CN115960242A

CN115960242A - Anti-cancer binding molecules and uses thereof

Info

Publication number: CN115960242A
Application number: CN202211066932.7A
Authority: CN
Inventors: 孟祥雪; 史继亚; 介淼星; 杨敏; 王雅秋; 任志衡; 陈立模; 李文佳
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Sunshine Lake Pharma Co Ltd
Priority date: 2021-09-09
Filing date: 2022-09-01
Publication date: 2023-04-14
Anticipated expiration: 2042-09-01
Also published as: WO2023036043A1; CN115960242B

Abstract

Disclosed herein are bispecific binding molecules for 4-1BB and GPC3, comprising a first domain that specifically binds 4-1BB, and a second domain that specifically binds GPC 3. Also disclosed herein are uses of the bispecific binding molecules to treat or prevent cancer.

Description

Anti-cancer binding molecules and uses thereof

Technical Field

The disclosure belongs to the field of biotechnology, and particularly relates to a binding molecule specifically binding 4-1BB and glypican 3 (GPC 3) and application thereof.

Background

4-1BB (also known as CD137, TNFRSF9, etc.) is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRS). The antibody aiming at the 4-1BB has the capacity of activating a 4-1BB signal path, and has potential medical values on tumor treatment, infection resistance, autoimmune disease resistance and the like. Glypican 3 (GPC 3) is a carcinoembryonic antigen belonging to the glypican family, and is highly expressed in various cancer cells, particularly hepatocellular carcinoma (HCC), melanoma, wilms' tumor, and hepatoblastoma. Therefore, there is a clinical need for novel protein drugs based on 4-1 BB-activating antibodies, which can target tumor cells and activate the 4-1BB pathway to treat tumors.

Disclosure of Invention

In order to solve one of the above technical problems in the prior art, the present disclosure provides a novel method for targeting a tumor associated antigen GPC3 (glypican 3) and simultaneously activating 4-1BB signaling pathway-related immune pathways, which can effectively solve the side effects of 4-1BB activating antibodies.

In one aspect of the present disclosure, there is provided a binding molecule comprising: a first domain that specifically binds 4-1BB or a fragment thereof, and a second domain that specifically binds glypican 3 (GPC 3) or a fragment thereof.

According to some embodiments of the disclosure, the binding molecule further comprises a third domain comprising an Fc fragment of an immunoglobulin.

According to some embodiments of the disclosure, the immunoglobulin is selected from IgA, igG, igM, igD, and IgE. In a particular embodiment of the present disclosure, the immunoglobulin is an IgG, e.g., an IgG1, an IgG2, an IgG3 or an IgG4.

According to some embodiments of the disclosure, the Fc fragment has one or more mutations in L234F, L235E, P331S, D356E and L358M, wherein the Fc fragment is numbered according to the EU index of Kabat.

In a specific embodiment of the present disclosure, the third domain has an amino acid sequence as set forth in SEQ ID NO 55, 62 or 69 or a variant thereof.

In some embodiments of the disclosure, the binding molecule may comprise a heavy chain constant region CH linked to the N-terminus of the Fc fragment ₁ 。

According to some embodiments of the disclosure, the first domain comprises a first heavy chain variable region VH ₁ And a first light chain variable region VL ₁ . According to some embodiments of the disclosure, the structure of the first domain is selected from Fab, fab ', F (ab') ₂ Fv or scFv. For the first domain to have Fv or scFv structure, the VH ₁ C-terminal of peptide chain and said VL ₁ The N-terminus of the peptide chain is linked directly or via a linker and vice versa.

In some embodiments of the disclosure, the VH is ₁ The method comprises the following steps: (a) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences shown as SEQ ID NO. 1, 2 and 3, respectively; or, (b) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences shown as SEQ ID NOs: 7, 8 and 9, respectively. In some embodiments of the disclosure, the VL is ₁ The method comprises the following steps: (c) Comprises thatLCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 4, 5 and 6, respectively; or, (d) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences shown as SEQ ID NOS: 10, 11 and 12, respectively.

According to some embodiments of the disclosure, the first domain comprises: an HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, an HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 2, an HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 3, an LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 4, an LCDR2 comprising the amino acid sequence shown as SEQ ID NO. 5, and an LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 6.

According to some embodiments of the disclosure, the first domain comprises: HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 7, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 8, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 9, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 10, LCDR2 comprising the amino acid sequence shown as SEQ ID NO. 11, and LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 12.

According to some embodiments of the disclosure, the first domain of the binding molecule of the disclosure comprises VH ₁ And VL ₁ . Said VH ₁ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO 31 or 33. The VL ₁ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO 32 or 34.

In particular embodiments of the present disclosure, the present disclosure binds to VH of a molecule ₁ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 31, and VL ₁ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO. 32.

In particular embodiments of the present disclosure, the present disclosure binds to VH of the molecule ₁ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO. 33, and VL ₁ Comprises and is asThe amino acid sequence shown as SEQ ID NO. 34 has at least 90% of sequence identity.

According to some embodiments of the disclosure, the second domain of the binding molecule of the disclosure comprises a second heavy chain variable region VH ₂ And a second light chain variable region VL ₂ . The second domain has a structure selected from the group consisting of Fab, fab ', F (ab') ₂ Fv or scFv. For the first domain to have Fv or scFv structure, the VH ₂ C-terminal of peptide chain and the VL ₂ The N-terminus of the peptide chain is linked directly or via a linker and vice versa.

In some embodiments of the disclosure, the VH is ₂ The method comprises the following steps: (e) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences as set forth in SEQ ID NO 13, 14 and 15, respectively; (f) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences set forth as SEQ ID NOS 19, 20 and 21, respectively; or, (g) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences shown as SEQ ID NOS: 25, 26, and 27, respectively. In some embodiments of the disclosure, the VL ₂ The method comprises the following steps: (h) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences shown as SEQ ID NO 16, 17 and 18, respectively; (i) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences set forth as SEQ ID NOs 22, 23 and 24, respectively; or, (j) an LCDR1, LCDR2, and LCDR3 comprising amino acid sequences shown as SEQ ID NOS: 28, 29, and 30, respectively.

According to some embodiments of the disclosure, the second domain comprises: HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 13, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 14, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 15, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 16, LCDR2 comprising the amino acid sequence shown as SEQ ID NO. 17, and LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 18.

According to some embodiments of the disclosure, the second domain comprises: an HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 19, an HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 20, an HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 21, an LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 22, an LCDR2 comprising the amino acid sequence shown as SEQ ID NO. 23, and an LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 24.

According to some embodiments of the disclosure, the second domain comprises: HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 25, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 26, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 27, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 28, LCDR2 comprising the amino acid sequence shown as SEQ ID NO. 29, and LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 30.

According to some embodiments of the disclosure, the second domain of the binding molecule of the disclosure comprises VH ₂ And VL ₂ . Said VH ₂ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO35, 37 or 39. The VL ₂ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO 36, 38 or 40.

In particular embodiments of the present disclosure, the present disclosure binds to VH of a molecule ₂ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 35, and VL ₂ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36.

In particular embodiments of the present disclosure, the present disclosure binds to VH of a molecule ₂ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 37, and VL ₂ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO 38.

In particular embodiments of the present disclosure, the present disclosure binds to VH of the molecule ₂ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO:39, and VL ₂ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 40.

According to some embodiments of the disclosure, in the binding molecules of the disclosure, the first domain, the second domain and/or the third domain are linked directly or via a linker. According to some embodiments of the present disclosure, the first heavy chain variable region and the first light chain variable region of the first domain may be directly linked or linked through a linker. According to some embodiments of the disclosure, the second heavy chain variable region and the second light chain variable region of the second domain are directly linked or linked through a linker.

In some embodiments of the disclosure, the linker used in the binding molecules of the disclosure has a structure as (G) _n S) _z Wherein n and z are each independently an integer from 1 to 4. For example, the linker sequence used in the present disclosure may be an amino acid sequence shown by GS, GSGS (SEQ ID NO: 57), GGGGS (SEQ ID NO: 58), GGSGS (SEQ ID NO: 59), GGGGSGGGGSGGGGGS (SEQ ID NO: 60), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61).

In some embodiments of the present disclosure, a binding molecule of the present disclosure comprises a first domain, a second domain, and a third domain connected in the following order: first domain-third domain-second domain; second domain-third domain-first domain; first domain-second domain-third domain; or, second domain-first domain-third domain.

According to some embodiments of the present disclosure, a binding molecule of the present disclosure may comprise two identical peptide chains that form a tetravalent homodimer. In some embodiments, one peptide chain of a binding molecule of the present disclosure has an amino acid sequence with at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO 41, 42, or 43.

In some embodiments, the binding molecules of the present disclosure may comprise a heterotetramer of a long peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 44 and a short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 45.

In some embodiments, the binding molecules of the present disclosure include heterotetramers that can include long peptide chains having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 46 and short peptide chains having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 47.

In some embodiments, the binding molecules of the present disclosure may comprise a heterotetramer formed by a long peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 48 and a short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 49.

In some embodiments, the binding molecules of the present disclosure may comprise a heterotetramer formed by a long peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 50 and a short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 51.

In some embodiments, the binding molecules of the present disclosure may comprise a heterotetramer of a long peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 64 and a short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 51.

In some embodiments, the binding molecules of the present disclosure may comprise a long peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 52, a first short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 54, and a second short peptide chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 53.

In some embodiments of the disclosure, a binding molecule of the disclosure may further comprise a heavy chain constant region, e.g., CH ₁ 、CH ₂ 、CH ₃ And/or CH ₄ . In some of the present disclosureIn embodiments, the binding molecules of the present disclosure may further comprise a light chain constant region CL.

The above functional fragments (e.g., VH) can be adjusted by one skilled in the art according to conventional techniques ₁ 、VL ₁ 、Fc、VH ₂ 、VL ₁ Etc.) while maintaining their respective binding characteristics.

In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding a binding molecule or fragment thereof described in the present disclosure.

In yet another aspect, the present disclosure provides a vector comprising a nucleic acid molecule of the present disclosure. According to some embodiments of the disclosure, the vector is an expression vector that can be transcribed and translated in a host cell to express the binding molecules of the disclosure or fragments thereof.

In yet another aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or vector of the present disclosure.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a binding molecule of the present disclosure, a nucleic acid molecule of the present disclosure, a vector of the present disclosure, or a host cell of the present disclosure and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure provides a method of treating or preventing cancer comprising administering to a subject in need thereof a pharmaceutical composition of the present disclosure.

In a further aspect, the present disclosure provides the use of a binding molecule of the present disclosure in the manufacture of a medicament for the treatment or prevention of cancer.

In some embodiments of the present disclosure, the cancer is selected from the group consisting of melanoma, glioma, renal cancer, breast cancer, liver cancer, wilms' tumor, hepatoblastoma, hematological cancer, and head and neck cancer.

In a further aspect, the present disclosure provides a binding molecule of the present disclosure, a nucleic acid molecule of the present disclosure, a vector of the present disclosure, a host cell of the present disclosure, or a pharmaceutical composition of the present disclosure for use in the preparation of a medicament for treating or preventing cancer.

The disclosure constructs 4-1BB activating antibody as Fab, scFv or single chain antibody, forms a binding domain of the bispecific antibody by fusion of linker sequence composed of flexible amino acids or inherent linking amino acids of the antibody, and constructs GPC3 specific antibody as Fab, scFv or single chain antibody, forms another binding domain of the bispecific antibody by fusion of linker composed of flexible amino acids or inherent linking amino acids of the antibody. The bispecific binding molecules of the present disclosure directed to 4-1BB and GPC3 are capable of generating 4-1BB signaling pathway-related T cell activation effects while targeted binding to GPC 3-positive tumor cells. In addition, the bispecific binding molecules of the present disclosure show excellent tumor growth inhibition effects in mouse tumor models, and the combination of binding molecules against 4-1BB and GPC3 has some synergistic effect in inhibiting tumor growth.

The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

Drawings

In order that the disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 shows binding of the binding molecules of the present disclosure to a CHO cell line stably transfected with human 4-1BB. (A) Binding of the binding molecules of the present disclosure to a CHO cell line highly expressing human 4-1 BB; (B) Binding of the binding molecules of the present disclosure to CHO cell lines highly expressing human 4-1BB and the corresponding EC50 values. MFI represents the mean fluorescence intensity.

FIG. 2 shows the binding of the binding molecules of the present disclosure to recombinant human 4-1BB protein. (A) Binding of the binding molecules of the present disclosure to recombinant human 4-1BB protein; (B) The binding molecules of the present disclosure bind to recombinant human 4-1BB protein and the corresponding EC50 values.

Figure 3 shows binding of the binding molecules of the present disclosure to activated T cells.

Figure 4 shows binding of the binding molecules of the present disclosure to recombinant human GPC3 protein ELISA. (A) Binding of the binding molecules of the present disclosure to recombinant human GPC3 protein; (B) Binding of the binding molecules of the present disclosure to recombinant human GPC3 protein and the corresponding EC50 values.

Figure 5 shows binding of the binding molecules to human Huh7 and HepG2 hepatoma cell lines. (A) Binding of the binding molecules of the present disclosure to human Huh7 hepatoma cell lines; (B) The binding of the binding molecules of the present disclosure to human HepG2 hepatoma cell lines and the corresponding EC50 values.

Figure 6 shows that binding molecules of the present disclosure activate T cells in the presence of GPC 3-positive liver cancer cells. (A) And (B) shows that the binding molecules of the present disclosure stimulate T cells to secrete interleukins IL2 and IFN γ, respectively, in the presence of GPC 3-positive hepatoma cells Huh 7. (C) (F) shows that the binding molecules of the present disclosure stimulate IL2 secretion from T cells and IFN γ detection in the presence of GPC3 positive hepatoma cells HepG2, respectively. (G) Binding molecules of the present disclosure are shown to stimulate T cells to secrete IFN γ in the presence of GPC 3-positive hepatoma cells Hep 3B.

Figure 7 shows that binding molecules of the present disclosure inhibit the growth of GPC 3-positive tumors. FIG. 7A shows the results of inhibition of GPC3 positive tumor growth by a 5mg/kg dose of binding molecule; FIG. 7B shows the results of inhibition of GPC3 positive tumor growth by a 20mg/kg dose of the binding molecule.

Fig. 8 shows a schematic of a binding molecule of the present disclosure.

Figure 9 shows that different concentrations of binding molecules of the present disclosure inhibit the growth of GPC 3-positive tumors.

FIG. 10 shows that different concentrations of HEC512-G1D inhibited the growth of GPC3 positive tumors.

Detailed Description

In order to make the objects, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below with reference to the embodiments. The specific embodiments described herein are merely illustrative of the disclosure and do not constitute any limitation on the disclosure. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure.

Definition of

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It is noted that the terms used herein should be interpreted as having a meaning that is consistent with the context of this specification and should not be interpreted in an idealized or overly formal sense.

The expression "about" as used herein is as understood by one of ordinary skill in the art and varies within certain ranges depending on the context in which it is used. If one of ordinary skill in the art would not understand the use of this term based on the context of its use, then "about" would mean that the particular value is at most plus or minus 10%.

As used herein, "4-1BB", also known as CD137, TNFRSF9 and the like, is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRS). 4-1BB is a costimulatory receptor expressed on various cells of the immune system, particularly on CD8+ T cells. Due to its broad expression, and the ability of 4-1BB to enhance potent and durable immune effects, 4-1BB is a clinical target for cancer immunotherapy.

As used herein, "glypican 3" or "GPC3" are used interchangeably and are carcinoembryonic antigens belonging to the glypican family, which is composed of glycosylphosphatidylinositol-anchored heparin sulfate proteoglycans. Glypicans regulate the activity of several growth factors, including Wnts, hedgehog (hedgehog), bone morphogenic proteins, and Fibroblast Growth Factors (FGFs). Glypicans are characterized by covalent attachment to polysaccharide chains known as heparin sulfate glycosaminoglycans. Glypicans are involved in cell signaling at the cell-extracellular matrix interface. To date, six different members of the human glypican family have been identified. Cell membrane bound GPC3 consists of two subunits linked by one or more disulfide bonds. GPC3 is expressed in the developing fetal liver and placenta and is down-regulated or silenced in normal adult tissues. GPC3 is highly expressed in various cancers, particularly hepatocellular carcinoma (HCC), melanoma, wilms' tumor, and hepatoblastoma.

The term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that differ in structure from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their accepted single-letter codes. As used herein, "polar amino acid" refers to an amino acid that comprises a side chain that prefers to reside in an aqueous environment. In some embodiments, the polar amino acid is selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and tyrosine. Polar amino acids can be positively charged, negatively charged, or neutrally charged. As used herein, a "non-polar amino acid" is selected from alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

As used herein, "substituted with one or more different amino acids" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with another, different "replacement" amino acid residue. "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Although the insert typically consists of an insertion of 1 or 2 amino acid residues, larger "peptide inserts" may also be prepared, for example an insertion of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residue may be naturally occurring or non-naturally occurring. "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

Antibodies suitable for use in the present disclosure may comprise conservative amino acid substitutions at one or more amino acid residues, for example at essential or non-essential amino acid residues. A "conservative amino acid substitution" is an amino acid substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, an essential or nonessential amino acid residue in an antibody is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, the amino acid segments may be replaced with segments that are structurally similar and differ in the order and/or composition of the side chain family members. Alternatively, in certain embodiments, mutations can be introduced randomly along all or a portion of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into binding polypeptides of the invention and screened for the ability of these binding polypeptides to bind to the desired target.

The term "anti-4-1 BB agonist antibody" or "specifically agonist antibody against 4-1BB" as used herein refers to an antibody that specifically binds 4-1BB and partially or completely promotes, induces, increases and/or activates 4-1BB bioactivity, response and/or downstream pathways mediated by 4-1BB signaling or other 4-1BB mediated functions. Tumor Necrosis Factor Receptor (TNFR) signaling, and in particular TNFRSF activation, requires receptor clustering and multimerization. 4-1BB is one of the TNFSF receptors known to require clustering to trigger downstream signaling. It is reported that the formation of 2 or more trimers by cross-linking of 4-1BB produces a more strongly activated protein. In some embodiments, the anti-4-1BB agonist antibody binds 4-1BB, inducing multimerization of 2 or more trimers of 4-1BB.

The terms "full-length antibody" and "intact antibody" of the invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a native antibody. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a native IgG-class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two light and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy or heavy chain variable domain, and three constant domains (CH 1, CH2, and CH 3), also known as heavy chain constant regions. From N-terminus to C-terminus, each light chain has a variable region (VL) (also known as a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also known as a light chain constant region). The heavy chain of an antibody may be one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), and may be further divided into subtypes, such as gamma 1 (IgG 1), gamma 2 (IgG 2), gamma 3 (IgG 3), gamma 4 (IgG 4), alpha 1 (IgA 1) and alpha 2 (IgA 2). The light chain of an antibody can be one of two types, a kappa light chain and a lambda light chain, based on the amino acid sequence of its constant domain.

Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The binding molecules of the present disclosure are homodimers comprising two identical peptide chains bonded by disulfide bonds. In some embodiments, the two identical peptide chains are long peptide chains comprising a first domain and a second domain that specifically bind 4-1BB and GPC3, respectively. The long peptide chain of the binding molecules of the present disclosure may include VL from the N-terminus ₂ -VH ₂ -Fc-VH ₁ -VL ₁ 、VH ₂ -VL _2- Fc-VH ₁ -VL ₁ 、VL ₂ -VH ₂ -Fc-VL ₁ -VH ₁ 、VH ₁ -VL ₁ -Fc-VH ₂ -VL ₂ 、VL ₁ -VH ₁ -Fc-VL ₂ -VH ₂ 、VL ₁ -VH ₁ -Fc-VH ₂ -VL ₂ Or other means of ligation known to those skilled in the art, with or without a linker sequence of the disclosure between each fragment.

The binding molecules of the present disclosure are heterotetramers comprising two long peptide chains that are disulfide-bonded, and two short peptide chains that are each disulfide-bonded to a corresponding long peptide chain. In some embodiments, the long peptide chain may comprise VH from the N-terminus ₂ -Fc-VH ₁ -VL ₁ And the short peptide chain may include VL from the N-terminus ₂ . In some embodiments, the long peptide chain may include VH from the N-terminus ₁ -Fc-VH ₂ -VL ₂ The short peptide chain may include VL from the N-terminus ₁ . In some embodiments, the long peptide chain may include VH from the N-terminus ₁ -Fc-VL ₂ -VH ₂ The short peptide chain may include VL from the N-terminus ₁ . Alternatively, the functional fragments of the long and short peptide chains may be combined in other ways known to those skilled in the art.

The binding molecules of the present disclosure are heterohexamersThe polypeptide comprises two long peptide chains, two first short peptide chains and two second short peptide chains, wherein the two long peptide chains are bonded through disulfide bonds, and the first short peptide chains and the second short peptide chains are respectively bonded with corresponding regions of the long peptide chains through disulfide bonds. In some embodiments, the long peptide chain may comprise VH ₁ -Fc-VL ₂ The first short peptide chain comprises VL ₁ The second short peptide chain comprises VH ₂ . Alternatively, the functional fragments of the long and short peptide chains may be combined in other ways known to those skilled in the art.

The terms "specifically binds," "selectively binds," and "specifically binds" as used herein refer to an antibody or antigen-binding portion thereof that binds to an epitope on a predetermined antigen. Typically, when assayed by Surface Plasmon Resonance (SPR) techniques using recombinant human 4-1BB as the analyte and an antibody as the ligand in a BIACORE 2000 instrument, the antibody is present at about less than 10 ^-6 M, e.g. less than about 10 ^-7 M、10 ^-8 M、10 ^-9 M or 10 ^-10 M an even lower equilibrium dissociation constant (K) _D ) Binds to a predetermined antigen with at least twice the affinity to non-specific antigens other than the predetermined antigen or closely related antigens (e.g. BSA, casein).

The term "subject" as used herein includes any human or non-human animal. For example, the methods and compositions of the invention can be used to treat a subject having cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

The terms "antibody fragment," "antigen-binding portion," or similar terms, as used herein, refer to a fragment of an antibody that remains bound to a target antigen (e.g., 4-1BB or GPC 3) and has the same activity as the full length of the antibody. Such fragments include, for example, single chain antibodies, single chain Fv fragments (scFv), fd fragments, fab 'fragments, or F (ab') 2 fragments. scFv fragments are single polypeptide chains, which fragments comprise the variable regions of the heavy and light chains of the antibody from which the scFv was derived. Furthermore, intraantibodies, minibodies, trifunctional antibodies, and bifunctional antibodies are also included within the definition of antibodies and are suitable for use in the methods described herein. See, e.g., todorovska et al (2001), J.Immunol.methods,248 (1): 47-66; hudson and Kortt, (1999), J.Immunol.methods,231 (1): 177-189; poljak, (1994), structure,2 (12): 1121-1123; rondon and Marasco, (1997), annu. Rev. Microbiol.,51, the disclosures of each of which are incorporated herein by reference in their entirety.

The term "Fc fragment" as used herein refers to the constant region of a full-length immunoglobulin. In some embodiments, the "Fc fragment" refers to the last two constant domains (CH) of IgA, igD, igG ₂ -CH ₃ ) Or the last three constant domains (CH) of IgE and IgM ₂ -CH ₃ -CH ₄ ) And N-terminal flexible hinges for these domains.

The percent identity between two sequences varies with the number of identical positions shared by the sequences (i.e.,% identity = number of identical positions/total number of positions x 100), where the number of gaps that need to be introduced and the length of each gap are to be considered for optimal alignment of the two sequences. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from http:// www. GCG. Com), using the NWSgapdna. CMP matrix with GAP weights 40, 50, 60, 70 or 80 and

length weights

1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithms of e.meyers and w.miller (cabaos, 4-11-17 (1989)) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, which algorithms have been incorporated into the ALIGN program (version 2.0). In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.mol. Biol. (48): 444-453 (1970)) algorithms, using either the Blossum 62 matrix or the PAM250 matrix, and

GAP weights

16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6, which have been incorporated into the GAP program in the GCG software package (available from http:// www. GCG. Com).

The term "variable region" or "variable domain" as used herein refers to a domain of an antibody heavy or light chain that is involved in the binding of an antigen binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved Framework Regions (FR) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" as used herein refers to certain segments of the variable domains that differ in sequence generally between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely in a β -sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the β -sheet structure. The HVRs in each chain are held tightly together by the FR region and, together with the HVRs of the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, sequences of immunological Interest, 5 th edition, national Institute of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding the antibody to the antigen, and have other effector functions, such as participation in antibody-dependent cellular cytotoxicity of the antibody.

The term "hypervariable region" or "HVR" as used herein refers to a region which is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops") in a region of an antibody variable domain. Typically, a native four-chain antibody comprises six HVRs: three are present in VH (H1, H2, H3) and three are present in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "Complementarity Determining Regions (CDRs)" which have the highest sequence variability and/or are involved in antigen recognition. Based on Chothia definition rules, exemplary CDRs (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR 3) are located at amino acid residues L26-L32 (L1), L50-L52 (L2), L91-L96 (L3), H26-H32 (H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al, J.mol.biol.196:901-917 (1. Mol.biol.3)987)). Exemplary CDRs (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR 3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2), and H95-H102 (H3) based on the Kabat definition rules (Kabat et al, sequences of proteins of immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991)). Based on

Defining rules, exemplary CDRs (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR 3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2) and H93-H102 (H3) (Honjo, T. And Alt, F.W. (1995) immunoglobulingenes. By way of comparison, the corresponding amino acid residues comprising the CDRs defined in the references cited above are listed in table 1. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in the art by a variety of methods, such as Kabat definition rules based on sequence variability, chothia definition rules based on the position of the structural loop regions, and reference tools for antibody humanization design based on CDR grafting (see J Mol Biol 273. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above-described known schemes described by this invention. Although the scope of the present disclosure is claimed based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present invention.

TABLE 1 amino acid numbering system for each CDR region (see http:// bioinf. Org. Uk/abs /)

As used herein, "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in the VH (or VL) as follows: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

According to some embodiments of the present disclosure, a binding molecule of the present disclosure may comprise a Fab arm comprising one heavy chain-light chain pair that specifically binds to an antigen. According to some embodiments of the present disclosure, a binding molecule of the present disclosure may comprise a recombinant IgG-like dual targeting molecule, wherein the molecule is flanked by Fab fragments or a portion of Fab fragments of at least two different antibodies; an IgG fusion molecule in which a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; an Fc fusion molecule in which a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, fc region, or portion thereof; a Fab fusion molecule in which different Fab fragments are fused together; heavy chain antibodies (e.g., domain antibodies, nanobodies) based on scFv and diabodies, where different single chain Fv molecules or different diabodies or different heavy chain antibodies are fused to each other or to another protein or carrier molecule.

As used herein, a "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: igA, igD, igE, igG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as 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.

As used herein, a "humanized antibody" comprises the complete sequence of a humanized antibody produced by the splicing of amino acid residues from a hypervariable region (HVR) of non-human origin to constant regions from the heavy and light chains of a human antibody. In certain embodiments, a humanized antibody comprises at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) 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. An antibody that is a "humanized form," such as a non-human antibody, refers to an antibody that has been humanized.

As used herein, "cross-reactive" refers to the ability of an antibody of the present disclosure to bind 4-1BB or GPC3 from different species. For example, an antibody of the present disclosure can bind human 4-1BB, while also binding 4-1BB of another species (e.g., mouse, rat, cynomolgus monkey, or dog). As used herein, cross-reactivity is measured by detecting cell binding or otherwise functionally interacting with purified antigen in a binding assay (e.g., SPR, ELISA), or with physiologically expressed cells.

As used herein, "at least 90% sequence identity" to a sequence as compared to the sequence may include at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence.

As used herein, "expression vector" and "expression construct" are used interchangeably and, when the above-described isolated nucleic acid molecule is ligated to a vector, the nucleic acid sequence may be directly or indirectly linked to regulatory elements on the vector, so long as the regulatory elements are capable of regulating translation, expression, etc. of the nucleic acid molecule. Of course, these regulatory elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. That is, the nucleic acid molecule is operably linked to a regulatory element. "operably linked" herein refers to the attachment of a foreign gene to a vector such that regulatory elements within the vector, such as transcriptional and translational regulatory sequences and the like, can exert their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody may be inserted separately into different vectors, usually into the same vector. Commonly used vectors may be, for example, plasmids, phages, etc.

As used herein, an expression vector may be included in a "cell" or "recombinant cell". The expression vectors can be introduced into mammalian cells to obtain recombinant cells, which can then be used to express the antibodies or antigen-binding portions provided by the present disclosure. The recombinant cell is cultured to obtain the corresponding antibody. These usable mammalian cells may be, for example, CHO cells or the like.

As used herein, "pharmaceutically acceptable carrier" can include any solvent, dispersion medium, coating, antibacterial, antifungal, isotonic and absorption delaying agent, and the like, which are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride and the like may be included in the pharmaceutical composition. Of course, the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which serve to prolong the shelf life or effectiveness of the antibody.

For example, an antibody or antigen-binding portion thereof of the present disclosure can be incorporated into a pharmaceutical composition suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in a variety of forms, such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibodies, or antigen-binding portions thereof, of the present disclosure can be administered by intravenous infusion, injection, intramuscular, or subcutaneous injection.

A "therapeutically effective amount" of a drug (e.g., a pharmaceutical composition) refers to that amount necessary to be effective in dosage and interval and time of administration to achieve the desired therapeutic or prophylactic effect. For example, a therapeutically effective amount of a drug eliminates, reduces/reduces, delays, minimizes or prevents the adverse effects of a disease.

The term "hematological cancer" as used herein includes lymphoma, leukemia, myeloma or lymphoid malignancies, and cancers of the spleen and lymph nodes. Exemplary lymphomas include B cell lymphomas (B cell hematologic cancers) and T cell lymphomas. B cell lymphomas include hodgkin's lymphoma and most non-hodgkin's lymphoma. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small-cell lymphocytic lymphoma, mantle Cell Lymphoma (MCL), burkitt's lymphoma, mediastinal large B-cell lymphoma, waldenstrom's macroglobulinemia, lymph node marginal zone B-cell lymphoma, spleen marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphoma-like granuloma. Non-limiting examples of T cell lymphomas include extranodal T cell lymphomas, cutaneous T cell lymphomas, anaplastic large cell lymphomas, and angioimmunoblastic T cell lymphomas. Hematological malignancies also include leukemias, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematological malignancies also include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematologic malignancy encompasses other hematologic and/or B-cell or T-cell related cancers.

The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. In the following description, descriptions of well-known technologies are omitted so as to avoid unnecessarily obscuring the concepts of the present disclosure. Such techniques are described in a number of publications, such as molecular cloning guidelines (fourth edition), cold spring harbor laboratory science publishers.

The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 HEK293 suspension cells were transiently transfected to obtain binding molecules.

The peptide chains in Table 2 below were subjected to whole-gene synthesis using whole-gene synthesis means, and inserted into pcDNA3.1 vectors, respectively. And respectively transiently transfecting the constructed vectors into HEK293 cells to perform protein expression. In addition, for a binding molecule comprising multiple peptide chains, vectors into which the multiple peptide chains are inserted, respectively, are co-transfected into HEK293 cells (e.g., vectors into which peptide chain 1, peptide chain 2, and peptide chain 3 of IOB1-FabIg-S2, respectively, are inserted, are co-transfected into HEK293 cells).

TABLE 2 Structure corresponding to each polypeptide sequence and its sequence

Note: the heavy chain variable region and the light chain variable region of anti-4-1BB are respectively derived from monoclonal antibody HEC511 (the amino acid sequence of the heavy chain is shown in SEQ ID NO:67, and the amino acid sequence of the light chain is shown in SEQ ID NO: 54) or monoclonal antibody HEC512 (the amino acid sequence of the heavy chain is shown in SEQ ID NO:68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 66). The heavy chain variable region and the light chain variable region of anti-GPC 3 are respectively derived from three monoclonal antibodies HS20 (the sequence of the heavy chain variable region is shown in SEQ ID NO:37, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38), 4A6 (the sequence of the heavy chain variable region is shown in SEQ ID NO:35, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36) or GC33 (the sequence of the heavy chain variable region is shown in SEQ ID NO:39, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40).

The binding molecules IOB1-S, IOB1-M, IOB1-ScFvIg-S, IOB1-FabIg-S2, IOB1-G-Fab1, IOB1-G-Fab2 and IOB1-G1D-Fab2 were obtained in greater than 95% purity, respectively, by purification of the expressed binding molecules in ProteinA medium, the schematic structures of which are shown in FIG. 8. These binding molecules were obtained for the following assay evaluation.

The expression titer of the above-mentioned binding molecule obtained per 100mL of the suspension cell culture medium was measured, and the results are shown in Table 3. In table 3, isotype IgG served as a negative control.

TABLE 3 titer of each binding molecule per 100mL of suspension cell culture.

Example 2 binding of the binding molecules of the present disclosure to CHO cell lines stably transfected with human 4-1BB.

This example uses flow cytometry to examine the binding capacity of binding molecules to CHO cell lines stably transfected with human 4-1BB. After the binding molecules were incubated with the cells, they were then bound using a FITC-labeled goat anti-mouse IgG Fc secondary antibody (Abcam: ab 97023), GC33 and isotype IgG antibodies as negative controls, and antibody HEC511 (the amino acid sequence of the heavy chain is shown in SEQ ID NO:67, the amino acid sequence of the light chain is shown in SEQ ID NO: 54) and antibody HEC512 (the amino acid sequence of the heavy chain is shown in SEQ ID NO:68, the amino acid sequence of the light chain is shown in SEQ ID NO: 66) which constructed to correspond to the binding molecules as positive control samples. The results are shown in FIG. 1. As can be seen in FIG. 1, the binding molecules can bind efficiently to the human 4-1BB (UniProtKB-Q07011) membrane-expressed protein.

Example 3 binding of the binding molecules of the present disclosure to human 4-1BB recombinant protein

This example examined the binding ability of the binding molecules to human 4-1BB (UniProtKB-Q07011) recombinant protein using ELISA. Coating the recombinant 4-1BB protein on an enzyme label plate at 4 ℃ overnight, washing for 3 times, then spin-drying, sealing for 1 hour at 37 ℃, washing for 3 times, and spin-drying for later use. The binding molecules of the present disclosure and the above-mentioned enzyme labeling plate were incubated at 4 ℃ for 2 hours, washed 3 times and then spin-dried, and then an HRP-labeled goat anti-mouse IgG-Fc secondary antibody was used for binding, an isotype IgG antibody was used as a negative control, and antibodies HEC511 and HEC512 corresponding to the construction of the binding molecules were used as control samples. The results are shown in FIG. 2.

As can be seen in FIG. 2, the binding molecules of the present disclosure can bind efficiently to human 4-1BB (UniProtKB-Q07011) recombinant protein.

Example 4 binding of binding molecules of the present disclosure to T cells isolated and activated in vitro from human blood

T cells are separated from a PBMC cell population derived from a human body, and the T cells are stimulated in vitro for 48 to 72 hours by using CD3 and CD28 antibodies coupled with magnetic beads, so that the stimulated T cells can highly express 4-1BB protein. The binding molecules of the present disclosure are then tested for their binding capacity to stimulated T cells using flow cytometry. After incubation of the binding molecules with cells, binding was followed using a FITC-labeled goat anti-mouse IgG Fc secondary antibody, using isotype IgG antibodies as a negative control. The results are shown in FIG. 3.

As can be seen in fig. 3, the binding molecules of the present disclosure can effectively bind to activated human T cells. That is, the binding molecules of the present disclosure can effectively bind to 4-1BB protein expressed by activated T cells.

Example 5 binding of binding molecules of the present disclosure to human GPC3 recombinant proteins

This example examined the binding ability of the binding molecules of the present disclosure to human GPC3 (XP — 022382036.1) recombinant protein using ELISA. Coating the recombinant GPC3 protein on an ELISA plate at 4 ℃ overnight, washing for 3 times, spin-drying, sealing at 37 ℃ for 1 hour, washing for 3 times, and spin-drying for later use. The antibody was incubated with the above-mentioned enzyme-labeled plate at 4 ℃ for 2 hours, washed 3 times and then spin-dried, followed by binding with an HRP-labeled goat anti-mouse IgGFc secondary antibody, using antibody HEC511, isotype IgG antibody as negative controls, and using antibodies HS20 and GC33 corresponding to the construction of the fusion protein as control samples. The results are shown in FIG. 4.

As can be seen from fig. 4, the binding molecules of the present disclosure can effectively bind to human GPC3 (XP _ 022382036.1) recombinant protein.

Example 6 binding of the binding molecules of the present disclosure to human hepatoma cell lines

The GPC3 protein is known to be highly expressed in liver cancer cells. In order to detect whether the binding molecules disclosed by the disclosure can bind to liver cancer cells, conventionally cultured Huh7 or HepG2 liver cancer cells are digested with trypsin and collected, and the binding capacity of the binding molecules disclosed by the disclosure to human liver cancer cell lines Huh7 or HepG2 is detected by using a flow cytometer. After incubation of the binding molecules of the present disclosure with cells, a FITC-labeled goat anti-mouse IgGFc secondary antibody was subsequently used for binding, isotype IgG antibodies were used as negative control, and antibodies GC33, HEC511 corresponding to the construction of the fusion proteins were used as control samples. The results are shown in FIG. 5.

As can be seen from fig. 5, the binding molecules of the present disclosure can effectively bind to GPC3 protein expressed by human liver cancer cell lines.

Example 7 affinity of the binding molecules of the present disclosure to human 4-1BB recombinant protein.

This example uses biofilm light interference technology (Bio-LayerInterferometer, BLI) to determine the binding affinity of the binding molecules of the present disclosure for 4-1BB. Briefly, human 4-1BB (UniProtKB-Q07011) protein was immobilized on the sensor for 300s, the sensor was then brought into PBST solution for 180s at equilibrium baseline, the probe was then immersed in the fusion protein solution for 600s at binding duration, the probe was then placed in PBST solution for 1200s at dissociation duration, and the probe was finally placed in a regenerating solution to complete the regeneration and preservation of the probe. The binding affinity of the binding molecules of the present disclosure to human recombinant protein 4-1BB was tested using BLI assay. The results are shown in Table 4.

TABLE 4 binding affinity of the binding molecules of the present disclosure to human recombinant 4-1BB protein

Example 8 binding affinity of binding molecules of the present disclosure to human GPC3 recombinant protein

This example uses the Bio-layer interference technique (BLI) to determine the binding affinity of the binding molecules of the present disclosure to GPC 3. Briefly, human GPC3 (XP _ 022382036.1) protein was immobilized on the sensor for 300s, the sensor was then put into PBST solution to equilibrate Baseline for 180s, the probe was then immersed into the fusion protein solution to bind for 600s, the probe was then put into the PBST solution to dissociate for 1200s, and the probe was finally put into the regenerating solution to complete the regeneration and preservation of the probe. The binding affinity of the binding molecules of the present disclosure to the human recombinant protein GPC3 was determined using the BLI assay. The results are shown in Table 5.

TABLE 5 binding affinity of binding molecules of the present disclosure to human recombinant GPC3 protein

Example 9 binding molecules of the present disclosure activate T cells in a GPC 3-positive hepatoma cell-dependent manner.

The CD3 antibody is coated on an enzyme label plate and is uniformly covered with tumor cells with certain density. After isolation of human T cells, the T cells and binding molecules of the disclosure are added to an elisa plate and the amount of IL2 expression in the supernatant is detected three days later, or the amount of IFN- γ expression in the supernatant is detected five days later. HEC511, HS20, 4A6 or isotype IgG antibodies were used as controls. The results are shown in FIG. 6.

Figures 6A and 6B show that the binding molecules of the present disclosure are capable of stimulating secretion of interleukins IL2 and IFN γ by T cells in the presence of GPC 3-positive hepatoma cells Huh 7. Figures 6C-6F show that the binding molecules of the present disclosure are capable of stimulating the secretion of IL2 by T cells and IFN γ detection in the presence of GPC 3-positive liver cancer cells HepG 2. Figure 6G shows that the binding molecules of the present disclosure are capable of stimulating secretion of IFN γ by T cells in the presence of GPC 3-positive hepatoma cells Hep 3B.

Example 10 validation of tumor-inhibiting Effect of binding molecules of the present disclosure in a tumor-bearing humanized mouse model of the MC38 high-expression GPC3 Stable cell line

PBS resuspended tumor cell line (MC 38-hGPC3 cells) with MC38 highly expressing human GPC3 protein at 5X 10 ⁵ One/0.1 mL/mouse, was inoculated subcutaneously into the right side of B-h4-1BB/h4-1BBL humanized mouse. When the average tumor volume reaches 100-150mm ³ In this case, the appropriate mice were selected into groups based on their tumor volume and body weight, and evenly distributed to 3 experimental groups of 6 mice each. Samples from each group were administered intraperitoneally at a dose of 5mg/kg (PBS was administered to the control group) twice weekly. After grouping, the tumor volume is measured for 2 times every week by using a vernier caliper, the tumor volume is measured before euthanasia, the long diameter and the short diameter of the tumor are measured, and the volume calculation formula is as follows: tumor volume =0.5 × major diameter × minor diameter ² . The results are shown in fig. 7A, where it can be seen that the bispecific antibodies of the present disclosure can significantly inhibit tumor growth after administration; and at the same dose, bispecific antibody inhibitionThe onset time of the tumor is earlier than that of the anti-4-1BB monoclonal body, and the tumor elimination time is also obviously earlier than that of the anti-41BB monoclonal body HEC512.

Example 11 evaluation of antitumor Activity of test antibody in MC38 high expression GPC3 Stable Trans cell line tumor-bearing humanized mouse model

Subcutaneous administration at 5X 10 on the right side of 41BB/41BBL double humanized C57BL/66 mice ⁵ The tumor cell line with MC38 highly expressing human GPC3 protein was inoculated at a concentration of 0.1 mL/cell. When the average tumor volume reaches 100-150mm ³ In the method, a suitable mouse is selected according to the weight and the tumor volume of the mouse and is put into a group, the test antibody IOB1-G-Fab2 is injected into the mouse body in an abdominal cavity according to the dose of 20mg/kg, the size of the tumor is measured periodically, and the growth or inhibition condition of the tumor is observed. The results are shown in fig. 7B, which shows that the test antibody has a significant effect of inhibiting the growth of GPC 3-positive tumors.

Example 12 evaluation of anti-tumor Activity of test antibodies in MC38 high expression GPC3 Stable Trans cell line tumor-bearing humanized mouse model

Subcutaneous administration at 5X 10 on the right side of 41BB/41BBL double humanized C57BL/66 mice ⁵ The cells were inoculated with a tumor cell line having MC38 highly expressing human GPC3 protein at a concentration of 0.1 mL/cell. When the average tumor volume reaches 100-150mm ³ Then, appropriate mice were selected according to the body weight and tumor volume of the mice and the test antibodies IOB1-G1D-Fab2 and HEC512-G1D (the amino acid sequence of the heavy chain is shown in SEQ ID NO:65 and the amino acid sequence of the light chain is shown in SEQ ID NO: 66) were injected intraperitoneally into the mice at doses of 20mg/kg, 5mg/kg and 1mg/kg, the size of the tumor was measured periodically, and the growth or inhibition of the tumor was observed. The results are shown in FIGS. 9 and 10, which show that the tested antibody has a significant effect of inhibiting the growth of GPC 3-positive tumors, and the inhibitory effect of IOB1-G1D-Fab2 is superior to that of HEC512-G1D.

The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solutions of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A binding molecule, wherein the binding molecule comprises:

a first domain, wherein the first domain specifically binds 4-1BB or a fragment thereof, and

a second domain that specifically binds glypican 3 (GPC 3) or a fragment thereof.

2. The binding molecule of claim 1, further comprising a third domain comprising an Fc fragment of an immunoglobulin,

preferably, the immunoglobulin is selected from IgA, igG, igM, igD and IgE, preferably IgG, more preferably IgG1, igG2, igG3 or IgG4;

preferably, the Fc fragment has one or more mutations in L234F, L235E, P331S, D356E and L358M, wherein the Fc fragment is numbered according to the EU index of Kabat;

more preferably, the third domain comprises an amino acid sequence as set forth in SEQ ID NO:55, 62 or 69 or a variant thereof.

3. The binding molecule of claim 1 or 2, wherein the first domain comprises a first heavy chain variable region VH ₁ And a first light chain variable region VL ₁ Wherein:

said VH ₁ The method comprises the following steps:

(a) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences shown as SEQ ID NO 1, 2 and 3, respectively; or

(b) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences shown as SEQ ID NO 7, 8 and 9, respectively, said VL ₁ The method comprises the following steps:

(c) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences as set forth in SEQ ID NO. 4, 5 and 6, respectively; or

(d) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences shown as SEQ ID NO 10, 11 and 12, respectively.

4. The binding molecule of any one of claims 1 to 3,

said VH ₁ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO 31 or 33, and/or

The VL ₁ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO 32 or 34.

5. The binding molecule of any one of claims 1 to 4, wherein said second domain comprises a second heavy chain variable region VH ₂ And a second light chain variable region VL ₂ Wherein

Said VH ₂ The method comprises the following steps:

(e) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences set forth as SEQ ID NOS 13, 14 and 15, respectively;

(f) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences set forth as SEQ ID NOS 19, 20 and 21, respectively; or

(g) HCDR1, HCDR2 and HCDR3 comprising amino acid sequences shown as SEQ ID NOS: 25, 26 and 27, respectively,

the VL ₂ The method comprises the following steps:

(h) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences shown as SEQ ID NO 16, 17 and 18, respectively;

(i) LCDR1, LCDR2 and LCDR3 comprising amino acid sequences as set forth in SEQ ID NO. 22, 23 and 24, respectively; or

(j) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO 28, 29 and 30, respectively.

6. The binding molecule of any one of claims 1 to 5,

said VH ₂ Comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO35, 37 or 39, and/or

The VL ₂ Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO 36, 38 or 40.

7. According to any of claims 1 to 6Means the binding molecule, wherein the structure of the first domain and/or the second domain is selected from the group consisting of Fab, fab ', F (ab') ₂ Fv or scFv.

8. The binding molecule of any one of claims 1 to 7, wherein the first domain, the second domain and/or the third domain are linked directly or via a linker,

the first heavy chain variable region and the first light chain variable region of the first domain are linked directly or through a linker, and/or

The second heavy chain variable region and the second light chain variable region of the second domain are linked directly or via a linker.

9. The binding molecule of claim 8, wherein the linker has a structure as (G) _n S) _z Wherein n and z are each independently an integer from 1 to 4.

10. The binding molecule according to any one of claims 1 to 9, wherein the binding molecule comprises a first domain, a second domain and a third domain connected in the following order:

first domain-third domain-second domain;

second domain-third domain-first domain;

first domain-second domain-third domain; or the like, or, alternatively,

second domain-first domain-third domain.

11. The binding molecule of any one of claims 1 to 10, wherein said binding molecule comprises a homodimer of a peptide chain,

preferably, the peptide chain has an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO 41, 42 or 43.

12. The binding molecule of any one of claims 1 to 10, wherein the binding molecule comprises a heterotetramer of a long peptide chain and a short peptide chain, wherein

(i) The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 44, and

the short peptide chain has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID NO. 45;

(ii) The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 46, and

the short peptide chain has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID NO. 47;

(iii) The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 48, and

the short peptide chain has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID NO. 49; or

(iv) The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 50, and

the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 51, or

(v) The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 64, and

the short peptide chain has at least 90 percent of sequence identity with the amino acid sequence shown in SEQ ID NO. 51.

13. The binding molecule of any one of claims 1 to 10, wherein the binding molecule comprises a long peptide chain, a first short peptide chain, and a second short peptide chain forming a heterotexamer, wherein

The long peptide chain has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID NO. 52,

the first short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:54, and

the second short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 53.

14. An isolated nucleic acid molecule encoding the binding molecule of any one of claims 1 to 13.

15. A vector comprising the nucleic acid molecule of claim 14, preferably an expression vector.

16. A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15.

17. A pharmaceutical composition comprising the binding molecule of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15 or the host cell of claim 16 and a pharmaceutically acceptable carrier.

18. Use of a binding molecule according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, a host cell according to claim 16 or a pharmaceutical composition according to claim 17 for the preparation of a medicament for the treatment or prevention of a cancer, preferably a cancer selected from the group consisting of melanoma, glioma, renal cancer, breast cancer, liver cancer, wilms' tumor, hepatoblastoma, hematological cancer and head and neck cancer.