WO2023000675A1 - Bispecific antibody targeting pd-l1 and 4-1bb - Google Patents

Abstract

Provided in the present application is a bispecific antibody targeting PD-L1 and 4-1BB. The bispecific antibody cannot only block the binding of PD-1 and PD-L1, but can also activate a human 4-1BB signal pathway and stimulate the activation of T cells, and can be used for an immunopotentiator or an immunomodulator of autoimmune diseases mediated by T cells.

Description

Bispecific antibodies targeting PD-L1 and 4-1BB technical field

The invention relates to the technical fields of tumor therapy and immunology, and specifically relates to a bispecific antibody targeting PD-L1 and 4-1BB.

Background technique

Bispecific antibody (BsAb), also known as bifunctional antibody, refers to an antibody that can recognize and bind to two different targets or two different epitopes of the same target at the same time, and can play some special biological functions. Even better than the synergistic effect of the combination of the two monoclonal antibodies. Compared with the combination therapy of two monoclonal antibody drugs, BsAb has stronger specificity, targeting and reduced off-target toxicity, and BsAb also reduces the cost of drug development and clinical trials.

Bispecific antibodies do not exist in the natural state, and their technical platforms can be divided into three categories according to their structure: non-IgG-like, symmetric IgG-like, and asymmetric IgG-like. Bispecific antibodies are currently a hot new drug research and development direction in the biopharmaceutical industry. From the perspective of disease field distribution, China's bispecific antibody projects are mainly concentrated in the direction of tumors, with breast cancer and gastric cancer in the majority.

In recent years, new targets for immunotherapy and new pathways of immune activation have been continuously discovered, and tumor immunotherapy has made significant progress. Programmed cell death protein 1 (PD-1) is a member of the CD28 superfamily. As a T cell inhibitory receptor, blocking the interaction between PD-1 and PD-L1 can effectively restore the tumor-killing function of T cells, promote the proliferation of tumor antigen-specific T cells, kill tumor cells, and inhibit tumor growth. In addition, PD-L1 can also bind to B7-1 in vivo. Studies have shown that the PD-L1/B7-1 complex is also a negative signal for T cell activation, and the combination of the two can lead to a decrease in the expression of T cell surface activation markers. Inhibition of T cell proliferation, etc. At present, the industry generally believes that antibodies targeting the PD-L1 pathway will bring breakthroughs in the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma. However, there are certain shortcomings in the therapy targeting PD-1/PDL1: some patients experience rapid and durable tumor regression, but most patients achieve little or no obvious effect. To increase the response rate of patients to immunotherapy, researchers attempt to develop new immunomodulatory targets and therapeutic strategies. One of the good strategies is to induce immune cell activation by immunostimulatory receptors. This "co-stimulatory" strategy provides the mechanistic basis for a variety of agents in clinical development, including targeting OX40, CD27, CD40, GITR and Antibodies to 4-1BB.

4-1BB (CD137/TNFRSF9) is a co-stimulatory molecule that belongs to the tumor necrosis factor receptor (TNFRSF9) superfamily member, mainly expressed in activated T cells, and can pass Enhance the function of tumor-specific CD8 ⁺ T cells to achieve anti-tumor effects, and can also enhance the anti-tumor immune response mediated by CD8 ⁺ T cells by enhancing the immune function of NK cells, DCs and CD4 ⁺ T cells. It has unique potential as a therapeutic target . In preclinical experiments, the anti-tumor activity of anti-4-1BB monoclonal antibody was verified in multiple mouse models of colon cancer (MC38, CT26), lung cancer (M109), breast cancer (EMT6) and B lymphoma (A20) . The first anti-4-1BB therapeutic drug Urelumab (BMS-663513) entering clinical trials is a fully human IgG4 monoclonal antibody. The drug has a good clinical effect, but it is accompanied by obvious liver toxicity, which limits its clinical development. The second drug to enter clinical research, Utomilumab (PF-05082566), is a humanized IgG2 monoclonal antibody that can block the binding to endogenous 4-1BBL while activating 4-1BB. Compared with urelumab, this antibody has better safety, but weak agonistic activity. Therefore, how to develop an anti-tumor drug with high efficiency and low toxicity against the 4-1BB target has become the focus of drug researchers.

Currently, there are no effective PD-L1/4-1BB bispecific antibody drug products on the market.

invention disclosure

The purpose of the present invention is to provide a bispecific antibody targeting PD-L1 and 4-1BB. The research and development of this bispecific antibody is expected to make up for the lack of PD-1/PD-L1 or 4-1BB targeting tumors in the current market, and expand new indications. At the same time, it can be used as a new generation of PD-L1 immunotherapy products. It is used to treat patients with clinical immune tolerance after existing treatments such as PD-1/PD-L1, and patients with a low response rate. It can also be used for cancers with low PD-L1 expression that have not yet had a good effect. kind.

In the first aspect, the present invention claims a bispecific antibody targeting PD-L1 and 4-1BB, which contains a PD-L1 antigen-binding domain and a 4-1BB antigen-binding domain.

The bispecific antibody can block the combination of PD-1 and PD-L1.

The bispecific antibody can activate T cells in MLR in vitro experiments, and enhance the secretion level of IL-2.

The T cell activation effect of the bispecific antibody is dependent on PD-L1, and has no activity without PD-L1.

The bispecific antibody can activate the 4-1BB signaling pathway, and this activation depends on crosslinking.

The bispecific antibody has an anti-tumor drug effect, and the drug effect of the double antibody is better than that of PD-L1 and 4-1BB in combination with two monoclonal antibodies.

The PD-L1 antigen-binding domain includes a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as shown in SEQ ID No.1 26- 32, 52-56, and 98-107; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as follows: 24-36, 52 of SEQ ID No.2 -shown at positions 58 and 93-100; wherein, the amino acid sequence of the HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain or the 98th- as in SEQ ID No.7 Shown in position 107 (the 98th-107th DRPDGAATNL of SEQ ID No.1 is mutated to DRPEGAATNL).

The 4-1BB antigen binding domain comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as shown in the 31-th sequence of SEQ ID No.3. 35, 50-65, and 98-106; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as follows: 24-34, 50 of SEQ ID No.4 -56 and 89-97 are shown.

Further, in the PD-L1 antigen-binding domain, the amino acid sequence of the heavy chain variable region is SEQ ID No.1 or SEQ ID No.7 1-118, or the same as SEQ ID No. 1 or the 1-118th positions of SEQ ID No.7 have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of the identity (the inconsistency is preferably in the framework region (FR) ). The amino acid sequence of the light chain variable region is SEQ ID No.2 or SEQ ID No.8 1-110, or has a 99% difference with SEQ ID No.2 or SEQ ID No.8 1-110 More than %, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% consistency (the inconsistency is preferably in the framework region (FR)).

Further, in the 4-1BB antigen binding domain, the amino acid sequence of the heavy chain variable region is SEQ ID No.3 or 586-702 of SEQ ID No.7, or the same as SEQ ID No. .3 or or the 586-702 of SEQ ID No.9 has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% (inconsistency is preferably in the framework region ( FR)). The amino acid sequence of the light chain variable region is SEQ ID No.4 or 459-565 of SEQ ID No.7, or the same as SEQ ID No.4 or 459-565 of SEQ ID No.7 It has a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% (the inconsistency is preferably in the framework region (FR)).

Furthermore, the structure of the bispecific antibody from the N-terminal to the C-terminal is named D, as follows:

1 ^st Fab-Fc-L-2 ^nd scFv;

Among them, - represents a peptide bond; L represents a connecting peptide (linker) or an independent peptide bond; Fc represents the Fc segment of an antibody; 1 ^{st Fab} represents a Fab domain that can specifically bind to the first antigen; A scFv domain that specifically binds to a second antigen.

The N-terminal of the scFv structural domain is a heavy chain variable region, and the C-terminal is a light-chain variable region; or the N-terminal is a light-chain variable region, and the C-terminal is a heavy-chain variable region. When the scFv structural domain is the 44th amino acid of the heavy chain variable region of the original antibody (the PD-L1 antigen-binding domain heavy chain variable region shown in SEQ ID No.1 or the 4th amino acid shown in SEQ ID No.3 -1BB antigen-binding domain heavy chain variable region) and light chain variable region amino acids (PD-L1 antigen-binding domain light chain variable region shown in SEQ ID No.2 or 4 shown in SEQ ID No.4 -1BB antigen-binding domain light chain variable region) is mutated to cysteine C at position 100; this mutation is a common mutation to increase disulfide bonds in the scFv format for increased stability.

The connecting peptide can be selected from the following: A(EAAAK) ₄ ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n; wherein, n is a positive integer (such as 1, 2, 3, 4, 5 or 6), preferably, n =4.

The bispecific antibody includes an Fc segment; the Fc segment includes a mutation or does not include a mutation site.

The Fc segment is IgG1, IgG2, IgG3 or IgG4 type, preferably IgG4 type.

Preferably, the 1st Fab of the bispecific antibody (named D1) of the structure D in the embodiment recognizes the PD-L1 antigen, the 2nd scFv recognizes the 4-1BB antigen, L is the amino acid sequence of "A(EAAAK)4ALE", and the Fc is preferably is IgG4.

Preferably, for the bispecific antibody of D structure (named D2) in another embodiment, the 1st ^Fab recognizes the PD-L1 antigen, the ^2nd scFv recognizes the 4-1BB antigen, and L is "(G4S)3" amino acid sequence, Fc is preferably IgG4. In an embodiment, the bispecific antibody is mutated to D6 on the basis of D2, and the 61st amino acid and the 101st amino acid from the N-terminal of the heavy chain SEQ ID No. 6 are mutated, which can be mutated singly or simultaneously to glutamic acid "E", or the 62nd amino acid and the 102nd amino acid mutation of the heavy chain SEQ ID No. 6 starting from the N-terminal, can be mutated singly or simultaneously to glycine "G" or alanine "A".

In a specific embodiment of the present invention, the bispecific antibody is any of the following:

(A) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No.5, and the amino acid sequences of the light chains are all as shown in SEQ ID No.8 (corresponding to struct D1);

(B) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No.6, and the amino acid sequences of the light chains are all as shown in SEQ ID No.8 (corresponding to struct D2);

(C) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No.7, and the amino acid sequences of the light chains are all as shown in SEQ ID No.8 (corresponding to Structure D6).

In the second aspect, the present invention claims to protect the nucleic acid molecule encoding the bispecific antibody described in the first aspect above.

In the nucleic acid molecule, the nucleotide sequences encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain are sequentially as shown in SEQ ID No. 19 from the 5' end to the 76th-96th position, 154-168, and 292-321; wherein, the nucleotide sequence of the HCDR3 in the heavy chain variable region encoding the PD-L1 antigen-binding domain or as shown in SEQ ID Shown in No.11 No. 292-321. The nucleotide sequences encoding LCDR1, LCDR2, and LCDR3 in the light chain variable region of the PD-L1 antigen-binding domain are sequentially as shown in SEQ ID No. 20 at positions 70-108 and 154-174 from the 5' end , No. 277-300 shown.

In the nucleic acid molecule, the nucleotide sequences encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the 4-1BB antigen-binding domain are sequentially as shown in SEQ ID No.21 from 5' end 91-105 Bit, No. 148-195, No. 292-318 are shown. The nucleotide sequences encoding LCDR1, LCDR2 and LCDR3 in the light chain variable region of the 4-1BB antigen-binding domain are sequentially as shown in SEQ ID No. 22 at positions 70-102 and 148-168 from the 5' end , Shown in bits 265-291.

Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region in the PD-L1 antigen-binding domain is the 1-354th positions of SEQ ID No.19 or SEQ ID No.11, Or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of the 1-354 of SEQ ID No.19 or SEQ ID No.11 Consistency (inconsistency Preferably in the framework regions (FR)). The nucleotide sequence encoding the light chain variable region in the PD-L1 antigen binding domain is SEQ ID No.20 or SEQ ID No.12 1-330, or the same as SEQ ID No.20 or SEQ ID Positions 1-330 of No. 12 have a identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% (the inconsistency is preferably in the framework region (FR)).

Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region in the 4-1BB antigen-binding domain is the 1756-2106 position of SEQ ID No.21 or SEQ ID No.11, Or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of the 1756-2106 of SEQ ID No.21 or SEQ ID No.11 Consistency (the inconsistency is preferred in the framework region (FR)). The nucleotide sequence encoding the light chain variable region in the 4-1BB antigen-binding domain is SEQ ID No.22 or SEQ ID No.11 1375-1695, or the same as SEQ ID No.22 or SEQ ID No.22 Positions 1375-1695 of ID No. 11 have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of identity (the inconsistency is preferably in the framework region (FR)).

Further, the nucleic acid molecule can be any of the following:

(a) is made up of the nucleic acid molecule a1 of the heavy chain in coding aforementioned (A) and the nucleic acid molecule a2 of the light chain in coding aforementioned (A); The nucleotide sequence of described nucleic acid molecule a1 is SEQ ID No.9 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.9 (the inconsistency is preferably in the framework region (FR)); The nucleotide sequence of the nucleic acid molecule a2 is SEQ ID No.12 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.12 ( The inconsistencies are preferably in the framework regions (FR)).

(b) is made up of the nucleic acid molecule b1 of the heavy chain in coding aforementioned (B) and the nucleic acid molecule b2 of the light chain in coding aforementioned (B); The nucleotide sequence of described nucleic acid molecule b1 is SEQ ID No.10 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.10 (the inconsistency is preferably in the framework region (FR)); The nucleotide sequence of the nucleic acid molecule b2 is SEQ ID No.12 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.12 ( The inconsistencies are preferably in the framework regions (FR)).

(c) is made up of the nucleic acid molecule c2 of the nucleic acid molecule c1 of the heavy chain in coding aforementioned (C) and the light chain in coding aforementioned (C); The nucleotide sequence of described nucleic acid molecule c1 is SEQ ID No.11 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.11 (the inconsistency is preferably in the framework region (FR)); The nucleotide sequence of the nucleic acid molecule c2 is SEQ ID No.12 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.12 ( The inconsistencies are preferably in the framework regions (FR)).

In the third aspect, the present invention claims a pharmaceutical composition.

The pharmaceutical composition claimed in the present invention comprises: (a1) the bispecific antibody described in the first aspect above; (a2) a pharmaceutically acceptable excipient, diluent or carrier.

In the fourth aspect, the present invention claims any of the following methods:

(C1) A method for detecting 4-1BB and/or PD-L1, comprising the steps of: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above to conduct a test sample detection;

(C2) A method for stimulating T cell activation, comprising the following steps: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or the drug described in the third aspect above The composition stimulates T cell activation;

(C3) A method for inhibiting the growth of colon cancer tumors, comprising the following steps: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or or the nucleic acid molecule described in the third aspect above The pharmaceutical composition inhibits colon cancer tumor growth;

(C4) A method for treating and/or preventing colon cancer, comprising the following steps: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or the nucleic acid molecule described in the third aspect above The above-mentioned pharmaceutical composition treatment and/or prevention colon cancer;

(C5) A method for preparing an immune modulator, comprising the following steps: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or the drug described in the third aspect above The composition is used as an active ingredient to prepare an immune modulator;

(C6) A method for treating and/or preventing and/or diagnosing tumors, comprising the steps of: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or the third aspect above The pharmaceutical composition described in the aspect treats and/or prevents and/or diagnoses tumors;

(C7) A method for treating and/or preventing and/or diagnosing an infectious disease, comprising the following steps: using the bispecific antibody described in the first aspect above or the nucleic acid molecule described in the second aspect above or the nucleic acid molecule described in the above The pharmaceutical composition described in the third aspect treats and/or prevents and/or diagnoses infectious diseases.

Wherein, the 4-1BB is human 4-1BB or monkey 4-1BB. The PD-L1 is human PD-L1 or monkey PD-L1.

Wherein, the tumor is a tumor with dysregulated expression of 4-1BB and/or PD-L1; the autoimmune disease is an autoimmune disease with dysregulated expression of 4-1BB and/or PD-L1; and the inflammatory disease is 4-1BB and/or an inflammatory disease with dysregulation of PD-L1 expression; the infectious disease is an infectious disease with dysregulation of 4-1BB and/or PD-L1 expression.

Further, the tumor can be selected from the following: colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumor or Bladder tumors.

Terms and Definitions

BsAb: bispecific antibody, referred to as double antibody.

ScFv: Single-chain variable region antibody fragment, also known as single-chain variable fragment.

FACS: Fluorescence-activated cell sorting, also known as flow cytometry (Fluorescence-activated cell sorting).

BSA: bovine serum albumin.

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, when referring to the amino acid sequence of CD137 protein or 4-1BB protein (UniProt Q07011), it includes the full length of 4-1BB protein, or the extracellular fragment 4-1BB-ECD of 4-1BB; Also included are fusion proteins of 4-1BB-ECD, for example, with a mouse or human IgG Fc protein fragment (mFc or hFc). When referring to the amino acid sequence of the PD-L1 protein (Uniprot #Q9NZQ7), it includes the full length of the PD-L1 protein, or the extracellular fragment PD-L1-ECD of PD-L1; also includes the fusion of PD-L1-ECD protein, such as a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG. In the present invention, the term "4-1BB protein" or "PD-L1 protein" shall include all such sequences, including their natural or artificial variants. And, when describing the sequence fragments of 4-1BB protein or PD-L1 protein, it also includes the corresponding sequence fragments in their natural or artificial variants.

As used herein, the term EC50 refers to the half-maximal effect concentration (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximum effect.

As used in this article, the term R2 is the correlation coefficient in statistics, which refers to the degree of agreement between the test data and the fitting function. ^The closer the R2 value is to ¹ , the higher the degree of agreement, and the closer to 0, the degree of agreement lower.

As used herein, the term MLR refers to mixed lymphocyte reaction (Mixed Lymphocyte Reaction), which refers to the detection of the stimulation of lymphocytes by antibodies or other drugs when normal lymphocytes of two unrelated individuals are mixed and cultured in vitro effect.

As used herein, the term Linker refers to a protein linker or linker element, which connects different target genes through an appropriate nucleotide sequence so that it can be expressed as a single peptide chain in a suitable organism.

As used herein, the term "antibody" or Antibody refers to an immunoglobulin, usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain Molecules. In a general sense, heavy chains can be understood as polypeptide chains with larger molecular weights in antibodies, and light chains refer to polypeptide chains with smaller molecular weights in antibodies. Light chains can be classified into κ and λ light chains. Heavy chains can usually be classified is μ, δ, γ, α, or ε, and defines the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes , in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, the terms "bispecific antibody heavy chain" and "bispecific antibody light chain" mean that when there are two chains in the bispecific antibody structure, the chain with the larger molecular weight is the bispecific The heavy chain of the antibody, and the chain with a smaller molecular weight is the light chain of the bispecific antibody.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses. In addition, the vector may also contain an origin of replication.

As used herein, the term "host cell" refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. In some embodiments of the invention, the term "targeting" refers to specific binding.

As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter abbreviations known in the art. For example, alanine can be represented by A.

As used herein, "Anti-4-1BB" or "parental antibody Anti-4-1BB", unless otherwise specified, is derived from the anti-human 4-1BB monoclonal antibody; "Anti-PD-L1" or "parental antibody Anti-PD-L1", unless otherwise specified, is derived from the Chinese patent application CN 201910174374.8 of Anhui Anke Bioengineering (Group) Co., Ltd. anti-human PD-L1 monoclonal antibody.

Description of drawings

Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention.

Figure 2 is the detection of the molecular weight and purity of the bispecific antibody of the present invention under SDS-PAGE reducing and non-reducing conditions (R means reduced, NR means non-reduced).

Figure 3 shows that the bispecific antibody of the present invention binds to human PD-L1.

Fig. 4 shows that the bispecific antibody of the present invention binds to human 4-1BB.

Figure 5 shows that the bispecific antibody of the present invention simultaneously binds to human PD-L1 and human 4-1BB.

Fig. 6 is a FACS detection of the binding activity of the bispecific antibody of the present invention to human PD-L1.

Fig. 7 is a FACS detection of the binding activity of the bispecific antibody of the present invention to human 4-1BB.

Fig. 8 is an ELISA detection of the binding activity of the bispecific antibody of the present invention to monkey PD-L1.

Fig. 9 is an ELISA detection of the binding activity of the bispecific antibody of the present invention to monkey 4-1BB.

Fig. 10 is MLR detection of IL-2 secretion level of T cells activated by the bispecific antibody of the present invention. In the figure, the concentration of each administration group bar graph from left to right is 10μg/ml, 2μg/ml, 0.4μg/ml, 0.08μg/ml, 0.016μg/ml; IgG concentration from left to right is 10μg/ml ml, 0.4μg/ml, 0.016μg/ml

Figure 11 is the detection of HuPD-L1-dependent activation of CD8 ⁺ T cells.

Fig. 12 is a reporter gene detection of activation of 4-1BB/NFκB activity by the bispecific antibody of the present invention.

Figure 13 is a reporter gene detection bispecific antibody blocking PD-1/PD-L1 activity of the present invention.

Figure 14 shows the anti-tumor efficacy of the bispecific antibody D1 of the present invention.

Figure 15 shows the anti-tumor efficacy of the bispecific antibody D6 of the present invention.

The best way to practice the invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry and immunology used in the following examples are all routine procedures widely used in the corresponding fields.

Example 1. Construction of recombinant vector of anti-human PD-L1/4-1BB bispecific antibody

The sequence of the anti-human PD-L1 monoclonal antibody comes from the patent CN 201910174374.8 of Anhui Anke Bioengineering (Group) Co., Ltd. Patent WO2021093753A1 of Biotech Ltd. The structural schematic diagram of the anti-human PD-L1/4-1BB bispecific antibody of the present invention is shown in Figure 1:

1 ^st Fab-Fc-L-2 ^nd scFv (D structure);

Among them, 1st Fab recognizes PD-L1 antigen, ^2nd scFv recognizes 4-1BB antigen, L is (A( ^EAAAK )4ALE, KVDKKVEPKSCDKTHT or (G4S)n) amino acid sequence, Fc is preferably IgG4.

In structure D, Fc is of the human IgG4 subtype, and the C-terminals of the two heavy chains of the anti-PD-L1 antibody are connected to a single-chain antibody of the anti-human 4-1BB antibody through a Linker. The other chain is the light chain of the anti-PD-L1 antibody.

D1: L is the amino acid sequence of "A(EAAAK)4ALE". The amino acid sequence of the heavy chain is shown in SEQ ID No.5 (the corresponding nucleotide sequence is shown in SEQ ID No.9), and the amino acid sequence of the light chain is shown in SEQ ID No.8 (the corresponding nucleotide sequence is shown in SEQ ID No.8) ID No.12).

D2: L is "(G4S)3" amino acid sequence. The amino acid sequence of the heavy chain is shown in SEQ ID No.6 (the corresponding nucleotide sequence is shown in SEQ ID No.10), and the amino acid sequence of the light chain is shown in SEQ ID No.8 (the corresponding nucleotide sequence is shown in SEQ ID No.8) ID No.12).

D6: Mutated to D6 on the basis of D2, the heavy chain SEQ ID No.6 is mutated from the 61st amino acid and the 101st amino acid at the N-terminal, and can be mutated to glutamic acid "E" individually or simultaneously, or the heavy chain SEQ ID No. ID No.6 is mutated from the 62nd amino acid and the 102nd amino acid at the N-terminal, and can be mutated singly or simultaneously to glycine "G" or alanine "A". The amino acid sequence of the heavy chain is shown in SEQ ID No.7 (the corresponding nucleotide sequence is shown in SEQ ID No.11), and the amino acid sequence of the light chain is shown in SEQ ID No.8 (the corresponding nucleotide sequence is shown in SEQ ID No.8 ID No.12).

The detailed construction process of the bispecific antibody recombinant expression vector is as follows:

The heavy chain nucleotide sequence of the bispecific antibody in the D structure (D1, D2 or D6) of the bispecific antibody was directly synthesized, and the XbaI enzyme cleavage site (TCTAGA) and the kozak consensus recognition sequence (5 '-GCCACC-3'), and signal peptide sequence (5'-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3'), a stop codon and a HindIII restriction site (AAGCTT) were introduced into the C-terminus, and inserted into the same enzyme after double digestion with XbaI and HindIII Cut pcDNA3.4 vector (Invitrogen, Cat: A14697), that is, the recombinant vector for obtaining the heavy chain target gene. The correct recombinant plasmids verified by sequencing were named pcDNA3.4-D1-H, pcDNA3.4-D2-H, and pcDNA3.4-D6-H respectively according to the inserted sequence.

At the same time, the bispecific antibody light chain gene was synthesized, and the XbaI enzyme cleavage site (TCTAGA), the kozak consensus sequence (5'-GCCACC-3'), and the signal peptide sequence (5' -ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGATCCACTGGT-3'), a stop codon and a HindIII restriction site (AAGCTT) were introduced into the C-terminus, the synthetic sequence was double-digested with XbaI and HindIII and then cloned into the pcDNA3.4 vector after the same double-digestion to obtain the The recombinant vector of the light chain gene of the bispecific antibody. The correct recombinant plasmids verified by sequencing were named pcDNA3.4-D1-L, pcDNA3.4-D2-L, and pcDNA3.4-D6-L respectively according to the inserted sequence.

Example 2. Expression and purification of bispecific antibodies

Use the Expi293 expression system (Thermo Fisher) with the recombinant vector corresponding to the structure described in Example 1, transfect and express according to the product manual operating procedures, culture for 5 days, take the supernatant, and use the obtained supernatant with a Protein A affinity chromatography column (GE Company) purification, after affinity purification, use superdex200pg gel (GE Company) to filter and purify, then pass the affinity elution collection liquid through the column at a ratio of 4%, collect the monomer peak, and then use a 30KD concentrated centrifuge tube to concentrate obtain the target molecule.

Purified antibodies were tested for molecular weight and purity by SDS-PAGE under reducing and non-reducing conditions. The results are shown in Figure 2. Under non-reducing conditions, the D structure appears as a substantially single band. Under reducing conditions, it appears as two bands with molecular weights of 80-100KD and 25-30KD, which are consistent with the theoretical molecular weight.

The sequences of bispecific antibodies with D1, D2, and D6 structures prepared above are consistent with those described in Example 1.

Example 3, ELISA determination of bispecific antibody binding activity to human PD-L1 antigen

The binding affinity of the bispecific antibody to human PD-L1 was assessed by ELISA. The full-length amino acid sequence of human PD-L1 is shown in SEQ ID No.15 (Uniprot#Q9NZQ7). Similar to the preparation of the above-mentioned antibody expression, the extracellular segment was used as the target sequence (SEQ ID No.15 from N-terminal 19-238), inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector, and it was verified to be correct by sequencing Afterwards, the target plasmid was obtained and expressed transiently through the Expi293 expression system. A 96-well plate (96-well ELISA plate, Nunc Company) was coated with human PD-L1 at a concentration of 350 ng/ml, 100 μl/well, overnight at 4°C. The plate was washed three times with PBS and blocked for 1 hour at 37°C. The bispecific antibody structures D2 and D6 obtained in Example 2 of the present invention were respectively diluted to 5 nM with PBS, and then diluted 4 times to 7 different concentrations, 100 μl/well, and incubated for 1 hour. The plate was washed three times with PBS, 100 μl of horseradish peroxidase-labeled goat anti-human (goat anti-human-HRP, ThermoFisher Company) diluted 8000 times was added to each well, and shaken for 0.5 hour. The plate was washed three times, and 100 μl TMB (tetramethylbenzidine, ThermoFisher Company) was added to each well. The color was developed in the dark, and 1M sulfuric acid was added to terminate the reaction. The OD value was measured at 450 nm with a Versamax microplate reader (Molecular). According to the antibody and antigen reaction curves, the 4-parameter Logistic fitting method was used to draw the graph, and the test results are shown in Table 1.

The result curve is shown in Figure 3, and there is a good binding between the bispecific antibody and the human PD-L1 antigen.

Table 1. ELISA detection of bispecific antibody binding activity to human PD-L1

Concentration (nM)	D6	D2
EC50(nM)	0.1148	0.1226
R 2	0.9997	0.9986

Example 4, ELISA determination of bispecific antibody binding activity to human 4-1BB antigen

The binding affinity of bispecific antibodies to human 4-1BB was assessed by ELISA. The full-length amino acid sequence of human 4-1BB is shown in SEQ ID No.13 (Uniprot#Q07011). The preparation method is similar to that of antibody protein and human PD-L1 protein, and its extracellular segment is the target sequence (amino acid sequence is shown in SEQ ID No. .13 from the N-terminal 24-186), inserted into the pCDNA3.4 vector to obtain the target plasmid, and expressed transiently through the Expi293 expression system. The concentration of human 4-1BB antigen coating plate was 350ng/ml. The bispecific antibodies D2 and D6 obtained in Example 2 of the present invention were diluted to 10 nM with PBS, and then diluted 4 times into 7 different concentrations. Other operations were the same as in Example 3. The test results are shown in Table 2.

The result curve is shown in Figure 4, there is a good binding between the bispecific antibody and the human 4-1BB antigen, and it is dose-dependent.

Table 2. Binding activity of bispecific antibody to human 4-1BB detected by ELISA

Concentration (nM)	D2	D6
EC50(nM)	0.1088	0.2108
R 2	0.9993	0.9998

Example 5, ELISA method to detect the simultaneous binding characteristics of bispecific antibodies to human PD-L1 and human 4-1BB antigens

The simultaneous binding activity of the bispecific antibody to human PD-L1 and human 4-1BB was evaluated by ELISA. Dilute the extracellular segment of human PD-L1 (the amino acid sequence is shown in the 19th-238th position from the N-terminal of SEQ ID No.15, with His tag, see Example 3 for the preparation method) with PBS to 500ng/mL and coat the plate, Operation is the same as embodiment 3. The bispecific antibodies D2 and D6 obtained in Example 2 of the present invention were diluted to 5 nM respectively, and then 5-fold gradients were made into 7 different concentration samples, 100 μl/well, and incubated for 1 hour. Then add the antigen human 4-1BB with a detection concentration of 500ng/ml (SEQ ID No.13 from N-terminal 24-186, with a mouse Fc tag, see Example 4 for the preparation method), 100 μl/well, and incubate at room temperature for 1 Hour. Dilute goat anti-mouse-HRP 1:2000 with 1% BSA, 100 μL/well, incubate with shaking at room temperature for 0.5 hour; wash the plate three times, add 100 μl TMB to each well. The color was developed in the dark, and 1M sulfuric acid was added to terminate the reaction. The OD value was measured at 450nm with a Versamax microplate reader. According to the antibody and antigen reaction curves, the 4-parameter Logistic fitting method was used to draw the graph, and the test results are shown in Table 3.

The result curve is shown in Figure 5. The bispecific antibody can simultaneously bind to human PD-L1 and human 4-1BB in a dose-dependent manner.

Table 3. Simultaneous binding activity of bispecific antibodies to human PD-L1 and human 4-1BB detected by ELISA

Concentration (nM)	D2	D6
EC50(nM)	0.1680	0.1608
R2	0.9993	0.9994

Example 6. FACS detection of binding properties of bispecific antibody to human PD-L1 antigen on the cell surface

Insert the full-length human PD-L1 (SEQ ID No.15) target sequence into the pCDNA3.4 vector according to the general method in the field to obtain a recombinant plasmid, and then use Lipofectamine 3000 transfection reagent (Invitrogen) to introduce it into wild-type CHO-K1 (ATCC ) cells to obtain the cell line CHO-K1/hPD-L1 highly expressing human PD-L1. Cultivate and collect CHO-K1/hPD-L1 cells in the logarithmic growth phase, wash with about 1ml of PBS, centrifuge and distribute to EP tubes, 2×10 ⁵ cells/tube. The bispecific antibody D6 was diluted to 50 nM with PBS, and then 3-fold serially diluted to 6 concentrations. Add the samples of each concentration to the EP tubes filled with cells in sequence, 100 μl/tube. After incubation for 1 hour, wash twice with 1ml cleaning solution (PBS+2% fetal bovine serum), add goat anti-human FITC secondary antibody (Invitrogen, Cat. No. H10301), incubate in the dark for 30 minutes, wash twice, add to each tube Resuspended in 500 μl PBS, and tested by flow cytometry. According to the antibody and antigen reaction curves, the 4-parameter Logistic fitting method was used to draw the graph, and the test results are shown in Table 4.

Table 4. FACS detection of bispecific antibody binding activity to human PD-L1

Concentration (nM)	D6
EC50(nM)	7.598
R 2	0.99

As shown in Figure 6, there is a good binding between the bispecific antibody and the human PD-L1 antigen, and it is dose-dependent.

Example 7, FACS method to detect the binding characteristics of bispecific antibody and human 4-1BB antigen on the cell surface

Insert the full-length human 4-1BB (SEQ ID No.13) target sequence into the pCDNA3.4 vector to obtain the recombinant plasmid according to the general method in the field, and then use Lipofectamine 3000 transfection reagent (Invitrogen Company) to introduce into wild-type CHO-K1 cells, A cell line CHO-K1/h4-1BB highly expressing human 4-1BB was obtained. Culture and collect CHO-K1/h4-1BB cells in logarithmic growth phase, wash with about PBS, centrifuge and distribute to EP tubes, 2×10 ⁵ cells/tube. The bispecific antibody D6 was diluted to 50 nM with PBS, and then 3-fold serially diluted to 6 concentrations. Add the samples of each concentration to the EP tube containing the cells in turn, 100 μl/tube, and set up a blank control. After incubation for 60 minutes, wash twice with 1ml cleaning solution (PBS+2% fetal bovine serum), add goat anti-human FITC secondary antibody, incubate in the dark for 30 minutes, wash twice, add 500μl PBS to each tube for resuspension, and flow machine detection. According to the antibody and antigen reaction curves, the 4-parameter Logistic fitting method was used to draw the graph, and the test results are shown in Table 5.

Table 5. FACS detection of bispecific antibody binding activity to human 4-1BB

Concentration (nM)	D6
EC50(nM)	16.39
R 2	0.99

As shown in Figure 7, there is a good binding between the bispecific antibody and the human 4-1BB antigen, and it is dose-dependent.

Example 8, ELISA detection of bispecific antibody and monkey PD-L1 antigen binding characteristics

The binding activity of the bispecific antibody to monkey PD-L1 was assessed by ELISA. The amino acid sequence of the extracellular segment of monkey PD-L1 is shown in SEQ ID No.16 (Uniprot#G7PSE7). Similar to the preparation of the above-mentioned antibody expression, the extracellular segment is used as the target sequence to construct a plasmid and express it transiently through the Expi293 expression system. The method is the same as that in Example 3, the specific differences are: the cladding antigen is 500ng/ml monkey PD-L1, the antibody to be tested is the bispecific antibody D6, the highest concentration is 1 μg/ml, and 4-fold gradient dilution is made into 7 different concentrations , and the test results are shown in Table 6. The parental antibody Anti-PD-L1 and Tercentriq (Roche) were selected as control antibodies in the experiment, and Tercentriq was produced and expressed according to Accession Number DB11595 in DrugBank (https://go.drugbank.com).

The result curve is shown in Figure 8. There is a good binding between the bispecific antibody and the monkey PD-L1 antigen, and it is dose-dependent, and the binding activity is not significantly different from that of the parental antibody.

Table 6. ELISA detection of bispecific antibody binding activity to monkey PD-L1

Concentration (μg/ml)	Anti-PD-L1	Tercentriq	D6
EC50(μg/ml)	2.732	3.509	2.659
R 2	0.9998	0.9996	0.9987

Example 9, ELISA detection of bispecific antibody and monkey 4-1BB antigen binding properties

The binding activity of the bispecific antibody to monkey 4-1BB was assessed by ELISA. The amino acid sequence of the extracellular segment of monkey 4-1BB is shown in SEQ ID No.14 (Uniprot#A9YYE7). Similar to the preparation of the above-mentioned antibody expression, the extracellular segment is used as the target sequence to construct a plasmid, which is transiently expressed by HEK293. The method is the same as in Example 3, the specific differences are: the cladding antigen is 500ng/ml monkey 4-1BB, the antibody to be tested is the bispecific antibody D6 obtained in Example 2, the highest concentration is 10nM, and the 4-fold gradient dilution is 7 For different concentrations, the test results are shown in Table 7. The parental antibody Anti-4-1BB was selected as the control antibody in the experiment, and human IgG4 (Sino Biological Company, catalog number HG4K) was used as the irrelevant control antibody.

The result curve is shown in Figure 9. There is a good binding between the bispecific antibody and the monkey 4-1BB antigen in a dose-dependent manner, and the binding activity is not significantly different from that of the parental antibody.

Table 7. Binding activity of bispecific antibody to monkey 4-1BB detected by ELISA

Concentration (nM)	Anti 4-1BB	human IgG	D6
EC50(nM)	0.1463	N/A	0.1672
R 2	0.9984	N/A	0.9988

Example 10. In Vitro Drug Efficacy Detection of the Activation Effect of Bispecific Antibodies on T Lymphocytes

Whole blood was obtained from healthy person A, and PBMC cells were separated using lymphocyte separation medium (Sigma Company) according to its instructions, and set aside. Obtain whole blood from healthy person B, use lymphocyte separation medium to separate PBMC cells, and then use EasySep ^TM Human CD14Positive Selection Kit II (Stemcell Company) to separate and obtain DC cells, and resuspend the cells in 10ng/ml IL-6 , 10ng/mL IL-1β, 10ng/mL TNF-α and 1μg/mL PGE2 medium to induce maturation.

Dilute the bispecific antibody D6 prepared in Example 2 and the parental antibodies Anti-4-1BB and Anti-PD-L1 to 10 μg/ml respectively, and dilute to 5 concentrations (10 μg/ml, 2 μg/ml, 2 μg/ml, 0.4μg/ml, 0.08μg/ml, 0.016μg/ml). Set irrelevant antibody human IgG4 (Sino Biological Company, Cat. No. HG4K), the concentration is set to 10 μg/ml, 0.4 μg/ml, 0.016 μg/ml. The obtained spare PBMC and DC cells were added to a 96-well plate at a ratio of 10:1, and the PBMC was 1×10 ⁵ per well. The volume is 100 μl/well, and then the diluted antibody to be tested is added, 100 μl/well. After 3 days of incubation, the secretion level of IL-2 in the supernatant was detected.

The results are shown in Figure 10. Compared with the two parental monoclonal antibodies and the negative unrelated control antibody, the bispecific antibody D6 can activate T cells in the mixed lymphoid reaction system and further increase the secretion level of IL-2 in the cell supernatant.

Example 11. In Vitro Drug Efficacy Test Bispecific Antibody T Cell Activation Effect Depends on PD-L1

As above, whole blood was obtained from a healthy person, and PBMCs were separated to obtain human CD8 ⁺ T cell magnetic beads (BD Company, product number 557766), and human CD8 ⁺ T cells were separated and purified according to the instructions, and then used for future use.

The anti-CD3 antibody (Biolegend, product number 317325) was diluted to 0.5 μg/ml with PBS, added to a 96-well plate (Corning) and incubated at 37° C. for 1 hour. Then CHO-K1/hPD-L1 (see Example 6) and CHO-K1 cells were added to the 96-well plate in different ratios (0:4; 1:3; 2:2; 3:1; 4:0), The total cells are 5000/well. After placing the 96-well plate in a cell incubator and incubating for 6 h, the cell supernatant was aspirated, and 100 μl of human CD8 ⁺ T cells were added to each well, 2.5×10 ⁴ cells/well. Dilute the antibody to be tested (D6) to 2 μg/ml with culture medium, and then dilute to 5 concentrations in a 20-fold gradient. Add the diluted antibody to a 96-well plate, 100 μl/well, and set 3 replicate wells for each concentration. The 96-well plate was placed in a cell culture incubator and incubated for 3 days, and the secretion of cytokine IFN-γ in the cell culture supernatant was detected by ELISA.

The results are shown in Figure 11, bispecific antibodies can activate CD8 ⁺ T cells, and this activation is dependent on PD-L1. When there is no PD-L1 in the system, the double antibody cannot activate CD8 ⁺ T cells. With the increase of the expression of PD-L1, the maximum ability of the double antibody to activate CD8 ⁺ T cells is getting higher and higher, but the half-effective concentration of the antibody does not change. Big.

Example 12, reporter gene method to detect bispecific antibody activity

Insert the full-length human 4-1BB sequence as the target gene between the XbaI and HindIII sites of the pCDNA3.4 vector (see Example 7), and obtain plasmid A after sequencing and verifying that it is correct. ID No.15) and the luciferase gene (sequence shown in SEQ ID No.16) plasmid B (pNFκB-luciferase, Youbao Bio, product number VT1588). Then, the two plasmids A and B were introduced into HEK293 cells (Shanghai Cell Bank, Chinese Academy of Sciences) with Lipofectamine 3000 transfection reagent (Invitrogen). Through pressure screening, HEK-293/NFκB-Luci/4-1BB was obtained.

HEK-293/NFκB-Luci/4-1BB cells and CHO-K1/hPD-L1 cells in the logarithmic growth phase (see Example 6) were taken, and 50 μl of each of the two cells was added to a 96-well plate (Corning, 3917 ), both cells were 3×10 ⁴ per well. Dilute the antibody to be tested (D6) to 20 μg/ml l (in the experiment, set the control with the combination of the parental antibody Anti 4-1BB and Anti PD-L1 at the same time, after 5-fold serial dilution of 9 concentrations, add to the 96-well plate sequentially, 100 μl After standing in the incubator for 18-24 hours, add 100 μl of ONE-Glo Luciferase assay sysytem reagent (Promega) and incubate at room temperature for 10 minutes to detect the chemiluminescence value.

The results are shown in Figure 12. The bispecific antibody can activate the NFκB signaling pathway downstream of 4-1BB depending on PD-L1, thereby enhancing the detection signal value, and then the two parental monoclonal antibodies are used in combination, because the Anti4-1BB monoclonal antibody loses Crosslinking can not activate 4-1BB downstream NFκB signaling pathway. PD-L1 monoclonal antibody can only bind CHO-K1/hPD-L1, and cannot act on this signaling pathway.

In a similar manner, Jurkat/NFAT-Luci/PD1 cells stably expressing human PD-1 and NFAT elements and CHO-K1/PDL1/TCR cells expressing human PD-L1 and TCR activating protein were constructed, and the two cells were divided into 50000:25000, Add to 96-well plate. The antibody D6 to be tested was diluted to 12 μg/ml, and after 3-fold serial dilution to 9 concentrations, it was sequentially added to a 96-well plate, 100 μl/well. After standing in the incubator for 18-24 hours, add 100 μl of ONE-Glo Luciferase assay sysytem reagent (Promega) and incubate at room temperature for 10 minutes to detect the chemiluminescence value.

The results are shown in Figure 13. The bispecific antibody can block the PD-1/PD-L1 signaling pathway, which means that the bispecific antibody can also exert the inhibitory function of the PD-L1 antibody.

Example 13. Inhibitory effect of bispecific antibody D1 on tumor growth

In the experiment, PD-1/4-1BB double humanized mice (B-hPD-1/h4-1BB mice) were selected to test the bispecific anti-tumor efficacy in vivo. B-hPD-1/h4-1BB mice are derived from Biocytogen (Cat. No. 110004), which is chimerized with human h4-1BB gene and human PD-L1 in the genome of C57BL/6 mice.

The murine colon cancer MC38 cell line was subcutaneously inoculated on the back (shaved) side of the tested mice (5×10 ⁵ cells per mouse, 100 μl). When the average tumor volume of the tumor-bearing mice reached 100 mm ³ , the mice were randomly divided into 3 groups according to the experimental design, with 5 mice in each group. Following tumor inoculation, dosing was administered twice weekly and tumor volumes were measured. The formula for calculating the volume is 1/2×length×width×width (mm ³ ). The day of group administration was defined as day 0. The grouping situation and dosing regimen are shown in Table 8:

Table 8, grouping and dosing regimen

Note: Anti PD-L1+Anti 4-1BB is the control for the combination of parental antibody Anti 4-1BB and Anti PD-L1.

The experimental results are shown in Figure 14. Compared with the normal saline group and the two monoclonal antibody combination groups, the tumor growth of the mice was effectively inhibited after administration of the bispecific antibody D1.

Example 14. Inhibitory effect of bispecific antibody D6 on tumor growth

As in Example 13, the same B-hPD-1/h4-1BB mice were selected to detect the anti-tumor efficacy of the bispecific antibody D6. The MC38 cell line (2×10 ⁶ cells, 100 μl) was inoculated in the tested mice. When the average tumor volume reached 150 mm , the mice ^were randomly grouped according to the experimental design, the grouping situation and the dosing regimen were as shown in Table 9, and other operations were the same as in Example 13:

Table 9, grouping and dosing regimen

The experimental results are shown in Figure 15, the bispecific antibody D6 has obvious anti-tumor efficacy, and the efficacy is dose-dependent. At a dose of 2 mg/kg, the tumor inhibition rate reached 99.87%.

Example 15. In vivo toxicological detection of bispecific antibody in rhesus monkeys

Four rhesus monkeys were selected for the experiment to evaluate the toxic and side effects of the bispecific antibody in monkeys. The rhesus monkeys were divided into two groups, high dose and low dose, one female and one male in each group, the high dose was 50 mg/kg, the low dose was 5 mg/kg, and the specific administration sample was D6, administered intravenously once a week times, continuous administration for 4 weeks, a total of 4 administrations. During the adaptation period, observe at least once a day in the morning and afternoon. On the day of administration, observe once before administration, observe once after administration, and observe at least once in the morning and afternoon each day on non-administration days. When animals show obvious symptoms of toxicity, the frequency of observation is increased, and the time is recorded. The results are shown in table 10, after 4 weeks of repeated administration of high and low doses, ALT (alanine aminotransferase) and AST (astpartate aminotransferase) of rhesus monkeys were not significantly increased compared with the adaptation period, showing well tolerated. On the other hand, the number of white blood cells in the peripheral blood of rhesus monkeys and the proportion of lymphocytes in them increased slightly, but they were all within the normal range. The general state, respiration, heart rate, hematology, liver function, The renal functions were normal, and the animals tolerated it well.

Table 10. Toxicological test of repeated administration in rhesus monkeys for 4 weeks

Remarks: On the first day (P1) of the adaptation period (Pretest Phase), the day of the first administration is defined as the first day (D1) of the dosing phase (Dosing Phase), WBC is white blood cells, and %LYMPH is the percentage of lymphocytes.

industrial application

The bispecific antibody of the present invention has good stability and high safety. It can not only block the combination of PD-1 and PD-L1, but also activate the human 4-1BB signaling pathway, stimulate T cell activation, and significantly increase IL-1. 2. The expression level of IFN-γ, indicating that the antibody can regulate the immune system by regulating the activity of immune cells, and can be used as an immune enhancer for anti-tumor or anti-viral immune response, or for autoimmune diseases mediated by T cells The immunomodulator can also be used to prepare medicines for treating tumors. Therefore, the use of the bispecific antibody provided by the present invention in the preparation of antibody-targeted drugs has great significance and application potential.

Claims (16)

1. A bispecific antibody targeting PD-L1 and 4-1BB, containing a PD-L1 antigen-binding domain and a 4-1BB antigen-binding domain;

   The PD-L1 antigen-binding domain includes a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as shown in SEQ ID No.1 26- 32, 52-56, and 98-107; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as follows: 24-36, 52 of SEQ ID No.2 -shown at positions 58 and 93-100; wherein, the amino acid sequence of the HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain or the 98th- as in SEQ ID No.7 107 as shown;

   The 4-1BB antigen binding domain comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as shown in the 31-th sequence of SEQ ID No.3. 35, 50-65, and 98-106; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as follows: 24-34, 50 of SEQ ID No.4 -56 and 89-97 are shown.
2. The bispecific antibody according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region is the 1-118th position of SEQ ID No.1 or SEQ ID No.7, or the same as SEQ ID The 1-118 positions of No.1 or SEQ ID No.7 have a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75%.
3. The bispecific antibody according to claim 1, characterized in that: in the PD-L1 antigen-binding domain, the amino acid sequence of the light chain variable region is SEQ ID No.2 or SEQ ID No.8 1-110, or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or 75% of the 1-110 positions of SEQ ID No.2 or SEQ ID No.8 Consistency of the above.
4. The bispecific antibody according to claim 1, characterized in that: in the 4-1BB antigen-binding domain, the amino acid sequence of the heavy chain variable region is SEQ ID No.3 or SEQ ID No.7 586-702, or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of the 586-702 of SEQ ID No.3 or SEQ ID No.7 consistency.
5. The bispecific antibody according to claim 1, characterized in that: in the 4-1BB antigen-binding domain, the amino acid sequence of the light chain variable region is SEQ ID No.4 or SEQ ID No.7 459-565, or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of the 459-565 of SEQ ID No.4 or SEQ ID No.7 consistency.
6. The bispecific antibody according to any one of claims 1-5, wherein the structure of the bispecific antibody from the N-terminal to the C-terminal is:

   1 ^st Fab-Fc-L-2 ^nd scFv;

   Among them, the 1st ^Fab recognizes the PD-L1 antigen, the ^2nd scFv recognizes the 4-1BB antigen, and L is the connecting peptide.
7. The bispecific antibody according to claim 6, wherein the connecting peptide is selected from the following: A(EAAAK) ₄ ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n; wherein, n is a positive integer, preferably, n=4.
8. The bispecific antibody according to any one of claims 1-7, characterized in that: the bispecific antibody comprises an Fc segment;

   The Fc segment contains a mutation or does not contain a mutation site;

   The Fc segment is IgG1, IgG2, IgG3 or IgG4 type, preferably IgG4 type.
9. The bispecific antibody targeting PD-L1 and 4-1BB is characterized in that: the bispecific antibody is any of the following:

   (A) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all shown in SEQ ID No.5, and the amino acid sequences of the light chains are all shown in SEQ ID No.8;

   (B) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all shown in SEQ ID No.6, and the amino acid sequences of the light chains are all shown in SEQ ID No.8;

   (C) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all shown in SEQ ID No.7, and the amino acid sequences of the light chains are all shown in SEQ ID No.8.
10. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1-9.
11. The nucleic acid molecule according to claim 10, characterized in that: in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region in the PD-L1 antigen binding domain is SEQ ID No.19 or SEQ ID No.19 1-354 of ID No.11, or have more than 99%, more than 95%, more than 90%, more than 85%, or 80% of SEQ ID No.19 or 1-354 of SEQ ID No.11 Above or above 75% consistency.
12. The nucleic acid molecule according to claim 10, characterized in that: in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region in the PD-L1 antigen-binding domain is SEQ ID No.20 or SEQ ID No.20. The 1-330th of ID No.12, or have more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% of the first-330th of SEQ ID No.20 or SEQ ID No.12 Or more than 75% agreement.
13. The nucleic acid molecule according to claim 10, characterized in that: in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region in the 4-1BB antigen binding domain is SEQ ID No.21 or SEQ ID No.21 1756-2106 of ID No.11, or have more than 99%, more than 95%, more than 90%, more than 85%, more than 80% of SEQ ID No.21 or 1756-2106 of SEQ ID No.11 Or more than 75% agreement.
14. The nucleic acid molecule according to claim 10, characterized in that: in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region in the 4-1BB antigen binding domain is SEQ ID No.22 or SEQ ID No.22 No. 1375-1695 of ID No.11, or more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of SEQ ID No.22 or 1375-1695 of SEQ ID No.11 Or more than 75% agreement.
15. A pharmaceutical composition, comprising: (a1) the bispecific antibody according to any one of claims 1-9; (a2) a pharmaceutically acceptable excipient, diluent or carrier.
16. Either of the following methods:

    (C1) A method for detecting 4-1BB and/or PD-L1, comprising the steps of: using the bispecific antibody described in any one of claims 1-9 or the bispecific antibody described in any one of claims 10-14 Detection of nucleic acid molecules or samples to be tested;

    (C2) A method for stimulating T cell activation, comprising the steps of: using the bispecific antibody according to any one of claims 1-9 or the nucleic acid molecule according to any one of claims 10-14 or claim 15 The pharmaceutical composition stimulates T cell activation;

    (C3) A method for inhibiting the growth of colon cancer tumors, comprising the steps of: using the bispecific antibody according to any one of claims 1-9 or the nucleic acid molecule according to any one of claims 10-14 or any one of claims The pharmaceutical composition described in 15 inhibits colon cancer tumor growth;

    (C4) A method for treating and/or preventing colon cancer, comprising the steps of: using the bispecific antibody described in any one of claims 1-9 or the nucleic acid molecule described in any one of claims 10-14 or The pharmaceutical composition according to claim 15 treats and/or prevents colon cancer;

    (C5) A method for preparing an immune modulator, comprising the steps of: using the bispecific antibody according to any one of claims 1-9 or the nucleic acid molecule according to any one of claims 10-14 or claim 15 The pharmaceutical composition is used as an active ingredient to prepare an immune modulator;

    (C6) A method for treating and/or preventing and/or diagnosing tumors, comprising the steps of: using the bispecific antibody described in any one of claims 1-9 or the bispecific antibody described in any one of claims 10-14 The nucleic acid molecule or the pharmaceutical composition according to claim 15 treats and/or prevents and/or diagnoses tumors;

    (C7) A method for treating and/or preventing and/or diagnosing infectious diseases, comprising the steps of: using the bispecific antibody according to any one of claims 1-9 or any one of claims 10-14 The nucleic acid molecule described in claim 15 or the pharmaceutical composition described in claim 15 can be used to treat and/or prevent and/or diagnose infectious diseases.

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