 antibody.
IX. methods and compositions for diagnosis and detection
In certain embodiments, any of the LAG-3 antibodies provided herein can be used to detect the presence of LAG-3 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays. In certain embodiments, the biological sample is blood, serum, or other bodily fluid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.
In one embodiment, LAG-3 antibodies for use in a diagnostic or detection method are provided. In another aspect, methods of detecting the presence of LAG-3 in a biological sample are provided. In certain embodiments, the method comprises detecting the presence of LAG-3 protein in the biological sample. In certain embodiments, LAG-3 is human LAG-3. In certain embodiments, the method comprises contacting the biological sample with a LAG-3 antibody as described herein under conditions that allow binding of the LAG-3 antibody to LAG-3, and detecting whether a complex is formed between the LAG-3 antibody and LAG-3. The formation of the complex indicates the presence of LAG-3. The method may be an in vitro or in vivo method. In one embodiment, the LAG-3 antibody is used to select a subject suitable for treatment with the LAG-3 antibody, e.g., where LAG-3 is a biomarker for selecting the subject.
In one embodiment, an antibody of the invention can be used to diagnose cancer or tumor, e.g., to assess (e.g., monitor) the treatment or progression of, diagnosis and/or staging of a disease (e.g., hyperproliferative or cancerous disease) described herein in a subject. In certain embodiments, a labeled LAG-3 antibody is provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32 P、 14 C、 125 I、 3 H and 131 fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceri)ferases), for example, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), fluorescein, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β -galactosidase, glucoamylase, lytic enzymes, saccharide oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize a dye precursor such as HR, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.
In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the LAG-3 antibody. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is a formalin fixed, paraffin coated (FFPE) sample. In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
In some embodiments, LAG-3 is detected prior to treatment, e.g., prior to initiation of treatment or prior to some treatment after a treatment interval.
In some embodiments, there is provided a method of treating a tumor or infection, the method comprising: testing a subject (e.g., a sample) (e.g., a sample of a subject comprising cancer cells) for the presence of LAG-3, thereby determining a LAG-3 value, comparing the LAG-3 value to a control value (e.g., a value for LAG-3 in a sample of a healthy individual), and if the LAG-3 value is greater than the control value, administering to the subject a therapeutically effective amount of a LAG-3 antibody (e.g., a LAG-3 antibody described herein), optionally in combination with one or more other therapies, thereby treating the tumor or infection.
It is to be understood that the various embodiments described in the various sections of the invention, such as the diseases, therapeutic agents, modes of treatment and administration and the like, are equally applicable to, or combinable with, the embodiments of the other sections of the invention. The embodiments described in the various sections of the invention apply to the properties, uses, methods, etc. of LAG-3 antibodies, as well as to compositions, conjugates, combination products, kits, etc. comprising LAG-3 antibodies.
Examples
Example 1: preparation and screening of anti-human LAG-3 antibody hybridoma
1. Preparation of hybridomas
Human LAG-3 extracellular domain (NCBI accession number: NP-002277.4, 1aa-450aa) was fused to mouse Fc to obtain hLAG-3-mFc fusion protein, and Balb/c mice were immunized with the hLAG-3-mFc fusion protein to stimulate immune response.
Human LAG-3 extracellular domain (NCBI accession number NP-002277.4, 1aa-450aa) was fused to human Fc to obtain hLAG-3-hFc fusion protein, and the anti-hLAG-3 antibody titer in serum of Balb/c mice was measured by enzyme-linked immunosorbent assay (ELISA) after 96-well ELISA plates were coated with the hLAG-3-hFc fusion protein.
When Balb/c mice are detected to have higher anti-hLAG-3 antibody titer and generate the required immune response, the hLAG-3-mFc fusion protein is used for carrying out impact immunization on the mice, and the spleens of the mice are taken aseptically after 3 days. Spleen cells of Balb/c mice are collected, cell suspension is prepared and fused with SP2/0 myeloma cells, and the fused cells are subpackaged into 96-hole cell culture plates after being resuspended by HAT culture medium. Standing at 37 deg.C for 5% CO 2 Culturing in an incubator to form hybridoma cell lines.
2. Positive hybridoma binding screen
10-14 days after the splenocytes of the Balb/C mice are fused with SP2/0 myeloma cells, the enzyme label plates are respectively coated with hLAG-3-hFc fusion protein and cynomolgus monkey LAG-3 recombinant protein (catalog number: 90841-C08H, Beijing Yi Qiao Shenzhou science and technology limited company) (20ng/ml) and are kept at 4 ℃ overnight; washing with PBS for three times, sealing with 4% skimmed milk powder-PBS, and standing at room temperature for 1 hr; washing with PBS for three times, adding hybridoma clone culture supernatant, and standing at room temperature for 1 hr. Let the following controls: (1) positive Control (PC): serum from mice immunized with hLAG-3-mFc fusion protein (diluted 1:1000 in PBS); (2) negative Control (NC): sera of mice (diluted 1:1000 in PBS) prior to immunization with hLAG-3-mFc fusion protein.
Washing with PBST (0.05% Tween-PBS) for three times, washing with PBS for two times, adding HRP-goat anti-mouse IgG (Fc γ), and heating at 37 deg.C for 0.5 hr; washing with PBST (0.05% Tween-PBS) for 3 times, adding TMB color development solution, developing in dark for 15-30min, adding ELISA stop solution, and stopping reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded, using 630nm as the reference wavelength. The first 15 clones with high absorbance reading were selected from high to low and cultured. And taking the clone culture supernatant every other day for secondary ELISA confirmation, and determining the clone number of the positive hybridoma.
Example 2: sequencing of murine anti-human LAG-3 antibodies
Murine anti-human LAG-3 antibody clones 7C5, 4C8, 7F1, 7F10, 3a10 and 10B4 were selected as exemplary candidate clones for sequencing.
Specifically, after the exemplary hybridoma cells 7C5, 4C8, 7F1, 7F10, 3a10 and 10B4 secreting anti-human LAG-3 antibody were expanded, total cellular RNA was extracted and reverse-transcribed into cDNA; amplifying antibody light chain variable region VL and heavy chain variable region VH sequences using degenerate primers; and sequencing was performed, and the results are shown in the following Table 2, in which the amino acid sequence of the heavy chain variable region (VH) of 7C5 murine antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the 4C8 murine antibody is shown in SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4; 7F1 murine antibody heavy chain variable region amino acid sequence shown in SEQ ID NO. 5, light chain variable region amino acid sequence shown in SEQ ID NO. 6; 7F10 murine antibody heavy chain variable region amino acid sequence shown in SEQ ID NO. 7, light chain variable region amino acid sequence shown in SEQ ID NO. 8; 3A10 murine antibody heavy chain variable region amino acid sequence shown in SEQ ID NO 9, light chain variable region amino acid sequence shown in SEQ ID NO 10; the amino acid sequence of the heavy chain variable region of the 10B4 murine antibody is shown in SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 12.
TABLE 2 variable region amino acid sequences of murine anti-LAG-3 antibodies
Figure BDA0003497456830000321
Figure BDA0003497456830000331
Example 3: preparation of anti-human LAG-3 chimeric antibody and control antibody
The 7C5, 4C8, 7F1, 7F10, 3a10 and 10B4 murine antibody light chain variable region and heavy chain variable region nucleotide sequences obtained in example 2 were cloned into eukaryotic transient expression vector pcdna3.1 (catalog No. V79520, Invitrogen) containing human-kappa light chain constant region and upstream of the human IgG4 heavy chain constant region encoding gene, to obtain human-murine chimeric light chains (ch7C5L, ch4C8L, ch7F1L, ch7F10L, ch3a10L and ch10B4L) and human-murine chimeric heavy chains (ch7C5H, ch4C8H, ch7F1H, ch7F10H, ch3a10H and ch10B4H) expression plasmids, respectively, and were transferred into HEK293 cells (ATCC) for recombinant expression. 5-6 days after transfection of HEK293 cells, culture supernatants were taken, and expression supernatants were purified using a ProteinA affinity column to obtain chimeric antibodies of 7C5, 4C8, 7F1, 7F10, 3A10 and 10B4 (ch7C5, ch4C8, ch7F1, ch7F10, ch3A10 and ch10B 4).
Similarly, the control antibody BMS-986016 light and heavy chain sequences were fully synthesized and cloned into eukaryotic transient expression vectors and expressed recombinantly in HEK293 cells. After 5-6 days of cell transfection, culture supernatant is taken and purified by a ProteinA affinity chromatography column to obtain BMS-986016 recombinant protein. Wherein, the amino acid sequence of the control antibody BMS-986016 is derived from WHO Drug Information (Vol.32, No.2,2018), the amino acid sequence of the heavy chain is shown as a sequence 13, and the amino acid sequence of the light chain is shown as a sequence 14.
13 in SEQ ID NO: control antibody BMS-986016 heavy chain amino acid sequence
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
14, SEQ ID NO: control antibody BMS-986016 light chain amino acid sequence
EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Example 4: chimeric antibody affinity assay
The affinity of the chimeric antibody of example 3 was measured by capturing the Fc fragment of the antibody with an anti-human antibody Fc fragment capture Antibody (AHC) biological probe using an Octet QKe system instrument from Fortebio. The specific operation is as follows.
The chimeric antibodies of ch7C5, ch4C8, ch7F1, ch7F10, ch3A10 and ch10B4 were each diluted to 4. mu.g/ml in PBS buffer and passed over the surface of an AHC probe (Cat:18-0015, PALL) for 120 s. hLAG-3-mFc fusion protein (60nM) obtained using human LAG-3 extracellular domain (NCBI accession number: NP-002277.4, 1aa-450aa) fused to mouse Fc was used as mobile phase with 300s binding time and 300s dissociation time. After the experiment, blank control response values were deducted, and the software was run for 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.
The kinetic constants are shown in table 3 below. The results show that all clones are combined with human LAG-3 recombinant protein, and the sequence is cloned correctly.
TABLE 3 affinity assay of chimeric antibodies with human LAG-3 recombinant protein
Name of antibody K D (M)
ch7C5 3.65E-10
ch4C8 4.27E-10
ch7F1 5.52E-09
ch7F10 1.17E-10
ch3A10 1.28E-10
ch10B4 8.84E-11
Example 5: FACS detection of binding of anti-human LAG-3 chimeric antibodies to LAG-3 on HEK293 cell surface
Construction of HEK293 cell line transiently expressing human LAG-3: the transient expression plasmid encoding full-length human LAG-3(NCBI accession No.: NP-002277.4) was transferred into HEK293 cells by the liposome method, and the obtained cells (hereinafter also referred to as 293/rhLAG-3 cells) were used for FACS detection 48 hours after the transfection.
293/rhLAG-3 cell suspensions were incubated with different concentrations of chimeric antibodies ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, and ch10B4, respectively, at room temperature for 30min, after washing the cells 3 times with PBS, goat anti-human IgG-FITC (Cat: F9512, Sigma) diluted 1:200 was added and incubated for 30 min.
After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry to examine the binding ability of the chimeric antibody to LAG-3 on the cell surface. The specific binding patterns of the ch7C5, the ch4C8, the ch7F1, the ch7F10, the ch3A10 and the ch10B4 are shown in FIG. 1, and the specific binding patterns are detected by FACS.
As can be seen in fig. 1, the ch7C5, ch4C8, ch7F1, ch7F10, ch3a10, ch10B4 chimeric antibodies showed specific binding to human LAG-3 on the surface of HEK293 cells compared to isotype controls.
Example 6: FACS detection of blocking Activity of anti-human LAG-3 chimeric antibody on binding of 293 cell surface HLA to LAG-3
Construction of 293 cell line transiently expressing human HLA: expression plasmids encoding full-length human HLA-A (Cat: HG13263-UT, Beijing Yi Qian Shen science and technology, Inc.) and HLA-B (Cat: HG16629-UT, Beijing Yi Qian Shen science and technology, Inc.) were co-transfected into HEK293 cells by liposome method, and the obtained cells (hereinafter also referred to as 293/rhHLA cells) were used for FACS detection 48 hours after transfection.
Different concentrations of ch7C5, ch4℃ 8, ch7F1, ch7F10, ch3A10, and ch10B4 chimeric antibody were incubated with 2ug/ml of hLAG-3-mFc fusion protein at room temperature for 60min, added to 293/rhHLA cell suspension, incubated at 4 ℃ for 60min, after washing the cells 3 times with PBS, added with goat anti-mouse IgG-FITC (Cat: F9006, Sigma) diluted 1:200 and incubated for 30 min. After washing the cells 3 times with PBS, the cells were examined for Mean Fluorescence Intensity (MFI) by flow cytometry to examine the ability of the chimeric antibody to block cell surface HLA binding to recombinant LAG-3 protein. The results of FACS measurements are shown in FIG. 2.
As can be seen from fig. 2, the chimeric antibodies of ch7C5, ch4C8, ch7F1, ch7F10, ch3a10 and ch10B4 all were able to significantly block the binding between cell surface HLA and recombinant LAG-3 protein, and the corresponding half effective inhibitory concentration IC50 values are shown in table 4.
TABLE 4 FACS detection of blocking IC50 of anti-human LAG-3 chimeric antibodies against binding of 293 cell surface HLA to LAG-3
Name of antibody ch4C8 ch7C5 ch10B4 ch3A10 ch7F1 ch7F10
IC50(ug/ml) 1.276 1.872 2.003 3.333 11.46 1.212
Example 7: humanization and recombinant expression of anti-human LAG-3 monoclonal antibodies
1. Humanization of murine monoclonal antibody 7F10
(1) CDR grafting
First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then based on the results of homology alignment, the most similar human antibody template VH1(1-46) was selected as the basic template, and the full-sequence blast result was combined to perform CDR grafting, and based on the CDR3 sequence, JH4(WGQGTLVTVSS) was selected as the J region sequence to perform CDR grafting, thereby achieving humanization of the 7F10 heavy chain variable region (VH) in the framework region. Similarly, the light chain framework regions were humanized by selecting VK I (O12) and VKII (L25) as basic templates, combining the full-sequence blast results, performing CDR grafting, and selecting JK2(FGQGTKLEIK) as JK region sequence according to the CDR3 sequence (GQSYSYPYT). The amino acid sequence of the humanized heavy chain variable region hz7F10_ VH1 of the 7F10 antibody CDR Grafted (CDR Grafted) is shown in SEQ ID NO: 15; the humanized light chain variable region hz7F10_ VL1 amino acid sequence is shown in SEQ ID NO. 16, and the humanized light chain variable region hz7F10_ VL2 amino acid sequence is shown in SEQ ID NO. 17.
15, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH1 amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMHWVRQAPGQGLEWMGRIDPEDVETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
16 in SEQ ID NO: humanized antibody hz7F10 light chain variable region VL1 amino acid sequence
DIQMTQSPSSLSASVGDRVTITCKASENVGTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPYTFGQGTKLEIK
17 in SEQ ID NO: humanized antibody hz7F10 light chain variable region VL2 amino acid sequence
EIVMTQSPATLSLSPGERATLSCKASENVGTYVSWYQQKPGQAPRLLIYGASNRYTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYSYPYTFGQGTKLEIK
(2) Antibody variable region mutation design
According to the sequence characteristics of murine 7F10, the humanized light heavy chain variable region sequence and light chain variable region sequence of CDR transplantation are subjected to mutation design, and the mutation sites are shown in the following table 5.
TABLE 5.7F 10 humanized sequence design
Figure BDA0003497456830000361
Note: the humanized hz7F10 heavy chain variable region is named herein as follows: hz7F10_ VH is followed by a numerical identification number, e.g. 1, 2, 3, 4, 5, 6, 7. Thus, following this nomenclature, for example, hz7F10_ VH1 represents the first hz7F10 heavy chain variable region, hz7F10_ VH2 represents the second hz7F10 heavy chain variable region, hz7F10_ VH7 represents the seventh hz7F10 heavy chain variable region;
the humanized hz7F10 light chain variable region is named herein as follows: hz7F10_ VL is followed by a numerical identification number, e.g. 1, 2, 3, 4, 5, 6, 7. Thus, following this nomenclature, for example, hz7F10_ VL1 represents the first hz7F10 light chain variable region, hz7F10_ VL2 represents the second hz7F10 light chain variable region, hz7F10_ VL7 represents the seventh hz7F10 light chain variable region;
Q1E in the table indicates that the amino acid Q at position 1 of the corresponding column of the "CDR-grafted" sequence SEQ ID NO. 15 was mutated to E;
Y36F denotes the mutation of amino acid Y at position 36 of the corresponding column of the "CDR-grafted" sequence SEQ ID NO 16 to F;
N53T shows the mutation of amino acid N at position 53 of the corresponding column of "CDR grafted" sequence SEQ ID NO 17 to T.
The humanized antibody hz7F10 heavy chain variable region and light chain variable region sequences shown below were obtained.
18, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH2 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMHWVRQAPGQGLEWIGRIDPEDVETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
19, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH3 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMHWVRQAPGQGLEWMGRIDPEDVETKYDPKFQGRVTITRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
20, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH4 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMHWVRQAPGQGLEWMGRINPEDVETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
21, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH5 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGYTIKDDYMHWVRQAPGQGLEWMGRIDPEDVETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
22, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH6 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGVNFTDDYMHWVRQAPGQGLEWMGRIDPEDVETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
23, SEQ ID NO: humanized antibody hz7F10 heavy chain variable region VH7 amino acid sequence
EVQLVQSGAEVKKPGASVKVSCKASGVNIKDDYMHWVRQAPGQGLEWIGRIDPEDVETKYDPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARSFYSNYVNYFDQWGQGTLVTVSS
24, SEQ ID NO: humanized antibody hz7F10 light chain variable region VL3 amino acid sequence
DIQMTQSPSSLSASVGDRVTITCKASENVGTYVSWFQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPYTFGQGTKLEIK
25 in SEQ ID NO: humanized antibody hz7F10 light chain variable region VL4 amino acid sequence
DIQMTQSPSSLSASVGDRVTITCKASENVGTYVSWYQQKPGKAPKLLIYAASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPYTFGQGTKLEIK
26, SEQ ID NO: humanized antibody hz7F10 light chain variable region VL5 amino acid sequence
EIVMTQSPATLSLSPGERATLSCRASENVGTYVSWYQQKPGQAPRLLIYGASNRYTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYSYPYTFGQGTKLEIK
27 of SEQ ID NO: humanized antibody hz7F10 light chain variable region VL6 amino acid sequence
EIVMTQSPATLSLSPGERATLSCKASENVGTYVSWYQQKPGQAPRLLIYGASTRYTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYSYPYTFGQGTKLEIK
28, SEQ ID NO: humanized antibody hz7F10 light chain variable region VL7 amino acid sequence
EIVMTQSPATLSLSPGERATLSCKASENVGTYVSWFQQKPGQAPRLLIYGASNRYTGIPDRFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYSYPYTFGQGTKLEIK
2. Recombinant expression of humanized monoclonal antibodies
The humanized light chain variable region sequences (hz7F10_ VL1, hz7F10_ VL2, hz7F10_ VH1) of the designed hz7F10 antibody were synthesized in their entirety, the humanized heavy chain variable region was cloned into eukaryotic transient expression vector pCDNA3.1 (catalogue: V79520, Invitrogen) upstream of the gene encoding the heavy chain constant region of human IgG4, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 29; the humanized light chain variable region is cloned into the upstream of the coding gene of the human light chain C kappa of the eukaryotic transient expression vector, the amino acid sequence of the light chain constant region is shown as a sequence 30, and the light chain expression vector and the heavy chain expression vector of the humanized hz7F10 antibody are constructed.
29 in SEQ ID NO: heavy chain constant region amino acid sequence
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
30 of SEQ ID NO: light chain constant region amino acid sequence
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
According to the mutation design of table 6, the site-directed mutagenesis is respectively carried out on the light chain expression plasmid pCDNA3.1 and the heavy chain expression plasmid pCDNA3.1, and the light chain expression plasmid and the heavy chain expression plasmid are transferred into escherichia coli DH5 alpha for amplification to obtain the hz7F10 antibody light chain and heavy chain mutation expression plasmids; the light and heavy chain plasmids of the hz7F10 humanized antibody are combined and simultaneously transferred into HEK293 cells for recombinant expression, and the combination information is shown in Table 6.
TABLE 6 table of hz7F10 combination information of light and heavy chains
Figure BDA0003497456830000391
5-6 days after cell transfection, the cells were cultured in FreeStyle293 medium (Cat. No. 12338-018, Gibco), and the culture supernatant was collected and purified by a ProteinA affinity column to obtain a humanized antibody.
The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the assay, the hz7F10 antibody and the chimeric antibody ch7F10 were diluted to 4ug/mL in PBS buffer and passed over the surface of an AHC probe (Cat:18-0015, PALL) for 300S. The hLAG-3-mFc recombinant protein was used as a mobile phase at a concentration of 60 nM. The binding time was 300s and the dissociation time was 300 s. After the experiment, the following steps are carried out by software: 1Langmuir binding pattern fit, kinetic constants for antigen-antibody binding were calculated.
Affinity measurement data of the humanized antibody was obtained by measuring the affinity of the hz7F10 combined antibody and the chimeric antibody ch7F10 to the hLAG-3-mFc recombinant protein by ForteBio (Table 7),
as can be seen from Table 7, after the antibody hz7F10-1 or the antibody hz7F10-2 was subjected to site mutation, the affinity of the antibodies hz7F10-3 to hz7F10-25 was improved by about 1-2 orders of magnitude as compared with that of the antibodies hz7F10-1 or hz7F 10-2. The antibody hz7F10-23 was selected as an example for the subsequent experiments.
TABLE 7 results of affinity assay of hz7F10 light and heavy chain combinations
Figure BDA0003497456830000392
Figure BDA0003497456830000401
Example 8: species-specific study of ELISA to detect binding of anti-LAG-3 humanized antibodies to LAG-3
Human LAG-3-mFc recombinant protein, cynomolgus monkey LAG-3 recombinant protein (Cat:90841-C08H, Beijing Yi Qiao Shenzhou science and technology, Inc.), mouse LAG-3 recombinant protein (Cat:53069-M08H, Beijing Yi Qiao Shenzhou science and technology, Inc.) are coated at 4 ℃ overnight, and the coating concentration is 1 mug/mL; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; adding hz7F10-23 (initial concentration is 10 mug/mL, 3 times of gradient dilution is performed for 12 concentrations in sequence) with different dilution times, incubating for 60min at 37 ℃, and washing the plate for 4 times by PBST; HRP-anti-human Fc (Cat: 109-; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded, using 630nm as the reference wavelength. Specific binding of hz7F10-23 and control antibodies to human, cynomolgus monkey, and mouse LAG-3 is shown in FIGS. 3, 4, and 5, respectively.
The results show that hz7F10-23 can specifically bind to human and cynomolgus monkey LAG-3 recombinant protein, the corresponding half effective binding concentration EC50 value is shown in Table 8, and the recombinant protein has no binding activity with mouse LAG-3 (figure 5), which provides a basis for the subsequent in vivo experimental model selection.
TABLE 8 ELISA detection of EC50 binding of anti-human LAG-3 humanized antibodies to different species of LAG-3
Name of antibody EC50 binding to human LAG-3 EC50 binding to cynomolgus monkey LAG-3
hz7F10-23 0.034nM 0.20nM
Example 9: anti-LAG-3 humanized antibody affinity assay
The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. The specific operation is as follows.
Antibodies (hz7F10-23 and control antibody BMS-986016) were diluted to 4ug/mL in PBS buffer and passed over the surface of AHC probe (Cat:18-0015, PALL) for 120 s. Human LAG-3-mFc recombinant protein as mobile phase. The binding time was 300s and the dissociation time was 300 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.
The binding reaction curves of antibody hz7F10-23 and control antibody BMS-986016 with human LAG-3 recombinant protein are shown in FIGS. 6 and 7, respectively. Curves were fitted and binding affinities, hz7F10-23 affinity (K) were calculated D ) Was 4.17E-11M, BMS-986016 affinity (K) D ) Is 1.61E-10M. Specific kinetic parameters are shown in table 9 below. The results indicate that hz7F10-23 has high affinity with human LAG-3, superior to control antibody BMS-986016.
TABLE 9 affinity assay results of anti-human LAG-3 humanized antibody and human LAG-3 extracellular region recombinant protein
K D Value (M) k on (1/Ms) k dis (1/s)
hz7F10-23 4.17E-11 2.45E+05 1.02E-05
BMS-986016 1.61E-10 2.72E+05 4.36E-05
Example 10: FACS detection of binding of anti-human LAG-3 humanized antibody to 293 cell surface LAG-3
Construction of 293 cell line transiently expressing human LAG-3: transient expression plasmids encoding full-length human LAG-3(NCBI accession No.: NP-002277.4) were transferred into HEK293 cells by liposome method, and 293/rhLAG-3 cells were used for FACS detection 48 hours after transfection.
293/rhLAG-3 cell suspensions were incubated with humanized antibody hz7F10-23 and control antibody BMS-986016 at different concentrations for 30min at room temperature, after washing the cells 3 times with PBS, goat anti-human IgG-FITC (Cat: F9512, Sigma) diluted 1:200 was added and incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry to examine the binding ability of the humanized antibody to LAG-3 on the cell surface.
The antibody hz7F10-23 was able to bind 293 cell surface LAG-3 as measured by FACS, and as can be seen from FIG. 8, the binding ability of antibody hz7F10-23 to cell surface LAG-3 was superior to that of the control antibody BMS-986016.
The half-effective binding concentration EC50 values of the antibody hz7F10-23 and the control antibody BMS-986016 were 1.6nM and 1.5nM, respectively, and the binding capacities of the two were equivalent.
Example 11: ELISA detection of blocking Activity of anti-LAG-3 humanized antibody on binding of LAG-3 to FGL-1
Coating a 96-well plate with human FGL-1 recombinant protein (Cat: CW55, a near-shore organism) at 4 ℃ overnight, wherein the coating concentration is 0.5 mu g/mL; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; pre-incubating different concentrations of hz7F10-23 and control antibody BMS-986016 with 0.02ug/ml LAG-3-mFc (NCBI accession number: NP-002277.4, 1aa-450aa) at 37 ℃ for 30min, adding into coated wells, incubating at 37 ℃ for 60min, and washing the plates with PBST for 4 times; HRP anti-mouse IgG (Cat: 115-; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength. Blocking of binding of LAG-3 to FGL-1 by humanized antibody hz7F10-23 is shown in FIG. 9.
As can be seen from FIG. 9, both the humanized antibody hz7F10-23 and the control antibody can specifically block the binding of human LAG-3 and FGL-1, and the half effective inhibitory concentrations IC50 of the humanized antibody and the control antibody are 0.038nM and 0.038nM, respectively, and the blocking activities are equivalent.
Example 12: FACS detection of blocking Activity of anti-human LAG-3 humanized antibody on binding of 293 cell surface HLA to LAG-3
Construction of 293 cell line transiently expressing human HLA: the expression plasmids encoding full-length human HLA-A (Cat: HG13263-UT, Beijing Yi Qian Shen science and technology GmbH) and HLA-B (Cat: HG16629-UT, Beijing Yi Qian Shen science and technology GmbH) were co-transfected into HEK293 cells by liposome method, and 293/rhHLA cells were used for FACS detection 48 hours after transfection.
Humanized antibody hz7F10-23 and control antibody BMS-986016 at different concentrations were incubated with 3ug/ml LAG-3-mFc recombinant protein at room temperature for 40min, added to 293/rhHLA cell suspension, incubated at 4 ℃ for 60min, cells were washed 3 times with PBS, added with goat anti-mouse IgG-FITC (Cat: F9006, Sigma) diluted 1:200 and incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry to examine the ability of the humanized antibody to block the binding of cell surface HLA to recombinant LAG-3 protein. The results of FACS-detected blocking of HLA-3 binding to LAG-3 by humanized antibody hz7F10-23 are shown in FIG. 10.
As can be seen from FIG. 10, the antibody hz7F10-23 was able to block the binding of cell surface HLA to recombinant LAG-3 protein with better blocking ability than the control antibody, and the half effective inhibitory concentrations IC50 of hz7F10-23 and BMS-986016 were 4.8nM and 9.9nM, respectively.
Example 13: pharmacodynamic evaluation of anti-LAG-3 antibodies in B6-huPD1huLAG3 humanized mouse subcutaneous graft tumor model
A6-8 week-old PD-1 and LAG-3 double humanized transgenic mouse B6-huPD1huLAG3 (Ji Jing Yao kang, catalog No.: T004621) was inoculated subcutaneously with 3X 10 6 MC38 murine Colon adenocarcinoma cells (ATCC) to allow tumor growth to 100mm 3 The random grouping is carried out on the left and the right, 6 animals are grouped, the grouping and the administration dosage and frequency are shown in the table 10, each group is administrated in the abdominal cavity twice a week for 6 times, the tumor volume and the mouse weight are measured when the administration is carried out, when the mouse weight is reduced by more than 15%, or the tumor volume of a single animal is more than 3000mm 3 Or the average tumor volume of a group of animals exceeds 2000mm 3 The experiment was stopped for the relevant mice and the mice were euthanized.
TABLE 10 grouping and dosing dose, frequency of B6-huLAG3 mice
Group of Medicine Dosage to be administered Frequency of administration
1 Isotype controls 10mg/kg Biw×6
2 hz7F10-23 10mg/kg Biw×6
3 BMS-986016 10mg/kg Biw×6
In the B6-huLAG3 humanized mouse subcutaneous graft tumor model, as can be seen from fig. 11, the humanized antibody hz7F10-23 was able to statistically significantly inhibit tumor growth, P ═ 0.0086, with a clear antitumor effect, compared to control mice using isotype antibody; the antitumor effect of the humanized antibody hz7F10-23 is better than that of the control antibody BMS-986016; as can be seen from FIG. 12, the body weight of the animals in each experimental group increased steadily without significant difference from the control.
Having described exemplary embodiments of the invention, it will be understood by those skilled in the art that this disclosure is illustrative only, and that various other substitutions, adaptations and modifications may be made within the scope of the invention. Accordingly, the present invention is not limited to the specific embodiments set forth herein.
<110> Miwei (Shanghai) Biotech Co., Ltd
<120> antibodies that bind LAG-3 and uses thereof
<130>
<150> CN202110171586.8
<151> 2021-02-08
<160> 90
<170> PatentIn version 3.3
<210> 1
<211> 124
<212> PRT
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<220>
<223> antibody 7C5 VH
<400> 1
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Tyr Pro Arg Ser Asp Asn Thr Tyr Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Phe Cys
85 90 95
Ala Arg Arg Ala Phe Tyr Tyr Tyr Gly Ser Asn Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
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<211> 112
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<223> antibody 7C5 VL
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Asp Val Leu Met Thr Gln Thr Pro Leu Phe Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Arg Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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<211> 118
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<223> antibody 4C8 VH
<400> 3
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ile Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Tyr Gly Thr Thr Tyr Thr Phe Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Ser
65 70 75 80
Leu Gln Met Ser His Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Arg Gly Glu Tyr Gly Ser Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 4
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<223> antibody 4C8 VL
<400> 4
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Asn Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 5
<211> 115
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 VH
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr
20 25 30
Ala Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Val Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60
Ser Arg Leu Ile Ile Thr Arg Asp Asn Ser Lys Arg Gln Val Phe Leu
65 70 75 80
Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys Gly Gly Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser
115
<210> 6
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<223> antibody 7F1 VL
<400> 6
Asp Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Val Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln His Tyr Asn Thr Asn Pro Pro Thr
85 90 95
Trp Thr Phe Gly Gly Gly Ser Lys Leu Glu Ile Lys
100 105
<210> 7
<211> 121
<212> PRT
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<220>
<223> antibody 7F10 VH
<400> 7
Glu Val Lys Leu Met Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Val Ser Gly Val Asn Ile Lys Asp Asp
20 25 30
Tyr Met His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ser Asp Thr Ser Ser Asn Thr Gly Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 8
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<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 VL
<400> 8
Asp Ile Val Met Thr Gln Ala Pro Lys Ser Met Ser Met Ser Val Gly
1 5 10 15
Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Phe Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 VH
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp
20 25 30
Tyr Met His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Glu Thr Glu Tyr Ala Ser Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Tyr Ser Asn Thr Val Tyr
65 70 75 80
Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Thr Ile Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val Ser Ser
100 105 110
<210> 10
<211> 112
<212> PRT
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<220>
<223> antibody 3A10 VL
<400> 10
Asp Ile Val Met Thr Gln Ala Pro Val Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Phe Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 11
<211> 124
<212> PRT
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<223> antibody 10B4 VH
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Ile Ser Trp Met Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Tyr Pro Arg Gly Gly Asn Thr Tyr Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Gln Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Ala Phe Tyr Tyr Phe Gly Ser Asn Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 12
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 10B4 VL
<400> 12
Asp Val Val Met Thr Gln Thr Pro Leu Phe Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Ala Ile Lys
100 105 110
<210> 13
<211> 447
<212> PRT
<213> Artificial sequence
<220>
<223> antibody BMS-986016 heavy chain amino acid sequence
<400> 13
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr
20 25 30
Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 14
<211> 214
<212> PRT
<213> Artificial sequence
<220>
<223> antibody BMS-986016 light chain amino acid sequence
<400> 14
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 15
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH1
<400> 15
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 16
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL1
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 17
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL2
<400> 17
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 18
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH2
<400> 18
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 19
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH3
<400> 19
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 20
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH4
<400> 20
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH5
<400> 21
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Lys Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH6
<400> 22
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Asn Phe Thr Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 23
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VH7
<400> 23
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Asn Ile Lys Asp Asp
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 24
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL3
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 25
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL4
<400> 25
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 26
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL5
<400> 26
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 27
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL6
<400> 27
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 28
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hz7F10_VL7
<400> 28
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 29
<211> 327
<212> PRT
<213> Artificial sequence
<220>
<223> antibody heavy chain constant region
<400> 29
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 30
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody light chain constant region
<400> 30
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 31
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 HCDR1
<400> 31
Ser Tyr Gly Ile Ser
1 5
<210> 32
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 HCDR2
<400> 32
Glu Ile Tyr Pro Arg Ser Asp Asn Thr Tyr Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 33
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 HCDR3
<400> 33
Arg Ala Phe Tyr Tyr Tyr Gly Ser Asn Tyr Tyr Ala Met Asp Tyr
1 5 10 15
<210> 34
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 LCDR 1; antibody 10B4 LCDR1
<400> 34
Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asp Thr Tyr Leu Glu
1 5 10 15
<210> 35
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 LCDR 2; antibody 10B4 LCDR2
<400> 35
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 36
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7C5 LCDR 3; antibody 10B4 LCDR3
<400> 36
Phe Gln Gly Ser His Val Pro Trp Thr
1 5
<210> 37
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 HCDR1
<400> 37
Asn Tyr Ala Met Ser
1 5
<210> 38
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 HCDR2
<400> 38
Thr Ile Thr Tyr Gly Thr Thr Tyr Thr Phe Tyr Ser Asp Asn Val Lys
1 5 10 15
Gly
<210> 39
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 HCDR3
<400> 39
Gly Glu Tyr Gly Ser Ser Phe Ala Tyr
1 5
<210> 40
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 LCDR1
<400> 40
Lys Ala Ser Gln Asn Val Arg Thr Ala Val Ala
1 5 10
<210> 41
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 LCDR2
<400> 41
Leu Ala Ser Asn Arg His Thr
1 5
<210> 42
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 4C8 LCDR3
<400> 42
Leu Gln His Trp Asn Tyr Pro Leu Thr
1 5
<210> 43
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 HCDR1
<400> 43
Asp Tyr Ala Val Ser
1 5
<210> 44
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 HCDR2
<400> 44
Val Val Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile Ser
1 5 10 15
<210> 45
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 HCDR3
<400> 45
Gly Gly Gly Gly Met Asp Tyr
1 5
<210> 46
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 LCDR1
<400> 46
Arg Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10
<210> 47
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 LCDR2
<400> 47
Ala Thr Ser Asn Leu Ala Ser
1 5
<210> 48
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F1 LCDR3
<400> 48
Gln His Tyr Asn Thr Asn Pro Pro Thr Trp Thr
1 5 10
<210> 49
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 HCDR 1; antibody 3a10 HCDR 1; humanized antibody hz7F10 HCDR1
<400> 49
Asp Asp Tyr Met His
1 5
<210> 50
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 HCDR 2; humanized antibody hz7F10 HCDR2
<400> 50
Arg Ile Asp Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 51
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 HCDR 3; humanized antibody hz7F10 HCDR3
<400> 51
Ser Phe Tyr Ser Asn Tyr Val Asn Tyr Phe Asp Gln
1 5 10
<210> 52
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 LCDR 1; humanized antibody hz7F10 LCDR1
<400> 52
Lys Ala Ser Glu Asn Val Gly Thr Tyr Val Ser
1 5 10
<210> 53
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 LCDR 2; humanized antibody hz7F10 LCDR2
<400> 53
Gly Ala Ser Asn Arg Tyr Thr
1 5
<210> 54
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 7F10 LCDR 3; humanized antibody hz7F10 LCDR3
<400> 54
Gly Gln Ser Tyr Ser Tyr Pro Tyr Thr
1 5
<210> 55
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 HCDR2
<400> 55
Trp Ile Asp Pro Glu Asn Gly Glu Thr Glu Tyr Ala Ser Lys Phe Gln
1 5 10 15
Gly
<210> 56
<211> 3
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 HCDR3
<400> 56
Phe Asp Tyr
1
<210> 57
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 LCDR1
<400> 57
Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 58
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 LCDR2
<400> 58
Leu Val Ser Lys Leu Asp Phe
1 5
<210> 59
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 3A10 LCDR3
<400> 59
Trp Gln Gly Thr His Phe Pro Gln Thr
1 5
<210> 60
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 10B4 HCDR1
<400> 60
Asn Tyr Gly Ile Ser
1 5
<210> 61
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 10B4 HCDR2
<400> 61
Glu Ile Tyr Pro Arg Gly Gly Asn Thr Tyr Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 62
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> antibody 10B4 HCDR3
<400> 62
Arg Ala Phe Tyr Tyr Phe Gly Ser Asn Tyr Tyr Ala Met Asp Tyr
1 5 10 15
<210> 63
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 HCDR2
<400> 63
Arg Ile Asn Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 64
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 HCDR2 consensus sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa is Asp or Asn
<400> 64
Arg Ile Xaa Pro Glu Asp Val Glu Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 65
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 LCDR1
<400> 65
Arg Ala Ser Glu Asn Val Gly Thr Tyr Val Ser
1 5 10
<210> 66
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 LCDR2
<400> 66
Ala Ala Ser Asn Arg Tyr Thr
1 5
<210> 67
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 LCDR2
<400> 67
Gly Ala Ser Thr Arg Tyr Thr
1 5
<210> 68
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 LCDR1 consensus sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa is Lys or Arg
<400> 68
Xaa Ala Ser Glu Asn Val Gly Thr Tyr Val Ser
1 5 10
<210> 69
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 LCDR2 consensus sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa is Gly or Ala
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa is Asn or Thr
<400> 69
Xaa Ala Ser Xaa Arg Tyr Thr
1 5
<210> 70
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR1
<400> 70
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys
20
<210> 71
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR1
<400> 71
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys
20
<210> 72
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR2
<400> 72
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 73
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR2
<400> 73
Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 74
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR2
<400> 74
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
1 5 10 15
<210> 75
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR2
<400> 75
Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
1 5 10 15
<210> 76
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR3
<400> 76
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30
<210> 77
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR3
<400> 77
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys
20 25 30
<210> 78
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR3
<400> 78
Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys
20 25 30
<210> 79
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 light chain variable region FR4
<400> 79
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 80
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR1
<400> 80
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 81
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR1
<400> 81
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 82
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR1
<400> 82
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Lys
20 25 30
<210> 83
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR1
<400> 83
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Asn Phe Thr
20 25 30
<210> 84
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR1
<400> 84
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Asn Ile Lys
20 25 30
<210> 85
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR2
<400> 85
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 86
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR2
<400> 86
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly
1 5 10
<210> 87
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR3
<400> 87
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 88
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR3
<400> 88
Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 89
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR3
<400> 89
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 90
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> humanized antibody hz7F10 heavy chain variable region FR4
<400> 90
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10

Claims (18)

1. An antibody or antigen-binding fragment that specifically binds LAG-3, comprising
(a) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 1 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 2; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(b) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 4; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(c) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 5 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 6; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(d) 3 CDRs in the amino acid sequence of heavy chain variable region shown in SEQ ID NO. 7 and 3 CDRs in the amino acid sequence of light chain variable region shown in SEQ ID NO. 8; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(e) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 9 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO. 10; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
(f) 11 and 12, 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO; or a variant having a single CDR or a plurality of CDRs with no more than 2 or 1 amino acid changes from the 6 CDR regions;
wherein the amino acid change is an addition, deletion or substitution of an amino acid.
2. An antibody or antigen-binding fragment that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein:
(a) the heavy chain variable region comprises HCDR1 as represented by SYGIS (SEQ ID NO:31), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRSDNTYYNGKFKG (SEQ ID NO:32) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYYGSNYYAMDY (SEQ ID NO:33) of HCDR3, or a variant of said HCDR3 that does not exceed 2 amino acid changes, or does not exceed 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown by KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(b) said heavy chain variable region comprises HCDR1 as indicated by NYAMS (SEQ ID NO:37), or a variant of said HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; TITYGTTYTFYSDNVKG (SEQ ID NO:38) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GEYGSSFAY (SEQ ID NO:39), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASQNVRTAVA (SEQ ID NO:40), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LASNRHT (SEQ ID NO:41), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and LQHWNYPLT (SEQ ID NO:42) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(c) the heavy chain variable region comprises HCDR1 as represented by DYASS (SEQ ID NO:43), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; VVWGDGSTNYHSALIS (SEQ ID NO:44) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and GGGGMDY (SEQ ID NO:45), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RASSSVSYMH (SEQ ID NO:46), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in ATSNLAS (SEQ ID NO:47), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and QHYNTNPPTWT (SEQ ID NO:48) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(d) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; RIDPEDVETKYDPKFQG (SEQ ID NO:50) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and SFYSNYVNYFDQ (SEQ ID NO:51), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in GASNRYT (SEQ ID NO:53), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and GQSYSYPYT (SEQ ID NO:54), or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
(e) the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO:49), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; WIDPENGETEYASKFQG (SEQ ID NO:55) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and FDY (SEQ ID NO:56) or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at KSSQSLLDSDGKTYLN (SEQ ID NO:57), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as represented by LVSKLDF (SEQ ID NO:58), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and WQGTHFPQT (SEQ ID NO:59) of LCDR3, or a variant of said LCDR3 having NO more than 2 amino acid changes or NO more than 1 amino acid change; or
(f) The heavy chain variable region comprises HCDR1 as shown in NYSIS (SEQ ID NO:60), or a variant of the HCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO:61) of HCDR2, or a variant of said HCDR2 that does not exceed 2 amino acid changes or does not exceed 1 amino acid change; and RAFYYFGSNYYAMDY (SEQ ID NO:62), or a variant of said HCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown at RSSQSIVHSNGDTYLE (SEQ ID NO:34), or a variant of the LCDR1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; LCDR2 as shown in KVSNRFS (SEQ ID NO:35), or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO:36) of LCDR3, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;
wherein the amino acid change is an addition, deletion or substitution of an amino acid.
3. The antibody or antigen-binding fragment of claim 2 that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein
The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); RIX 1 PEDVETKYDPKFQG (SEQ ID NO:64), optionally, wherein X is 1 Is D or N; and SFYSNYVNYFDQ (SEQ ID NO:51) of HCDR 3;
the light chain variable region comprises X 2 ASENVGTYVS (SEQ ID NO:68) LCDR1, optionally wherein X 2 Is K or R; x 3 ASX 4 RYT (SEQ ID NO:69) LCDR2, optionally; wherein X 3 Is G or A; x 4 Is N or T; and LCDR3 as shown in GQSYSYPYT (SEQ ID NO: 54).
4. The antibody or antigen-binding fragment of claim 3 that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein
The heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) is shown as HCDR 2; and SFYSNYVNYFDQ (SEQ ID NO:51) of HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at RASENVGTYVS (SEQ ID NO:65), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RIDPEDVETKYDPKFQG (SEQ ID NO:50) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASNRYT (SEQ ID NO:53), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54);
the heavy chain variable region comprises HCDR1 as shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at AASNRYT (SEQ ID NO:66), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54); or
The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); RINPEDVETKYDPKFQG (SEQ ID NO:63) HCDR2, SFYSNYVNYFDQ (SEQ ID NO:51) HCDR 3; the light chain variable region comprises LCDR1 as shown at KASENVGTYVS (SEQ ID NO:52), LCDR2 as shown at GASTRYT (SEQ ID NO:67), and LCDR3 as shown at GQSYSYPYT (SEQ ID NO: 54).
5. The antibody or antigen-binding fragment of claim 1 or 2 that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein
(a) The heavy chain variable region comprises the sequence of SEQ ID NO 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(b) the heavy chain variable region comprises the sequence of SEQ ID NO 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(c) the heavy chain variable region comprises the sequence of SEQ ID NO 5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(d) (i) the heavy chain variable region comprises the sequence of SEQ ID NO:7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO:8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(ii) the heavy chain variable region comprises the sequence of SEQ ID NO 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or
(iii) The heavy chain variable region comprises the sequence of SEQ ID NO 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 17 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(e) the heavy chain variable region comprises the sequence of SEQ ID NO 9 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain variable region comprises the sequence of SEQ ID NO 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(f) the heavy chain variable region comprises the sequence of SEQ ID NO 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
6. The antibody or antigen-binding fragment of claim 5 that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment comprises
(a) The heavy chain variable region shown in SEQ ID NO. 1 and the light chain variable region shown in SEQ ID NO. 2;
(b) the heavy chain variable region shown in SEQ ID NO. 3 and the light chain variable region shown in SEQ ID NO. 4;
(c) the heavy chain variable region shown in SEQ ID NO. 5 and the light chain variable region shown in SEQ ID NO. 6;
(d) the heavy chain variable region shown in SEQ ID NO. 7 and the light chain variable region shown in SEQ ID NO. 8;
the heavy chain variable region shown in SEQ ID NO. 15 and the light chain variable region shown in SEQ ID NO. 16;
the heavy chain variable region shown in SEQ ID NO. 15 and the light chain variable region shown in SEQ ID NO. 17;
the heavy chain variable region shown by SEQ ID NO. 18 and the light chain variable region shown by SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 18 and the light chain variable region shown in SEQ ID NO. 25;
the heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 25;
the heavy chain variable region shown in SEQ ID NO. 18 and the light chain variable region shown in SEQ ID NO. 26;
the heavy chain variable region shown in SEQ ID NO. 18 and the light chain variable region shown in SEQ ID NO. 27;
the heavy chain variable region shown by SEQ ID NO. 19 and the light chain variable region shown by SEQ ID NO. 26;
the heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 27;
the heavy chain variable region shown in SEQ ID NO. 20 and the light chain variable region shown in SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 20 and the light chain variable region shown in SEQ ID NO. 25;
the heavy chain variable region shown in SEQ ID NO. 20 and the light chain variable region shown in SEQ ID NO. 27;
the heavy chain variable region shown in SEQ ID NO. 20 and the light chain variable region shown in SEQ ID NO. 28;
the heavy chain variable region shown in SEQ ID NO. 21 and the light chain variable region shown in SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 21 and the light chain variable region shown in SEQ ID NO. 25;
the heavy chain variable region shown by SEQ ID NO. 21 and the light chain variable region shown by SEQ ID NO. 27;
the heavy chain variable region shown in SEQ ID NO. 21 and the light chain variable region shown in SEQ ID NO. 28;
the heavy chain variable region shown in SEQ ID NO. 22 and the light chain variable region shown in SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 22 and the light chain variable region shown in SEQ ID NO. 25;
the heavy chain variable region shown in SEQ ID NO. 22 and the light chain variable region shown in SEQ ID NO. 27;
the heavy chain variable region shown in SEQ ID NO. 22 and the light chain variable region shown in SEQ ID NO. 28;
the heavy chain variable region shown in SEQ ID NO. 23 and the light chain variable region shown in SEQ ID NO. 24;
the heavy chain variable region shown in SEQ ID NO. 23 and the light chain variable region shown in SEQ ID NO. 25; or
The heavy chain variable region shown in SEQ ID NO. 23 and the amino acid sequence of SEQ ID NO. 28;
(e) the heavy chain variable region shown in SEQ ID NO. 9 and the amino acid sequence of SEQ ID NO. 10; or
(f) The heavy chain variable region shown in SEQ ID NO. 11 and the amino acid sequence of SEQ ID NO. 12.
7. The antibody or antigen-binding fragment of any one of claims 1 to 6 that specifically binds LAG-3, which is an IgG1, IgG2, IgG3, or IgG4 antibody; optionally, it is an IgG1 or IgG4 antibody; optionally, it is a humanized IgG1 or a humanized IgG4 antibody.
8. The antibody or antigen-binding fragment of any one of claims 1 to 7 that specifically binds LAG-3, wherein the antigen-binding fragment is a Fab, Fab ', F (ab') 2, Fv, single chain Fab, or diabody (diabody).
9. The antibody or antigen-binding fragment of any one of claims 1 to 8 that specifically binds LAG-3, having one or more of the following properties:
(1) binding LAG-3 with high affinity, e.g., human LAG-3 and cynomolgus monkey LAG-3, e.g., K binding between the anti-LAG-3 antibody or antigen binding fragment thereof and LAG-3 D Is about 10 -7 M to about 10 -12 M, preferably, about 10 -8 M to about 10 -12 M, as measured by ForteBio kinetic binding assay;
(2) specifically block the binding of LAG-3 to FGL-1;
(3) specifically block binding of LAG-3 to cell surface MHC class II molecules (e.g., human HLA);
(4) maintaining the T cells in an activated state;
(5) has antitumor effect in vivo.
10. An isolated nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1-9.
11. A vector comprising the nucleic acid of claim 10, preferably said vector is an expression vector.
12. A host cell comprising the nucleic acid of claim 10 or the vector of claim 11, preferably said host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of e.coli cells, yeast cells, mammalian cells or other cells suitable for the production of antibodies or antigen binding fragments thereof, most preferably said host cell is 293 cells or CHO cells.
13. A method of making the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1-9, the method comprising culturing the host cell of claim 12 under conditions suitable for expression of a nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1-9, optionally isolating the anti-LAG-3 antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the anti-LAG-3 antibody or antigen-binding fragment thereof from the host cell.
14. A pharmaceutical composition comprising the anti-LAG-3 antibody, or antigen-binding fragment thereof, of any one of claims 1-9, and optionally a pharmaceutically acceptable carrier.
15. A pharmaceutical composition comprising the anti-LAG-3 antibody, or antigen-binding fragment thereof, of any one of claims 1-9, and an additional therapeutic agent, and optionally a pharmaceutically acceptable carrier; preferably, the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, additional antibodies (e.g., anti-PD-1 antibody or anti-PD-L1 antibody), cytotoxic agents.
16. Use of the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the pharmaceutical composition of claim 14 or 15, for the manufacture of a medicament for preventing or treating a tumor in a subject.
17. The use of claim 16, wherein the tumor is a cancer, such as hematological cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular cancer, bone cancer, colon cancer (e.g., colon adenocarcinoma), non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B-cell lymphoma, and melanoma.
18. A kit for detecting LAG-3 in a sample, the kit comprising an anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, for performing the steps of:
(a) contacting the sample with an anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9; and
(b) detecting formation of a complex between the anti-LAG-3 antibody or antigen-binding fragment thereof and LAG-3; optionally, the anti-LAG-3 antibody or antigen-binding fragment thereof is detectably labeled.
CN202210118342.8A 2021-02-08 2022-02-08 LAG-3 binding antibodies and uses thereof Pending CN114907478A (en)

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