CN113150150B - TIM3 binding molecules and uses thereof - Google Patents

TIM3 binding molecules and uses thereof Download PDF

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CN113150150B
CN113150150B CN202110200647.9A CN202110200647A CN113150150B CN 113150150 B CN113150150 B CN 113150150B CN 202110200647 A CN202110200647 A CN 202110200647A CN 113150150 B CN113150150 B CN 113150150B
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antibody
tim3
amino acid
variable region
chain variable
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CN113150150A (en
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吴炯
杨斌
潘燕峰
夏玉龙
唐英杰
金昕
孙馨
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Suzhou Hengkang Life Science Co ltd
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Abstract

The present invention provides a TIM3 binding molecule which specifically recognizes endogenous and exogenous TIM3 and has T cell activating activity, which is useful therapeutic for the treatment of different cancers, especially hematological tumors, such as leukemia and lymphoma.

Description

TIM3 binding molecules and uses thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a molecule combined with TIM 3. .
Background
T cell immunoglobulin and mucin family-3 (T-cell immunoglobulin and mucin-domain containing-3, tim 3) proteins, are TMembers of the im family, also known as HAVCR2 (Hepatitis A virus cellular receptor-2) proteins, are cell membrane receptors encoded by the HAVCR2 gene. It was found that Tim-3 was expressed in activated CD4 + Th1、Th17、CD8 + Tc1 cells, and maintain immune tolerance by negatively regulating the function of the cells.
T cell depletion is known in the art to be mediated by several immune checkpoint inhibitory receptors (e.g., PD1, TIM3, CTLA-1, LAG3, etc.), TIM3 and PD1 signaling pathway interactions play an important role in T cell depletion. Immune checkpoints play an important role in maintaining autoimmune tolerance and avoiding the immune system from attacking the self organs, while many cancers achieve immune evasion by exactly deregulating immune checkpoint protein expression. By blocking immune checkpoints, restoring the body's own anti-tumor immune response, and clearing cancer cells in the body by means of body's immune function has been one of the directions of oncologists ' research.
Inhibitory monoclonal antibodies directed against immune checkpoints CTLA-1 and PD-1 have achieved objective efficacy in the clinical treatment of various tumors such as melanoma, renal cancer, lung cancer, etc. However, there are many patients who cannot benefit from these two target therapies, and therefore, it is desirable to find new drugs against immune checkpoints, such as Tim-3, to achieve treatment of diseases including tumors by inhibiting immune checkpoints.
Disclosure of Invention
The present invention relates to molecules, such as antibodies or antigen binding fragments thereof, that bind to TIM3 and which are rapidly and strongly internalized on cells expressing TIM3, such as tumor cells, and have a significant therapeutic effect in the treatment of different diseases, such as tumors, especially hematological tumors, like leukemia and lymphoma. Moreover, they show strong immunostimulatory cytokine release in Mixed Lymphocyte Reaction (MLR), and thus have significant application value as immunostimulatory drugs.
In one aspect, the present invention provides a TIM3 binding molecule comprising one or more amino acid sequences selected from the group consisting of: as set forth in SEQ ID NO:6, and the amino acid sequence is shown as SEQ ID NO: 7. the amino acid sequence shown in SEQ ID NO: 8. the amino acid sequence shown in SEQ ID NO: 14. the amino acid sequence shown in SEQ ID NO: 15. the amino acid sequence shown and the sequence shown in SEQ ID NO: 16. the amino acid sequence shown. For one embodiment, a TIM3 binding molecule of the invention comprises a sequence as set forth in SEQ ID NO:5 and/or the amino acid sequence shown as SEQ ID NO:13, and a nucleotide sequence shown in seq id no.
In one aspect, the invention relates to an antibody that binds TIM3, comprising: light chain CDR1 (LCDR 1), comprising SEQ ID NO:6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, light chain CDR2 (LCDR 2), comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, light chain CDR3 (LCDR 3), comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, heavy chain CDR1 (HCDR 1), comprising the amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, heavy chain CDR2 (HCDR 2), comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, and a heavy chain CDR3 (HCDR 3), comprising the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto. In one embodiment, the invention provides an antibody that binds TIM3, comprising an amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, and/or an amino acid sequence as set forth in SEQ ID NO:13 or a heavy chain variable region amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto.
For one embodiment, the molecule that binds TIM3 is a TIM3 antagonistic protein. For one embodiment, the above-described molecule that binds TIM3 is a TIM3 antagonistic antibody or antibody fragment. For one embodiment, the molecule that binds TIM3 is a fusion protein that blocks the TIM3 signaling pathway.
For one embodiment, the above-mentioned molecule that binds TIM3 is an antibody or antigen-binding fragment thereof that binds TIM3, such as a murine, chimeric, human or humanized antibody or antigen-binding fragment thereof. In one non-limiting embodiment, the antibody or antigen-binding fragment thereof that binds TIM3 described above is a monoclonal antibody, scFv, fab fragment, fab 'fragment, F (ab)' fragment, bispecific antibody, immunoconjugate, or a combination thereof. For one non-limiting embodiment, the isolated antibodies or fragments thereof that bind TIM3 specifically recognize endogenous and exogenous TIM3.
For one non-limiting embodiment, the molecules, antibodies, or antigen binding fragments thereof that bind to TIM3 described above have the activity of promoting an immune response, activating immune cells, such as activating T cells.
For one embodiment, the molecule or antibody that binds TIM3 is a monospecific molecule or antibody fragment that binds TIM3. For one embodiment, the above-described molecules or antibodies that bind TIM3 are multispecific antibodies or antibody fragments. In one embodiment, the multispecific antibody described above is a bispecific antibody. In one embodiment, the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen, such as a tumor antigen, e.g. selected from the group consisting of: tumor antigens of CD3, CD20, fcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, step1 and TenB 2.
In one aspect, the invention relates to an immunoconjugate comprising a therapeutic agent, e.g., a cytotoxic agent, linked to a molecule or antibody that binds to TIM3 as described above.
In one aspect, the invention relates to a pharmaceutical composition comprising a molecule, antibody, immunoconjugate as described above that binds TIM3, and a pharmaceutically acceptable carrier.
In one aspect, the invention relates to an article of manufacture comprising a container having the pharmaceutical composition described above contained therein and a package insert, wherein the package insert describes the use of the pharmaceutical composition.
In one embodiment, the above-described article of manufacture further comprises one or more containers containing one or more other medicaments. In one embodiment, the other drug is an immunostimulatory antibody or a chemotherapeutic agent.
In one aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16.
in one aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:5 and/or SEQ ID NO:13.
In one aspect, the invention also relates to the use of these isolated nucleic acid molecules in the preparation of immunotherapeutic-related drugs or cells (e.g., CAR-T cells, TCR-T cells).
The present invention relates to vectors, host cells comprising the above isolated nucleic acid molecules, and their use in the preparation of molecules and antibodies that bind to TIM 3.
In one aspect, the invention relates to a method of promoting an immune response in a subject, comprising contacting an immune cell in said subject with a molecule or antibody that binds TIM3 as described above, thereby promoting an immune response in the subject. In one embodiment, the subject is a tumor-bearing subject or a virus-bearing subject.
In one aspect, the invention relates to a method of inhibiting tumor cell growth in a subject, comprising administering to the subject a molecule or antibody that binds TIM3 as described above.
In one aspect, the invention relates to a method of treating a viral infection in a subject, comprising administering to the subject a molecule or antibody that binds TIM3 as described above. For some embodiments, the above-described molecules or antibodies that bind TIM3 are used in combination with one or more other agents, such as in combination with other antibodies (including anti-PD-1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, and other TIM3 antibodies), anti-cancer agents, or anti-viral agents.
In one aspect, the present invention relates to the use of a TIM 3-binding molecule or antibody as described above for the preparation of a medicament.
In particular, the invention relates to:
an antibody that binds TIM3, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the following:
light chain CDR1 (LCDR 1), amino acid sequence as set forth in SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,
light chain CDR2 (LCDR 2), amino acid sequence as set forth in SEQ ID NO:7, and
light chain CDR3 (LCDR 3), amino acid sequence as set forth in SEQ ID NO: shown as 8;
the heavy chain variable region comprises:
heavy chain CDR1 (HCDR 1), amino acid sequence as set forth in SEQ ID NO: as shown in the drawing 14,
heavy chain CDR2 (HCDR 2), amino acid sequence as set forth in SEQ ID NO:15, and
heavy chain CDR3 (HCDR 3), amino acid sequence as set forth in SEQ ID NO: shown at 16.
The antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
The antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:13 or comprises an amino acid sequence as set forth in SEQ ID NO:13, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
The antibody is a chimeric antibody or a humanized antibody.
The antibody described above, which is an antibody fragment that binds TIM 3.
The antibody is Fab, fab '-SH, scFv or (Fab') 2.
The antibody described above is a full-length antibody.
The antibody is an IgG antibody.
The antibody described above is a monospecific antibody that binds to TIM 3.
The antibody described above is a multispecific antibody.
The antibody of the above, wherein the multispecific antibody is a bispecific antibody.
The antibody described above, wherein the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen.
The antibody, wherein the cell surface antigen is a tumor antigen.
The antibody, wherein the tumor antigen is selected from the group consisting of: CD3, CD20, fcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, step1 and TenB2.
An immunoconjugate comprising a therapeutic agent linked to an antibody as described above.
The immunoconjugate described above, wherein the therapeutic agent is a chemotherapeutic agent.
The immunoconjugate described above, wherein the therapeutic agent is a cytotoxic agent.
An immunologically active polypeptide comprising the light chain variable region and the heavy chain variable region of an antibody as described above.
A pharmaceutical composition comprising an antibody or immunoconjugate or immunologically active polypeptide as described above, and a pharmaceutically acceptable carrier.
An article of manufacture comprising a container containing the pharmaceutical composition described above and a package insert, wherein the package insert describes the use of the pharmaceutical composition.
The article of manufacture described above further comprising one or more containers containing one or more other medicaments.
The above product, wherein the other drug is antibody, hormone drug or chemotherapeutic agent.
An isolated nucleic acid comprising nucleotide sequences encoding the light chain variable region and the heavy chain variable region of the antibody described above.
A vector comprising the isolated nucleic acid described above.
A host cell comprising the vector described above.
The host cell described above, wherein the host cell is a mammalian cell.
A method of producing the above antibody, comprising culturing the above host cell and recovering the above antibody that binds TIM 3.
Drawings
FIGS. 1A-1B show Western blot analysis of anti-human TIM3 mab (clone 3G 11). Wherein, left panel (A) is incubation with TIM3 mab (1:2000), right panel (B) is incubation with anti-human Fc mab, while anti-human GAPDH is used as spotting control. In the figure, A is a cell lysate encoding TIM3 (22-202 aa) -Fc transfected with 293-6E cells; b is 293-6E cells transfected with cell lysates encoding TIM1 (21-290 aa) -Fc; C293-6E cells transfected with cell lysates encoding TIM4 (25-314 aa) -Fc; d is 293-6E cells transfected with cell lysates encoding empty vector plasmids (control).
FIG. 2 shows the results of western blotting examining the expression of TIM3 in the following lysates recognized by anti-human TIM3 mab (3G 11). Wherein a is an activated human T cell (T cell after activation with CD3 mab); b is Jurkat cells; c is 293A cells; d is 293A cells transfected with a plasmid encoding TIM3 (1-205 aa) -GFP, the upper panel was incubated with TIM3 mab (1:2000), the middle panel was incubated with GFP antibody, and the lower panel was incubated with GAPDH antibody as a spotting control.
FIG. 3 is a result of immunostaining detection of IF (immunofluorescence) for identifying TIM3 mab specifically recognizing human exogenously transfected TIM 3. Wherein, the upper row of cells are dyed into HEK293 cells and only transfected with a vector skeleton (empty vector); the next row of cells was stained for HEK293 cells transfected with eukaryotic expression vectors encoding TIM 3.
FIG. 4 shows the results of a flow assay of TIM3 expression using human peripheral blood to determine whether TIM3 mab recognizes endogenously expressed TIM 3. Wherein whole blood of the sample used for the flow results is from donor number D2015, D2015-Isotype as Isotype antibody negative control; D2015-5B4, -6C7, -3G11A (3G 11) is TIM3 mab 5B4, 6C7, 3G11 staining; d2015-BD is TIM3 mab positive control (BD product).
FIGS. 5A-5B are results of an ELISA assay for affinity of TIM3 mab. The upper panel (A) shows the results of TIM3 mab 3G11, and the lower panel (B) shows the results of TIM3 mab 5B 4.
FIG. 6 shows the results of determining the activation of T cell activity by TIM3 mAb 3G11 and 5B4 using a real-time fluorescent quantitative nucleic acid amplification detection system (QPCR) to detect IL-2 and IFNgamma expression in real time. Wherein Ctrl: mIgG (10 ug/ml); TIM3 mAb clones, 10ug/ml each; anti-Tim3, positive control (Biolegend); gal-9, galectin-9, 1ug/ml; ctrl-Gal-control IgG isotype without Galectin-9.
FIG. 7 shows the results of determining the dose-dependent activation of T cells by TIM3 mab 3G11 using QPCR to detect IL-2 and IFNgamma expression in real time. Wherein Ctrl-Gal, control IgG isotype without Galectin-9.
FIG. 8 shows the results of determining the dose-dependent activation of T cells by using QPCR to detect IL-2 and IFNgamma expression in real time. Wherein Ctrl-Gal, control IgG isotype without Galectin-9.
FIG. 9 is a graph showing the results of enhancing the cytotoxic effect of CIK (cytokine induced killer cells, cytokine-induced killer cells) on leukemia cells using TIM3 mab 3G11 and 5B 4. Wherein, E: T refers to the ratio of effector cells to target cells.
FIG. 10 shows the results of the inhibition of CT26 colon cancer cell growth by TIM3 monoclonal antibody alone or in combination with PD1 monoclonal antibody.
Detailed Description
Definition of the definition
The term "antibody" herein is used in a broad sense to encompass a variety of antibody structural molecules comprising one or more CDR domains disclosed herein that bind TIM3, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., fv, fab, fab ', fab ' -SH, F (ab ') 2 ) Linear antibodies, single chain antibody molecules (e.g., scFv), etc., provided that they areExhibit the desired binding activity to TIM 3.
One skilled in the art may fuse one or more CDR domains disclosed herein with one or more other polypeptide sequences to prepare functional fusion proteins or polypeptide molecules that bind to TIM3 molecules, such as vaccines, cell membrane receptor antagonists, signal pathway modulators, and chimeric antigen receptor molecules, among others. For example, one or more CDR domains disclosed herein can be used to prepare TIM3 CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy) molecules. Such fusion protein molecules derived, prepared based on the teachings of the present disclosure are also encompassed within the scope of the present invention.
The modifier "monoclonal" in the term "monoclonal antibody" as used herein means that the antibody is obtained from a substantially homogeneous population of antibodies, containing only minor amounts of naturally occurring mutations or mutations that occur during the preparation of monoclonal antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. Monoclonal antibodies of the invention can be made by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.
The terms "full length antibody", "intact antibody" refer to an antibody having a structure substantially similar to the structure of a native antibody, which terms are used interchangeably herein.
"class" of antibodies refers to the type of constant domain or constant region that the heavy chain possesses. Antibodies are of 5 general classes: igA, igD, igE, igG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, igG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
An "antibody fragment" refers to a portion of an intact antibody that comprises the antigen-binding or variable regions of the intact antibody. Antibody fragments such as Fab, fab'F (ab') and Fv fragments; diabodies (diabodies); single chain antibody molecules, such as single chain Fv (scFv) molecules. Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each having one antigen binding site and a residual "Fc" fragment. Pepsin treatment to produce F (ab') 2 Fragments which have two antigen binding sites and which are still capable of cross-linking the antigen.
The "Fab" fragment contains the constant region of the light chain and the first constant region of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that they have added several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines of the antibody hinge region. Fab '-SH refers to Fab' in which the cysteines of the constant region have free sulfhydryl groups. F (ab') 2 Antibody fragments were initially produced as pairs of Fab 'fragments with hinge cysteines between the pairs of Fab' fragments.
An antibody fragment in which a "single chain Fv" or "scFv" antibody fragment exists as a single polypeptide chain comprises the VH and VL regions of the antibody. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL regions.
"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. In a double-chain Fv, this region consists of a dimer of non-covalently tightly linked light and heavy chain variable regions. In single chain Fv, the heavy and light chain variable regions may be covalently linked by a flexible peptide linker, so that the light and heavy chains can be linked in a structure similar to a "dimer" in a double chain Fv, in which the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer. The 6 CDRs together confer antigen binding specificity to the antibody.
The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is associated with binding of the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 complementarity determining regions (CDR regions). (see, e.g., kindt et al, kuby Immunology, 6 th edition. A single VH or VL domain may be sufficient to confer antigen binding specificity.
The term "hypervariable region" or "HVR" as used herein refers to regions of an antibody variable domain that have sequence hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen contacts"). Generally, an antibody comprises 6 HVRs (CDR regions): 3 are in VH (H1, H2, H3) and 3 are in VL (L1, L2, L3). Exemplary HVRs (CDR regions) herein include:
(a) Hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)) occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3);
(b) HVR (CDR regions) occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991));
(c) Antigen contacts at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al, J. Mol. Biol. 262:732-745 (1996)); and
(d) Combinations of (a), (b) and/or (c) including HVR (CDR region) amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).
Unless otherwise indicated, HVR (CDR region) residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al (supra).
A "chimeric antibody" is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody may comprise a murine variable region and a human constant region.
"humanized" antibody refers to chimeric antibodies comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domain, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.
A "human co-framework" is a framework representing the amino acid residues most commonly found in a selected human immunoglobulin VL or VH framework sequence. Generally, the human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. In general, the subgroup of sequences is as in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, NIH Publication 91-3242, bethesda MD (1991), vol.1-3. In one embodiment, for VL, the subgroup is subgroup κI as described by Kabat et al (supra). In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al (supra).
"human antibodies" may also be referred to as "human antibodies", "fully human antibodies" or "fully human antibodies", as antibodies whose amino acid sequences correspond to amino acid sequences produced by humans or human cells. This human antibody definition specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be prepared using a variety of techniques known in the art, including phage display library techniques, and the like.
A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including hybridoma fusion or ligation of Fab' fragments.
"homology" or "identity" between two amino acid sequences or nucleotide sequences refers to the percentage of identical amino acid residues or nucleotide residues between the two sequences. If the two sequences to be compared differ in length, the sequence "homology" or "identity" preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the amino acid residues or nucleotide residues of the longer sequence. Sequence identity can be routinely determined using sequence analysis software commonly used in the art, such as the Wisconsin sequence analysis package.
"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an inherent binding affinity that reflects a 1:1 interaction between a binding partner member (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative embodiments for measuring binding affinity are described below.
"tumor antigen" refers to an antigen that is newly presented during the course of a cell's cancerous changes and that is overexpressed. For example, the tumor antigen may be a newly produced protein during canceration, a specific degradation product of a protein, a protein with a changed structure, a masked epitope-exposed protein, an abnormal aggregation of various membrane protein molecules or an abnormally high expression embryo antigen or differentiation antigen. Wherein the antigen specific for the tumor cells is referred to as "Tumor Specific Antigen (TSA)"; rather than being specific for a tumor, antigenic molecules that are also present on other tumor cells or normal cells are often referred to as "tumor-associated antigens (TAAs)", e.g., embryonic proteins, glycoprotein antigens, squamous cell antigens.
"antagonism" refers to the phenomenon in which one substance is inhibited or restrained by another substance. For example, a molecule or antibody that binds to TIM3 may inhibit or restrict the growth of a cell (e.g., a tumor cell) that expresses TIM3, e.g., inhibit the growth of the tumor cell.
"fusion protein" refers to a protein molecule made up of different proteins or polypeptides. The different proteins or polypeptides may be chemically linked or may be obtained by recombinant expression of DNA sequences of different origin by genetic recombination techniques. "fusion proteins" prepared by recombinant expression of DNA sequences of different origins by genetic recombination techniques are sometimes also referred to as "chimeric proteins".
An "immunocompetent peptide (polypeptide)" refers to a polypeptide or peptide that has the activity of stimulating an immune response in the body, such as a polypeptide or peptide that has the activity of stimulating lymphocyte proliferation, cytokine secretion, or enhancing killing or phagocytosis of an antigen in the body.
An "immune response" refers to the process by which immune cells, upon exposure to an antigen (e.g., autoantigen, antigenic foreign body, mutant cell or tumor cell), recognize the antigen, self-activate proliferation, differentiation, form effector cells or molecules to cope with the antigen or eliminate the antigen.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes; chemotherapeutic agents or drugs (e.g., methotrexate, aldrich, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; various antineoplastic or anticancer agents known in the art.
An "immunoconjugate" is a conjugate of an antibody with one or more heterologous molecules (including, but not limited to, a cytotoxic agent).
"tumor" refers to a generic term for various malignant or benign tumors. "malignancy" and "cancer" are used interchangeably herein.
A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals, primates, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.
The phrase "therapeutically effective amount" refers to an amount of a drug (e.g., a TIM3 binding molecule or antibody of the invention) that, when administered to a subject, is an amount sufficient to produce a therapeutic effect in the subject. For example, a tumor (cancer) is treated by administering to a subject a TIM3 binding molecule or antibody of the invention, a "therapeutically effective amount" of a TIM3 binding molecule or antibody to the subject reduces tumor cells (cancer cells); shrinking tumor cells (cancer cells); and/or inhibit tumor growth to some extent. For tumor (cancer) treatment, the effect can be determined by measuring the size of the tumor. The therapeutically effective amount can be readily determined by one of ordinary skill in the art following routine methods.
The term "package insert" is used to refer to instructions, typically included in commercial packages of therapeutic products, that contain information about the indication, usage, dosage, administration, combination therapy, contraindications, and/or warnings regarding the use of such therapeutic products.
Antibodies, methods of preparation, compositions and articles of manufacture
1) Antibodies to
The present invention relates to anti-TIM 3 antibodies. In certain embodiments, the invention provides an anti-TIM 3 antibody comprising a binding domain comprising at least 1, 2, 3, 4, 5, or 6 hypervariable regions (HVRs), or so-called Complementarity Determining Regions (CDRs), selected from the group consisting of: (a) HVR-L1 (which may also be referred to as light chain CDR 1) comprising the amino acid sequence shown in SASSSVSSSHLY (SEQ ID NO: 6) or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% homologous to that sequence; (b) HVR-L2 (which may also be referred to as light chain CDR 2) comprising the amino acid sequence shown in GTSNLAS (SEQ ID NO: 7) or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% homologous to that sequence; (c) HVR-L3 (which may also be referred to as light chain CDR 3) comprising the amino acid sequence shown in HQWSSFPLT (SEQ ID NO: 8) or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% homologous to that sequence; (d) HVR-H1 (which may also be referred to as heavy chain CDR 1) comprising the amino acid sequence shown in GFTFTDY (SEQ ID NO: 14) or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; (e) HVR-H2 (which may also be referred to as heavy chain CDR 2) comprising the amino acid sequence shown in RNKANGYT (SEQ ID NO: 15) or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; and (f) HVR-H3 (which may also be referred to as heavy chain CDR 3) comprising the amino acid sequence shown in DLDY (SEQ ID NO: 16) or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% homology thereto. In some cases, an anti-TIM 3 antibody may have a light chain Variable (VL) domain (region) comprising an amino acid sequence having at least 80% sequence homology (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to the amino acid sequence shown in VDIVLTQTPAIMSASPGEKVTLTCSASSSVSSSHLYWYQQKPGSSPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSFPLTFGAGPSHL (SEQ ID NO: 5), and/or a heavy chain Variable (VH) domain (region) comprising an amino acid sequence having at least 80% sequence homology (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to the amino acid sequence shown in LIGAC-AWGFSETLLCTSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTKYSASVKGRFTISRDYSQSILYLQMNTLTAEDSATYFCARDLDYWGQGTFLTVSSKK (SEQ ID NO: 13).
For some embodiments, the anti-TIM 3 antibody comprises the amino acid sequence set forth in SEQ ID NO:5 and SEQ ID NO:13 and a heavy chain variable region amino acid sequence as set forth in seq id no.
2) Antibody fragments
In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, (Fab') 2 Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat. Med.9:129-134 (2003). See, for example, WO 93/16185 for scFv fragments.
A bifunctional antibody is an antibody fragment having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161. Trifunctional and tetrafunctional antibodies are described, for example, in Hudson et al, nat.Med.9:129-134 (2003).
A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (see, e.g., U.S. Pat. No. 6,248,516 B1).
Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant host cell (e.g., E.coli or phage) production.
3) Chimeric and humanized antibodies
In certain embodiments, the antibodies provided herein are chimeric antibodies. Chimeric antibodies can be prepared, for example, as described in U.S. Pat. No. 4,816,567.
In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In general, humanized antibodies comprise one or more variable domains, in which all HVR (CDR) regions, or portions thereof, are derived from a non-human antibody and FR (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be substituted with corresponding residues from a non-human antibody to repair or improve the affinity of the antibody.
Humanized antibodies and methods of making the same are described in U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409.
4) Human antibodies
In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art.
Human antibodies can be prepared by administering an immunogen to a modified transgenic animal and then challenging with an antigen to produce a whole human antibody or a whole antibody having human variable regions. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated in the animal chromosome. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For methods of obtaining human antibodies from transgenic animals, see, e.g., U.S. Pat. nos. 6, 075,181 and 6,150,584 (describing XENOMOUSETM technology); U.S. patent No. 5,770,429; U.S. patent No. 7,041,870 (describing K-M techniques); U.S. application publication No. US 2007/0061900. The human variable regions from the whole antibodies produced by such animals may be further modified, e.g., by combining them with different human constant regions.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybridoma cell lines for the production of human monoclonal antibodies have been described, see, e.g., boerner et al, j.immunol.,147:86 (1991). Human antibodies produced via human B cell hybridoma technology are also described in Li et al, proc.Natl. Acad. Sci. USA,103:3557-3562 (2006). Other methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, xiandai Mianyixue,26 (4): 265-268 (2006) (describing human-human hybridomas).
Human antibodies can also be made by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences may then be combined with the desired human constant domain.
In particular, antibodies of the invention having high affinity may be isolated by screening the combinatorial library for antibodies having activity to bind TIM 3. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such Methods can be referred to, for example, by Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004).
In some phage display methods, VH and VL gene lineages are cloned individually by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, followed by screening for antigen-binding phages. Phages typically present antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Patents describing human antibody phage libraries include, for example: U.S. patent No. 5,750,373 and U.S. patent No. 2005/0079774.
Antibodies or antibody fragments isolated from a human antibody repertoire are considered herein to be human antibodies or human antibody fragments.
5) Multispecific antibodies
In any of the above aspects, the anti-TIM 3 antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. For certain embodiments, one binding specificity is for TIM3 and the other binding specificity is for any other antigen (e.g., a second biomolecule, such as a cell surface antigen, such as a tumor antigen). Accordingly, bispecific anti-TIM 3 antibodies may have binding specificity for TIM3 and tumor antigens, such as CD3, CD20, fcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, step1, or TenB 2. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities, see WO 93/08829, WO2009/08025, and WO 2009/089004A1, among others.
6) Antibody variants
The antibodies of the invention encompass amino acid sequence variants of the anti-TIM 3 antibodies of the invention. For example, antibody variants prepared to further improve the binding affinity and/or other biological properties of the antibodies may be desired. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as binding properties to the TIM3 antigen.
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Substitution-type mutants (including conservative substitution mutants or non-conservative substitution mutants) may be made at one or more sites in the HVR (CDR) region and/or the FR region.
Amino acids can be grouped according to common side chain properties:
(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;
(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;
(3) Acid: asp, glu;
(4) Alkaline: his, lys, arg;
(5) Residues that affect chain orientation: gly, pro;
(6) Aromatic: trp, tyr, phe.
Conservative substitutions are defined as substitutions between the same set of amino acids, non-conservative substitutions are defined as substitutions of an amino acid of one of the different classes with an amino acid of another class. Amino acid substitutions may be introduced into an antibody of the invention and the product screened for the desired activity (e.g., retention/improvement of antigen binding or improvement of ADCC or CDC) to obtain an antibody variant of the invention.
The present invention encompasses antibody variants containing non-conservative mutations and/or conservative mutations obtained from antibodies according to the present disclosure, provided that the variants still have the desired TIM3 binding activity.
One type of substitution variant involves an antibody variant that replaces one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). In general, the resulting variants selected for further investigation will be modified (e.g., improved) with respect to the parent antibody in terms of certain biological properties (e.g., increased affinity) and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR (CDR) residues are mutated and the mutated antibodies are displayed on phage, and the mutated antibodies are screened for a particular biological activity (e.g., binding affinity).
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (CDRs) so long as such changes do not substantially impair the ability of the antibody to bind to TIM 3. For example, conservative changes may be made in the HVR (CDR) without substantially decreasing binding affinity. For example, such changes may occur outside of antigen-contacting residues in the HVR, e.g., conservative or non-conservative amino acid substitutions may occur at 1, 2, 3, 4, 5 amino acid residues in the FR region.
7) Recombination method
The anti-TIM 3 antibodies of the invention may be prepared using recombinant methods, for example, as described in U.S. patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-TIM 3 antibody described herein is provided. Such nucleic acids may encode the VL amino acid sequence and/or the amino acid sequence of VH of the antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed to have): (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp20 cell). In one embodiment, a method of making an anti-TIM 3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of anti-TIM 3 antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).
Host cells suitable for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237. After expression, the antibodies in the soluble fraction may be isolated from the bacterial cytoplasm and may be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with a partially or fully human glycosylation pattern. See Li et al, nat. Biotech.24:210-215 (2006).
Host cells suitable for expressing glycosylated antibodies may also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used to bind insect cells, particularly for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts. For example, U.S. patent No. 6,417,429 describes the plantibeties technique for producing antibodies in transgenic plants.
Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be suitable. Another example of a suitable mammalian host cell strain is the monkey kidney CV1 cell strain transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 cells); baby hamster kidney cells (BHK); mouse sertoli cells (e.g., TM4 cells); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); brutro rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NS0, and Sp2/0.
8) Immunoconjugates
The invention also provides immunoconjugates comprising an anti-TIM 3 antibody herein in combination with one or more cytotoxic agents such as chemotherapeutic agents or agents, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes.
In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody binds to one or more drugs, including but not limited to maytansine, orlistat, dolastatin, methotrexate, vindesine, a taxane, trichothecene (trichiohece), and CC1065.
In another embodiment, the immunoconjugate comprises a conjugate of an anti-TIM 3 antibody as described herein with an enzymatically active toxin, or fragment thereof, including, but not limited to, the diphtheria a chain, non-binding active fragments of diphtheria toxin, the exotoxin a chain, trichothecene, and the like.
In another embodiment, the immunoconjugate comprises a radioactive conjugate formed by the binding of an anti-TIM 3 antibody as described herein to a radioactive atom. A variety of radioisotopes may be used to produce the radio conjugate. Examples include At 211 、 I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 And radioactive isotopes of Lu.
Conjugates of antibodies and cytotoxic agents may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate hydrochloride), active esters (such as suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-nitrogen derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene).
9) Pharmaceutical preparation
The pharmaceutical formulations of the anti-TIM 3 antibodies of the invention are prepared by mixing the antibody of the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexa hydrocarbon quaternary ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt forming counter ions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).
Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908.
The formulations herein may also contain more than one active ingredient which is necessary for the particular indication being treated, preferably active ingredients having complementary activities which do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and/or anti-hormonal agents). Such active ingredients are suitably present in combination in an amount effective for the intended purpose.
10 Article of manufacture)
In another aspect of the invention, there is provided an article of manufacture comprising an antibody or pharmaceutical composition of the invention. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Such containers may be formed from a variety of materials, such as glass or plastic. The container contains the composition of the invention itself or a combination of the composition and another composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is to be used to treat a selected tumor. In addition, the article of manufacture may comprise (a) a first container comprising a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises another tumor therapeutic agent or another antibody. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that such compositions are useful for treating tumors. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
The present invention will be described in detail below.
The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced in view of the general description provided above.
Example 1: preparation of anti-TIM 3 monoclonal antibodies
Immunization of mice
1. Animals: balb/c mice each group had 3 animals and had a weight of 20 g/animal. A total of 2 groups of mice were immunized.
2. Immunogens: the nucleotide sequence encoding the extracellular region of TIM3 (amino acids 1-201 or amino acids 31-180) was inserted into the vector pcDNA3.1 and PET-32a (Biovector, cat# 3683689) by PCR, respectively, to prepare the plasmids pcDNA3.1-TIM3 (1-201 aa) and the recombinant protein of the extracellular region of TIM3 (amino acids 31-180).
3. Hyperimmunization (SuperImmune ™): a0.6. Mu.g/. Mu.l PBS solution was prepared from the plasmid pcDNA3.1-TIM3 (1-201 aa) and the mice were immunized according to the hyperimmune technique (Supper Immune ™) described in the literature (Immune adherence and the processing of soluble complement-fixing anti-body/DNA immune complexes in mice JC Edberg, L Tosic, RP Taylor-Clinical immunology and immunopathology, 1989-Elsevier). Once every two weeks, 20-30 μl of blood was collected from the tail of the mice after 3 times of immunization, and serum was separated by centrifugation for ELISA to detect the titer of the antibodies after immunization. If the antibody titer is higher than 1:10000, the fusion is performed after one boost. If this titer is not achieved, immunization needs to be continued until the titer is higher than 1:10000. Immunization process: the first group was immunized 5 times with pcDNA3.1-TIM3 (1-201 aa) and the second group was immunized 3 times with pcDNA3.1-TIM3 (1-201 aa) and then boosted 1-2 times with the above-mentioned recombinant protein of the extracellular region of TIM 3.
(II) fusion screening
Spleens of the immunized mice were taken and prepared into single suspension cells for fusion with SP2/0 cells, followed by clonal selection.
Spleen and myeloma cell fusion:
(1) The SP2/0 cells in the logarithmic growth phase were discarded from the culture supernatant, the cells were collected by adding D-Hank's solution, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 45ml of the D-Hank's solution was added to prepare a cell suspension.
(2) The spleens of the immunized mice were prepared into single suspension cells, and the spleen cells were added to the above SP2/0 suspension (SP 2/0: spleen cells=1:5-10) and thoroughly mixed, and centrifuged at 1000rpm for 5min.
(3) The supernatant was discarded and the bottom of the tube was flicked to disperse the cells.
(4) 1ml PEG4000 was added to the mixed cells of step "3", slowly and dropwise added to the centrifuge tube and the tube was slowly rotated, slightly more rapidly (PEG 4000 had to be added over 1 minute). Standing for 1.5min, and finally immediately adding D-Hank's solution to 45ml. The operation of this step (4) was completed within 3 minutes.
(5) Centrifuging at 1000rpm for 5min, rapidly reversing the centrifuge tube, removing supernatant, blowing off the cell mass with a suction tube, adding D-Hank's solution to 45ml, mixing, and centrifuging at 1000rpm for 5min.
(6) The supernatant was discarded, and the cells were blown off with a pipette, and further, HAT1640 medium containing 10% calf serum was added to the mixture and dispersed in a previously prepared 96-well plate at 100. Mu.l/well.
(7) HT1640 medium was changed starting 7-8 days after cell fusion.
8 96-well plates were fused each time, for a total of 5 fusions per group of immunized mice. After each fusion, positive clones were subcloned 3 more times in succession.
Screening: clones were screened using stable transfection of CHO cells expressing TIM3 protein, using CHO cells as negative control. Clones so screened ensure identification of eukaryotic expressed TIM3.
The whole screening gives a total of 47 clones, and the 10 clones with the highest titer among the 47 clones are selected for further biochemical and immunological identification.
Example 2: identification of anti-TIM 3 monoclonal antibodies
1. The 10 clones with the highest titers were selected and subjected to a series of biochemical and immunological assays, the results of which are shown in Table 1 below (+positive; negative).
TABLE 1
Of the 10 selected clones described above, 3G11 and 5B4 clones were selected for further study as follows.
2. Specificity for human TIM3 recognition
Western immunoblotting detection procedure:
A. sample lysate preparation
(1) Collecting cells growing to about 80% and having good conditions, discarding the culture medium, adding sample storage solution, mixing well, and collecting cells;
(2) Ultrasonically crushing cells for 8 minutes until the sample is not sticky;
(3) And (3) carrying out dry heating on the sample EP tube at 100 ℃ for 10min, cooling, centrifuging at 12000 rpm for 10min, sucking 60 mu L of supernatant, subpackaging and preserving to-20 ℃ to avoid repeated freezing and thawing.
(4) Protein denaturation: placing the lysed cells into a dry heater, heating and preserving heat at 100deg.C for 5-10 min.
(5) Collecting the lysate: the cooked protein was centrifuged at 12000 rpm at 4℃for 10 min. The supernatant was pipetted into small EP tubes, 60. Mu.L per tube, and stored at-20deg.C, avoiding repeated freeze thawing.
B. Loading: thawing the treated sample, centrifuging for 2min, mixing, and collecting supernatant.
C. Electrophoresis: the voltage is 200V, and the electrophoresis is carried out for about 50 minutes.
D. Transferring: voltage 60v,1-2 h.
E. Closing: on a shaker, 1 h was slowly shaken at 70rpm at room temperature.
F. TIM3 antibody to be detected: slowly shaking at 70rpm on a shaker, and standing at room temperature for 1-2h or at 4deg.C overnight; PBST was rinsed 3 times.
G. Goat anti-mouse secondary antibody: slowly shaking the table at room temperature for 1 h; PBST was rinsed 3 times.
H. And (5) developing.
FIG. 1 shows the result of Western blot analysis of anti-human TIM3 mab (clone 3G 11). Wherein, left panel (A) is incubation with TIM3 mab (1:2000), right panel (B) is incubation with anti-human Fc mab, while anti-human GAPDH is used as spotting control. In the figure, A is a cell lysate encoding TIM3 (22-202 aa) -Fc transfected with 293-6E cells; b is 293-6E cells transfected with cell lysates encoding TIM1 (21-290 aa) -Fc; C293-6E cells transfected with cell lysates encoding TIM4 (25-314 aa) -Fc; d is 293-6E cells transfected with cell lysates encoding empty vector plasmids (control). As can be seen from the Western blot analysis results of FIG. 1, anti-human TIM3 mab (clone 3G 11) specifically recognizes only human TIM3, but not human TIM1 and human TIM4, its cognate family members.
3. TIM3 mab recognizes human endogenous and exogenous TIM3
Detection of TIM3 at a by western blot detection anti-TIM 3 mab (3G 11) with the same procedure as described above: activated T cells; b: jurkat cells; c:293A cells; d: expression in cell lysates of 293A cells transfected with plasmids encoding TIM3 (1-205 aa) -GFP. As shown in FIG. 2, the upper panel was incubated with TIM3 mab (1:2000), the middle panel was incubated with GFP antibody, and the lower panel was incubated with GAPDH antibody as a spotted control, which indicated that TIM3 mab was able to recognize both human endogenous and exogenous TIM3.
4. TIM3 mab specific recognition of human exogenously transfected TIM3
Immunostaining detection: A. fixing: the cell climbing sheet is lightly washed by PBS, 4% paraformaldehyde is fixed for 15-20 min at room temperature, and the cell climbing sheet is washed twice by PBS. And B, sealing: 400 μl of blocking buffer/well was blocked at room temperature for 45min. C, TIM3 mab: standing at room temperature for 1h or overnight in a refrigerator at 4 ℃. D, goat anti-mouse secondary antibody: incubate for 1h in the dark. E. 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) staining: discarding the secondary antibody, washing with a flushing buffer solution, sucking out residual liquid, adding DAPI working solution, and standing at room temperature in a dark place for 2-5min. F, sealing piece observation: DAPI was discarded, the residual liquid was dried by washing with PBS and double distilled water for 5min, and the slide was inversely fastened to the slide glass with anti-quenching sealing tablet, sealed, and observed.
The results are shown in FIG. 3. TIM3 mab (3G 11) was used 1: 1000. diluted, incubated for 1 hour at room temperature, incubated with Alexa 568 labeled secondary antibody for 1 hour, and photographed under a condocol microscope. In FIG. 3, the upper row of cells stained HEK293 cells transfected only the vector backbone (empty vector); the next row of cells was stained for HEK293 cells transfected with eukaryotic expression vectors encoding TIM3. TIM3 mab (3G 11) was used 1: diluted 1000, incubated for 1 hour at room temperature, incubated with Alexa 568 labeled secondary antibody for 1 hour, and photographed under a condocol microscope. DAPI staining is staining of nuclei. The results indicate that TIM3 mab specifically recognizes human exogenously transfected TIM3.
Example 3: monoclonal antibody recognition endogenous TIM3 detection
Human peripheral blood was used for flow detection of TIM3 expression to determine whether TIM3 mab recognizes endogenously expressed TIM3. The experimental procedure was as follows: 1) PBMC were prepared from 5-10 mL whole blood cells. 2) If necessaryErythrocytes can be lysed by addition of erythrocyte lysis buffer. 3) PBMC cells were slowly transferred to 15ml sterile tubes (labelling). 4) Slowly add 5ml of wash buffer (room temperature). 5) Centrifuge 500g for 5 min. 6) The supernatant was discarded. 7) The cells were resuspended and then 6-10 ml of wash buffer was added. 8) Gently mix cells and then aspirate 2mL into another 3-5 15mL tubes (each tube contains at least 1x10 6 Individual cells) and then labeled, and centrifuged at 500g for 5 minutes at room temperature. 9) The supernatant was decanted using a pipette to remove the remaining supernatant on the open end of the tube (100. Mu.l remaining in the tube), and 11. Mu.l of 1:10 diluted mouse anti-human TIM3 mAb, or 11. Mu.l of 1:10 diluted mouse anti-GFP mAb was added to each tube as a control. Incubate for 60 minutes at room temperature. 10 2-3mL of wash buffer was added to each tube and centrifuged at 500g for 5 min at room temperature. 11 Decanting the supernatant using a pipette to remove the remaining supernatant at the open end of the tube (100 μl remaining in the tube), and adding 11 μl of 1:40 diluted goat anti-mouse IgG (h+l) secondary antibody Alexa Fluor 568 (Life Technology lot number) to each tube: 1793903 Incubation in the dark for 30 minutes at room temperature. 12 2mL of wash buffer was added to each tube and centrifuged at 500g for 5 min at room temperature. 13 Pouring the supernatant and adding 200. Mu.L of 1% paraformaldehyde in PBS. Vortex to resuspend pellet and store in the dark at 4 ℃ prior to flow cytometry analysis. Analysis was performed within 24 hours.
The flow results indicate that: various TIM3 mab clones (3G 11, 5B4 and 6C 7) recognize TIM3 endogenously expressed by human monocytes, and the results are shown in fig. 4.
Example 4: affinity assay for TIM3 mab
The coating protein was 1.0ug/ml of the recombinant protein of the extracellular region of TIM3 prepared as described above. ELISA assays were performed with gradient diluted TIM3 mab. EC50 values were calculated. TIM3 mab 3G11 and 5B4 have good affinity, and the EC50 values measured were 0.733, nM and 0.82, nM, respectively. The results are shown in FIG. 5.
Example 5: functional assay of TIM3 mab
1. Blocking TIM3 mab activation of T cell activity
Normal human PBMC were cultured for 3 days in RPMI supplemented with 10% fetal bovine serum, 50U/ml recombinant IL2 (Cat#68-8779-82, thermoFisher, USA) and anti-CD 3 antibodies (Cat# 317301, OKT, bioLegend, san Diego, USA) to up-regulate Tim3 expression. anti-CD 3 stimulated PBMC were incubated overnight at 37 degrees C with either 10. Mu.g/ml anti-Tim 3 monoclonal antibodies 3G11 and 5B4 or IgG isotype control or 10. Mu.g/ml anti-Tim 3 positive control (Cat#345001 (F38-2E 2), bioLegend, san Diego, USA) to block the interaction of Tim3 with Gal-9. The following day was incubated with Galectin-9 (Gal-9, tim3 ligand) (Cat# 754802, bioLegend, san Diego, USA) (1 ug/ml) for 2 hours and IL2 and IFNgamma mRNA was assayed by QPCR to detect IL-2 and IFNgamma expression in real time. Ctrl is control, galectin 9 was not added, and Ctrl+Gal is control, galectin 9 was added.
The experiment adopts the Real-time expression (QPCR, real-time Quantitative PCR Detecting System, i.e. a Real-time fluorescence quantitative nucleic acid amplification detection system) of IL-2 and IFNg to judge the biological effect of the TIM3 monoclonal antibody. Experimental results show that TIM3 monoclonal antibodies 3G11 and 5B4 have the functions of blocking the combination of TIM3 receptor and Galectin 9 and activating T cells. The results are shown in FIG. 6.
2. TIM3 mab exhibits a dose-dependent effect on activating T cells
Three different doses of TIM3 mab (2, 10, 50 ug/ml) were used for the experiment, and the experimental procedure was as above. Experimental results show that TIM3 monoclonal antibodies 3G11 and 5B4 block the combination of TIM3 and ligand Galectin 9 in a dose-dependent manner, and activate T cell functions. The results are shown in fig. 7 and 8, respectively.
3. Blocking TIM3 mab enhances the cytotoxic effect of CIK on leukemia cells
Human peripheral blood T cells were cultured in vitro to obtain CIK (cytokine induced killer cells, cytokine-induced killer cells), and the CIK cells activated by this culture were used as Effector cells (effect) and leukemia cells U937 (Target) were cultured in a mixed manner in vitro, and the killing effect of the Effector cells on the Target cells was examined with the addition of TIM 3. The percentage of specifically lysed target cells was judged by detecting the release of lactate dehydrogenase LDH. The results are shown in FIG. 9. T refers to the ratio of effector cells to target cells. Experimental results show that TIM3 monoclonal antibodies 3G11 and 5B4 have the effect of enhancing the killing effect of CIK on tumor cells.
Example 6: humanized expression and detection of TIM3 mab
TIM3 mab 3G11 clones were sequenced using Sanger dideoxy termination sequencing, with the sequencing results shown in table 2 below.
TABLE 2
Humanized TIM3 mab 3G11 was prepared by embedding the six CDR regions of the 3G11 monoclonal antibody heavy and light chains into human IgG1 and LC kappa constant backbones. Specifically, a humanized chimeric antibody VL light chain plasmid pTT-LC (EcoR 1-Leader-Sal 1-VL-BsiW 1-LC kappa) and a VH heavy chain plasmid pTT-HC (EcoR 1-Leader-Sal 1-VH-Nhe 1-CH1-CH2-CH 3) were constructed, the plasmids were transfected in HEK293-6E in proportion, 100. Mu.l of the supernatant was taken after the culture and tested, and the ELISA results were shown in Table 3 below:
TABLE 3 Table 3
The above results show that TIM3 mab (3G 11) expressed after humanization (chimera, or semi-humanization) can specifically recognize TIM3 recombinant proteins as before humanization.
Example 7: in vivo experiments of inhibition of tumor growth by TIM3 mab
To test the ability of TIM3 (3G 11) monoclonal antibodies to inhibit tumor growth in vivo, tumor transplantation models seeded with human CT26 colon cancer cells on NSG mice were used. mu.L of RPMI1640 medium (containing 2X 10) 6 Individual CT26 colon cancer cells) were mixed with 50 μl of basement membrane matrigel, and each NSG mouse was subcutaneously injected on day 0. On days 6, 9, 12 and 15, mice were intraperitoneally injected with 100. Mu.g of different antibodies, respectively TIM3 monoclonal antibody, PD1 monoclonal antibody (nivolumab (Opdivo, bristol-Myers Squibb GmbH) &Co), TIM3 monoclonal antibody+PD 1 monoclonal antibody (TIM 3 monoclonal antibody and PD1 monoclonal antibody were Co-administered), control antibody (mouse IgG, sigma, # I5381). During the experiment, the size of the tumor in the mice was determined, wherein the tumor volume was calculated using the following formula: tumor volume = 1/2 x tumor length x tumor width 2 。
The results of inhibition of tumor growth in mice by the different antibodies are shown in fig. 10, which shows that TIM3 antibody alone inhibited CT26 colon cancer growth very significantly compared to the control group (mouse IgG). It is worth emphasizing that TIM3 antibodies are more pronounced than PD1 antibodies alone in anticancer effect. TIM3 in combination with PD1 antibody treatment plays a synergistic anticancer role, further significantly inhibiting tumor growth and consistently reducing tumor burden, compared to TIM3 or PD1 antibody alone.
Although the embodiments of the present application have been described above with reference to the accompanying drawings, the present application is not limited to the above-described specific embodiments and application fields, and the above-described specific embodiments are merely illustrative and instructive examples, and do not constitute a limitation on the scope of the claims of the present application. Many substitutions and changes may be made by one of ordinary skill in the art in light of the present disclosure without departing from the scope of the application as set forth in the appended claims.
Sequence listing
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Claims (26)

1. An antibody that binds TIM3, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the following:
light chain CDR1 (LCDR 1), amino acid sequence as set forth in SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,
light chain CDR2 (LCDR 2), amino acid sequence as set forth in SEQ ID NO:7, and
light chain CDR3 (LCDR 3), amino acid sequence as set forth in SEQ ID NO: shown as 8;
the heavy chain variable region comprises:
heavy chain CDR1 (HCDR 1), amino acid sequence as set forth in SEQ ID NO: as shown in the drawing 14,
heavy chain CDR2 (HCDR 2), amino acid sequence as set forth in SEQ ID NO:15, and
heavy chain CDR3 (HCDR 3), amino acid sequence as set forth in SEQ ID NO: shown at 16.
2. The antibody of claim 1, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
3. The antibody of claim 1, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:13 or comprises an amino acid sequence as set forth in SEQ ID NO:13, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
4. The antibody of any one of claims 1-3, which is a chimeric or humanized antibody.
5. The antibody of claim 3, which is an antibody fragment that binds TIM 3.
6. The antibody of claim 5, which is Fab, fab '-SH, scFv or (Fab') 2 。
7. The antibody of any one of claims 1-3, which is a full length antibody.
8. The antibody of claim 7, which is an IgG antibody.
9. The antibody of claim 8, which is a monospecific antibody that binds TIM 3.
10. The antibody of claim 8, which is a multispecific antibody.
11. The antibody of claim 10, wherein the multispecific antibody is a bispecific antibody.
12. The antibody of claim 11, wherein the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen.
13. The antibody of claim 12, wherein the cell surface antigen is a tumor antigen.
14. An immunoconjugate comprising a therapeutic agent linked to the antibody of any one of claims 4-13.
15. The immunoconjugate of claim 14, wherein said therapeutic agent is a chemotherapeutic drug.
16. The immunoconjugate of claim 15, wherein said therapeutic agent is a cytotoxic agent.
17. An immunologically active polypeptide comprising the light chain variable region and the heavy chain variable region of the antibody of any one of claims 4-11.
18. A pharmaceutical composition comprising the antibody of any one of claims 1-13 or the immunoconjugate of any one of claims 14-16, or the immunologically active polypeptide of claim 17, and a pharmaceutically acceptable carrier.
19. An article of manufacture comprising a container containing the pharmaceutical composition of claim 18 and a package insert, wherein the package insert describes the use of the pharmaceutical composition.
20. The article of manufacture of claim 19, further comprising one or more containers containing one or more other medications.
21. The article of manufacture of claim 20, wherein the additional agent is an antibody, a hormonal agent, or a chemotherapeutic agent.
22. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain variable region and the heavy chain variable region of the antibody of any one of claims 1-8.
23. A vector comprising the isolated nucleic acid of claim 22.
24. A host cell comprising the vector of claim 23.
25. The host cell of claim 24, wherein the host cell is a mammalian cell.
26. A method of making the antibody of any one of claims 1-8, comprising culturing the host cell of claim 25 and recovering the antibody that binds TIM 3.
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