WO2022089418A1

WO2022089418A1 - Fusion protein of recombinant truncated flt3 ligand and human antibody fc

Info

Publication number: WO2022089418A1
Application number: PCT/CN2021/126370
Authority: WO
Inventors: 胡思怡; 付凤根; 伍伟伟
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2020-10-26
Filing date: 2021-10-26
Publication date: 2022-05-05

Abstract

Provided is a fusion protein applicable in a tumor treatment and a hematopoietic stem cell transplantation. Specifically, provided is a fusion protein having excellent pharmaceutical properties and stability and formed by a truncated FLT3 ligand and an antibody Fc region. Also provided are nucleic acids encoding the fusion protein, a vector comprising the nucleic acids, a host cell, and a method for producing the fusion protein. Also provided are a pharmaceutical composition and a pharmaceutical concomitant comprising the fusion protein, and medicinal uses thereof.

Description

Fusion protein of recombinant truncated FLT3 ligand and human antibody Fc

technical field

The present invention relates to fusion proteins suitable for tumor therapy and hematopoietic stem cell transplantation. More specifically, the present invention relates to a fusion protein formed by a truncated FLT3 ligand and an antibody Fc region with good druggable properties and stability. The present invention also relates to nucleic acids encoding the fusion proteins, vectors comprising the nucleic acids, host cells, and methods of producing the fusion proteins. The present invention also relates to pharmaceutical compositions and pharmaceutical combinations comprising the fusion proteins, and their medical uses.

Background technique

Fms-like tyrosine kinase 3 (FLT3) is widely expressed in hematopoietic stem cells (HSCs), precursor cells, immature lymphocytes and dendritic cells (DCs). FLT3 ligand (FLT3L, also known as SL cytokine) is the only ligand that has been found to specifically bind to FLT3. The expression of FLT3 ligand is the highest in human peripheral blood mononuclear cells, and it is also highly expressed in the heart, placenta, lung, spleen, thymus and other tissues. FLT3 ligands are cytokines that promote the proliferation and differentiation of hematopoietic stem cells and precursor cells, and play a key role in the differentiation, proliferation and mobilization of dendritic cells (DCs). Therefore, FLT3 ligands have good application prospects in tumor immunotherapy.

Numerous studies have shown that the main antitumor mechanism of FLT3 ligands is to promote the proliferation of DCs throughout the body and within the tumor, and to improve the ability of DCs to present antigens expressed by the tumor. FLT3 ligands can be used in combination with radiotherapy, chemotherapy, targeted drugs, immune checkpoint inhibitors, RNA vaccines and other drugs, and their effectiveness has been tested in various animal models such as leukemia, fibrosarcoma, melanoma, breast cancer, and lung cancer. be proven. In particular, FLT3L combined with local tumor radiotherapy can induce tumor cell death, release a large number of tumor antigens, and the expanded DC cells effectively present antigens, stimulate antigen-specific T cell proliferation, activation and migration, and ultimately kill tumor cells.

Human FLT3 ligand is a transmembrane protein with a total of 235 amino acids, consisting of a signal peptide (26aa), an extracellular domain (156aa), a transmembrane domain (23aa) and a cytoplasmic domain (30aa). The region contains multiple N-glycosylation sites, but the glycosylation sites are not directly involved in binding to FLT3. The homology of human and mouse FLT3L is 72%, and its function is cross species. FLT3 ligands exist in the human body in two forms: full-length protein and secreted protein, both of which can bind to FLT3 and exert biological functions. In the early stage, Immunex selected the extracellular region gene of FLT3 ligand, removed 3 amino acids at the C-terminal, and constructed a recombinant FLT3 ligand protein drug, named CDX301. The results of the phase I clinical study showed that CDX301 has a good safety profile in humans, with no dose-limiting toxicity, but the effect of single-use tumor therapy is very limited. CDX301 combined with intratumoral in situ radiotherapy (SBRT) has achieved good results in the treatment of advanced non-small cell lung cancer (NSCLC) patients who have relapsed after PD-1/PD-L1 monoclonal antibody therapy, and progression-free survival (PFS) and overall survival (OS) was significantly prolonged. In addition, CDX301 combined with CD40 monoclonal antibody also has a certain anti-tumor effect for advanced NSCLC patients. Compared with CD40 monoclonal antibody, it does not increase the safety problem.

The recombinant FLT3 ligand protein drug CDX301 contains 153 amino acids and forms a homodimer through non-covalent interactions with a molecular weight of about 35KDa. The biggest disadvantage of CDX301 is its small molecular weight and short half-life (t1/2) in the human body, about 13 hours under the condition of 10ug/kg/day and about 28 hours under the condition of 75ug/kg/day. For tumor treatment, CDX301 needs to be injected subcutaneously once a day for 5-10 consecutive days, and the convenience of administration is poor.

Therefore, linking the FLT3 ligand with the antibody Fc fragment to construct a cytokine fusion protein will be beneficial to prolong the half-life of the FLT3L ligand, reduce the frequency of administration, and improve the anti-tumor effect. Tu Hua et al. linked mouse FLT3 ligand with human IgG1 Fc fragment to form a fusion protein (msFLT3L-huFc). After i.p. injection into mice, compared with recombinant mouse FLT3 ligand protein, peak drug concentration (Cmax), half-life ( t1/2), the drug exposure from 0 to 24 hours (AUC0→24h) was significantly increased (J Immunol Methods. 2014, 413: 69-73). CITIC State Construction Corporation linked the amino acids of the extracellular region of the FLT3 ligand to the human IgG1 Fc fragment through a 15-amino acid polypeptide (3xSer3Ala2) sequence. Compared with the fusion protein without the addition of the polypeptide sequence, the affinity of the constructed fusion protein was increased by 5 times. However, none of these studies paid attention to the stability and druggability of the fusion protein composed of FLT3 ligand and Fc.

In view of the good application prospects of FLT-3 ligands in tumor immunotherapy, there is still a need in the art to continue to develop FLT-3 ligand proteins with good anti-tumor effects and improved druggability and stability.

SUMMARY OF THE INVENTION

Through in-depth research, the inventors found that the C-terminal near-membrane sequence of the FLT3 ligand was removed by truncating the extracellular region of the FLT3 ligand, and the truncated FLT3 ligand was fused to the Fc region of the antibody through a flexible linker with a length of 10-20 amino acids, The resulting fusion protein satisfies the above requirements, and the molecule has enhanced druggability and stability while having good biological activity.

Based on this finding, therefore, in one aspect, the present invention provides a novel FLT3 ligand-human antibody Fc fragment fusion protein comprising, from the N-terminus to the C-terminus: a C-terminally truncated FLT3L extracellular domain (in the Also referred to herein as truncated FLT3L, or FLT3L-D), linker sequences, and antibody Fc regions (FLT3L-linker-Fc).

In some preferred embodiments, the fusion protein of the present invention has the following characteristics:

(1) The truncated FLT3L has removed the C-terminal near-membrane region amino acid sequence from the extracellular region of the FLT3 ligand, i.e., according to the amino acid residue numbering of SEQ ID NO: 1, and after amino acid position 160 of SEQ ID NO: 1 The corresponding amino acid sequence of the sequence DSSTLPPPWSPRPLEATAPTAP;

(2) the linker is 5-25 amino acids in length, preferably 10-20, such as 10 or 15 amino acids, and more preferably has the sequence (G4S)3 or (G4S)2;

(3) The truncated FLT3L is fused to the N-terminus of a human IgG Fc region through the linker, preferably the Fc region has amino acid mutations that remove binding to Fc receptors, such as L234A and L235A mutations.

Therefore, in some preferred embodiments, the fusion protein of the present invention has the following characteristics:

(1) When expressed in mammalian cells, bivalent molecules are formed through disulfide bonds in the Fc hinge region. Compared with natural FLT3 ligands, the molecular weight is increased by about 2.5 times, and the in vivo half-life is prolonged, which is conducive to reducing the frequency of administration;

(2) Retain the affinity and biological activity similar to natural FLT3 ligands, effectively stimulate DC cell proliferation in vitro and anti-tumor immune response in vivo;

(3) Compared with fusion proteins that retain the intact FLT3 ligand extracellular domain, the stability of the molecule is significantly enhanced, for example, fragmentation and degradation under high temperature conditions are reduced.

In yet another aspect, the present invention also provides nucleic acid sequences encoding fusion proteins according to the present invention, vectors and host cells comprising the same; and methods of producing fusion proteins of the present invention using said nucleic acid sequences.

In yet another aspect, the present invention also provides a pharmaceutical composition and a pharmaceutical combination comprising the fusion protein of the present invention or the nucleic acid sequence encoding it, optionally, the pharmaceutical composition or the pharmaceutical combination further comprises an anti-PD-1 antibody.

In a further aspect, the present invention also provides in vitro and in vivo applications of the fusion proteins of the present invention or their encoding nucleic acid sequences, including therapeutic applications, such as applications in tumor treatment and hematopoietic stem cell transplantation.

Brief Description of Drawings

Figure 1 shows the schematic structure (A) of the FLT3 ligand in the form of a full-length protein and the non-membrane-bound molecule CDX-301; and the amino acid sequence of the human FLT3 ligand (B).

Figure 2 shows the Zenix chromatographic detection results of the three candidate molecules and Benchmark.

Figure 3 shows the HIC chromatographic detection results of the three candidate molecules and Benchmark.

Figure 4 shows changes in the purity of candidate molecules and Benchmark molecules detected by SEC-HPLC after 0, 1, and 2 weeks in the accelerated stability test at 40°C.

Figure 5 shows the change in biological activity of candidate molecules determined by CCK8 staining method after accelerated stability test at 40°C.

Figure 6 shows the results of in vitro stimulation of DC expansion and phenotypic analysis by candidate fusion protein molecules.

Figure 7 shows the in vivo antitumor effects of candidate fusion protein molecules and Benchmark molecules after a single dose (A); and the in vivo antitumor effects of candidate molecules under conditions of reduced dosing frequency relative to Benchmark molecules (B).

Figure 8 shows that, in in vivo experiments, candidate fusion protein molecules significantly stimulate the number of immune cell DCs within the tumor.

Figure 9 shows the results of in vivo PK studies in mice of candidate fusion protein molecules.

Detailed description of the invention

The present invention provides a FLT3 ligand fusion protein, which has a unique FLT3 ligand truncated structure, has high affinity and high specificity for binding to the human FLT3 receptor, and exhibits good pharmacological activity, good druggability and stability sex. The fusion proteins provided by the present invention can be used as therapeutic means for tumors and cancers.

definition

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill in the art.

For easier understanding of the present invention, certain scientific and technical terms are specifically defined as follows. Unless explicitly defined otherwise elsewhere herein, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including in the claims), the singular includes its corresponding plural unless the context clearly dictates otherwise.

The term "about" refers to a value within an acceptable error range for a particular value as determined by one of ordinary skill in the art, the error range being dependent upon the measurement means used to measure or determine the value, i.e., the limitations of the measurement system . For example, "about" can mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" may refer to a range of up to 5%, 10%, or 20% (ie, ±5%, ±10%, or ±20%).

The term "and/or" when used in conjunction with two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps. Herein, when the term "comprising" or "comprising" is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps. For example, reference to a fusion protein "comprising" a particular sequence is also intended to encompass fusion proteins consisting of that particular sequence.

As used herein, the term "FLT3" refers to the FMS like tyrosine kinase 3 receptor. It acts as a cell surface-bound receptor for FLT3L ligands, regulating the differentiation, proliferation and survival of hematopoietic precursor cells and dendritic cells. As used herein, unless otherwise stated, the term refers to any native FLT3 from any vertebrate (including mammals such as primates (eg, humans) and rodents (eg, mice and rats), but preferably is a FLT3 protein from human, such as the human FLT3 protein disclosed by UniProtKB Accession No. P36888.

As used herein, the term "FLT3L fusion protein" refers to an immunofusion protein comprising a non-membrane-bound FLT3 ligand fused to the Fc region of an antibody. With regard to the fusion proteins of the present invention, in some preferred embodiments, truncated non-membrane-bound FLT3 ligand-linker-antibody Fc regions are included in order from the N-terminus to the C-terminus. In some embodiments, the FLT3L ligand is a truncated FLT3 ligand polypeptide from human, or a variant thereof. In certain preferred embodiments, the FLT3L-Fc fusion protein comprises a truncated human FLT3L polypeptide linked to a human IgG Fc region. In certain embodiments, the FLT3L-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:3 or 4. However, it is to be understood that the present invention also encompasses minor sequence variations in the FLT3L and/or Fc regions, such as insertions, deletions, substitutions, especially conservative amino acid substitutions, that do not affect the function and/or activity of the FLT3L or FLT3L-Fc fusion protein . The FLT3L-Fc fusion proteins of the present invention can bind to the FLT3L receptor, which can lead to downstream signaling of the FLT3L receptor. FLT3L-Fc fusion protein function and/or activity can be assayed by methods known in the art including, but not limited to, ELISA, ligand-receptor binding assays, and CCK8 staining assays. In certain embodiments, the present invention provides a FLT3L-Fc fusion protein that binds to the FLT3L receptor, wherein the binding can result in downstream signaling of the FLT3L receptor, the FLT3L-Fc fusion protein comprising and SEQ ID NO: 3 or 4 amino acid sequences with at least 95% sequence identity. In certain embodiments, the Fc region of the FLT3L fusion protein has no effector activity (eg, does not bind to FcyIIIR) or exhibits substantially lower effector activity than an intact (eg, wild-type) IgG antibody. In certain other embodiments, the Fc region of the FLT3L-Fc fusion protein does not trigger cytotoxicity, such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

As used herein, the expression "numbering of amino acid residues according to SEQ ID NO: 1" means that the FLT-3 ligand polypeptide (including full length, extracellular region and truncated forms) to be numbered is compared with SEQ ID NO: 1 Carry out amino acid sequence alignment, thereby according to the numbering of amino acid residues on SEQ ID NO:1, determine the numbering of the amino acid at the corresponding position on the polypeptide. Sequence alignments can be performed using the BLAST algorithm publicly available from the NCBI website, using default parameters.

As used herein, the term "conservative substitution" means amino acid substitutions that do not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to the substitution of one amino acid for another with similar chemical properties (eg, charge or hydrophobicity). Conservative substitution tables for functionally similar amino acids are well known in the art. In the present invention, conservatively substituted residues are derived from the following conservative substitutions Table X, especially the preferred conservative amino acid substitution residues in Table X.

Table X

原始残基original residue	示例性取代Exemplary substitution	优选保守氨基酸取代Preferred conservative amino acid substitutions
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp；Lys；ArgGln; His; Asp; Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn;Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp;Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val；SerVal; Ser	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

For example, the fusion proteins of the present invention may have conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 3 or 4, or only conservative amino acid substitutions, and in a preferred embodiment, conservative substitutions do not exceed, for example, 30, 20, or 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residues.

The term "isolated" FLT3L fusion protein herein refers to the purified state of the molecule. For example, "isolated" can mean that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cellular debris and growth media. However, as known to those skilled in the art, the term "isolated" does not mean the complete absence of such substances or the absence of water, buffers or salts unless they would significantly interfere with experimental or therapeutic applications of the antibodies described herein amount exists. In some embodiments, the isolated fusion protein can have a purity of greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC).

Herein, the terms "Fc region" or "antibody Fc" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass naturally occurring various immunoglobulin Fc sequences, such as Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi : 10.3389/fimmu.2014.00520.) In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, such as Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National As described in Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. In some embodiments, the antibody Fc region may carry a hinge sequence or part of a hinge sequence at the N-terminus, eg, according to EU numbering, the sequence E216 to T225 or the sequence D221 to T225.

The terms "Fc region variant" or "variant Fc region" are used interchangeably herein to refer to an Fc region polypeptide comprising modifications relative to a native sequence Fc region. The Fc region variants of the present invention are defined in terms of the amino acid modifications that compose them. Thus, for example, L234A is an Fc region variant with an alanine substituted for leucine at position 234 relative to the parent polypeptide, wherein numbering is according to the EU index. Modifications can be additions, deletions or substitutions. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may contain unnatural amino acids. The purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector function elicited thereby.

The term "Fc receptor" or "FcR" herein describes a receptor that binds the Fc region of an antibody. In some embodiments, the FcR is a native human FcR, eg, an FcyR (gamma receptor). In some embodiments, the antibody Fc region of the fusion proteins of the invention comprises mutations that reduce or eliminate binding to one or more FcRs to reduce side effects that may arise from the Fc region and FcRs on the surface of immune cells. In a preferred embodiment, the present invention comprises mutations that reduce the binding of the Fc region to FcyRs, eg, L234A and L235A.

As used herein, a linker refers to a flexible polypeptide amino acid sequence, usually within 50 amino acids in length.

As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. "Percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences. ) or the same amino acid residue (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to yield the percent sequence identity. Optimal alignment for determination of percent sequence identity can be achieved in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.

As used herein, "affinity" or "binding affinity" may be used to reflect the intrinsic binding capacity of the interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant ( _kdis and _kon , respectively ₎ . Binding affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio affinity assay technology herein.

The term "vector" herein refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors," which refer to nucleic acids capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.

Aspects of the present invention are described in further detail in the following subsections.

1. FLT3L fusion protein of the present invention

Favorable biological properties of the FLT3L fusion proteins of the invention

The inventors have found through intensive research that by deleting the C-terminal membrane proximal region sequence of FLT3L (for example, in the case of SEQ ID NO: 2, DSSTLPPPWSPRPLEATAPTAP) and fusing it with the Fc region through a flexible linker, not only can the truncated FLT3L- The properties of D-Linker-Fc molecule are improved in drugability and stability; and it can fully achieve biological efficacy equivalent to or better than that of natural FLT3 ligand, and at the same time, it is beneficial to prolong the molecular half-life and reduce the frequency of administration.

In some embodiments, therefore, the present invention provides FLT3L fusion proteins having at least one or more of the following biological activities:

(a) specifically binds to the FLT3 receptor expressed on the surface of immune cells;

(b) Activation of the cellular FLT3 receptor signaling pathway through the FLT3 receptor;

(c) promoting hematopoietic stem cells and precursor cells to proliferate and differentiate into DC cells;

(d) promoting dendritic cell (DC) proliferation, optionally, showing a synergistic effect on increasing DC proliferation efficiency after combined use with stem cell stimulating factor;

(e) improving the ability of DC cells to present tumor antigens;

(f) enhancing antigen-specific T cell immune responses;

(g) It has tumor-suppressive activity, and exhibits synergistically enhanced anti-tumor effect when used in combination with other anti-tumor drugs (eg, anti-PD1 antibodies).

In some embodiments, the fusion protein of the present invention binds to the human FLT3 receptor with high affinity based on the determination of biofilm layer interferometry, wherein,

(a) The equilibrium dissociation constant KD value is less than 1×10 ⁻⁷ M, preferably 1-5×10 ⁻⁸ M, especially 1-3×10 ⁻⁸ M, or the affinity is substantially lower than that of the corresponding human FLT3 ligand unfused to the Fc region The same, or higher, for example, the KD value is about 2-3 times lower; and (b) optionally, the dissociation rate constant Kdis is less than 5×10 ⁻² s ⁻¹ , preferably 1×10 ⁻² s ⁻¹ to 1×10 ⁻³ s ^-1 , eg, 3-65x10 ^-3 s ^-1 , preferably about 5x10 ^-3 s ^-1 , or substantially the same dissociation rate, or slower than the corresponding human FLT3 ligand unfused to an Fc region, eg The Kdis value is about 2-3 times lower.

In still other embodiments, the fusion proteins of the present invention also have the following advantageous properties:

(1) It has good storage stability, for example, in the accelerated stability test at 40°C, it still maintains a colorless and clear appearance and at least 95% SEC-HPLC purity after being placed at 40°C for 2 weeks;

(2) Compared with the FLT3 ligand not fused to the Fc region, the half-life of the drug is prolonged and the frequency of administration is effectively reduced.

Fusion protein of the present invention

In one aspect, the invention provides FLT3 ligand fusion proteins comprising a truncated FLT3 ligand fused to the N-terminus of an antibody Fc region via a linker.

(a). Truncated FLT3 ligand

The native full-length FLT-3 ligand is a transmembrane protein that can function in both membrane-bound and soluble extracellular (ie, non-membrane-bound) forms. As used herein, for the purposes of the present invention, the FMS-like tyrosine kinase 3 ligand, "FLT-3 ligand", which is a component of the fusion protein of the present invention, refers to a ligand that can independently bind to the FLT3 receptor and form a Complex, non-membrane-bound FLT-3L polypeptide without transmembrane and cytoplasmic domains. Therefore, in this document, unless specifically stated otherwise, the terms "FLT-3 ligand", "FLT-3L" or "FLT3L" should generally be understood to mean, non-membrane-bound FIL-3 ligands, not Transmembrane and cytoplasmic regions with full-length FIL-3 ligand polypeptides.

Illustrated in Figure 1 is a full-length human FLT3L polypeptide sequence (SEQ ID NO: 1) consisting of a signal peptide (aa1-26), an extracellular region (aa27-182), and a transmembrane region (aa183-205) ) and the cytoplasmic region (aa206-235). According to the amino acid residue numbering of SEQ ID NO: 1, the FLT-3L polypeptide as a fusion protein component of the present invention, therefore, there will be no transmembrane region and cytoplasm corresponding to the positions of amino acids 183-235 of SEQ ID NO: 1 region sequence. In some embodiments, the polypeptide may further lack the amino acid sequence of the juxtamembrane region at the C-terminus of the extracellular region of the FLT-3 ligand.

As used herein, the amino acid sequence of the juxtamembrane region of a FLT3 ligand refers to, or corresponds to, the amino acid sequence located after the C-terminal cysteine residue in the extracellular region of the wild-type full-length FLT3 ligand and before the transmembrane region segment of the amino acid sequence. Thus, for human FLT3 ligands, the amino acid sequence of the juxtamembrane region is, according to the amino acid residue numbering of SEQ ID NO: 1, located after approximately cysteine amino acid residue 158 at the C-terminus of the extracellular region of the FLT3 ligand and Amino acid sequence preceding the transmembrane region amino acid residue at approximately position 183. As will be apparent to those of skill in the art, due to differences between sequences, the positions described may differ by ±1 or 2 amino acids, but are still encompassed within the scope of the present invention.

As used herein, a C-terminally truncated FLT3 ligand refers to most or substantially all (eg, about 18 amino acids or more or 19- 22 amino acid residues) non-membrane-bound FLT3 ligands having a juxtamembrane region amino acid sequence, preferably, the ligand retains about two residues after the C-terminal cysteine in the extracellular region. For example, for a human FLT3 ligand, according to the amino acid residue numbering of SEQ ID NO: 1, the FLT3 ligand polypeptide with the amino acid sequence of the near-membrane region after the C-terminal amino acid residue 160 of the extracellular region of the FLT3 ligand is removed, Referred to as truncated FLT3 ligand, or "FLT3L-D ligand" or "FLT3L-D". For example, in some embodiments, the truncated FLT-3 ligand comprises the extracellular region corresponding to aa27-160 of SEQ ID NO: 1, but lacks the juxtamembrane region corresponding to aa161-182 of SEQ ID NO: 1 non-membrane-bound FLT-3 ligand polypeptides, especially FLT-3 ligand polypeptides having human sequences.

In this context, as can be understood by those skilled in the art, FLT-3 ligand or FLT-3 may have the amino acid sequence of native FLT-3L protein and allelic variants thereof from different species, or may have Homologs that are somewhat homologous and retain the desired functional activity. Preferably, however, in the present invention, the term refers to FLT-3 ligands having human sequences. "Having a human sequence" or "having a sequence derived from a human", as used herein, means having a natural amino acid sequence derived from a human, but also encompasses having one or more, such as 1-10, or 1, on the natural sequence - In the case of 5, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, only the sequence remains bound to FLT-3 and causes the desired effector function (e.g., Binding to the FLT-3 receptor, and the functions of promoting DC proliferation and antigen presentation).

Thus, in some embodiments, the present invention encompasses the use of C-terminal truncated forms of the extracellular region of native or wild-type FLT-3 ligands. As used herein, wild-type or native "FLT3 ligand" refers to a naturally occurring "FLT3 ligand protein, such as a native FLT3 ligand protein derived from human, mouse, rat, non-human primate. Does not include A non-membrane bound native human FLT3 ligand sequence for the signal peptide is shown in SEQ ID NO:2.

In still other embodiments, the present invention also contemplates the use of FLT3L that has a high degree of similarity or identity to SEQ ID NO: 2 (ie, the amino acid sequence of aa27-160 of SEQ ID NO: 1) and retains binding to the FLT3 receptor the extracellular region of the protein. Such FLT3L protein extracellular domains have biological activity to transmit stimulatory signals to FLT3 expressing cells through membrane-bound FLT3 receptors. Such FLT3L proteins with 96%-99% or more identity to SEQ ID NO:2 are known in the art. Examples include, but are not limited to, eg, the ectodomain sequences of the FLT3 ligand proteins from NCBI with the following accession numbers: XP_034800759.1, XP_016792031.1, XP_018870103.1, 2009389B, XP_032025102.1, XP_032025099.1, XP_032025097.1, XP_024093881.1,XP_024093880.1,XP_024093877.1,XP_024093878.1,XP_007995745.1,XP_033079936.1,XP_007995742.1,XP_033079937.1,XP_007995738.1,XP_017737679.1,XP_011801760.1,XP_033079934.1,XP_017737676. 1, XP_033079931.1, XP_033079933.1, XP_033079932.1, CAC33116.1, ADA27923.1, ADA27922.1, ADA27926.1, ADA27919.1, AAE82530.1, AAE82525.1, ADA27931.1, AAE8255 ADA27916.1,ADA27918.1,ACS06928.1,ADA27928.1,ADA27921.1,ADA27932.1,AAE82522.1,ADA27927.1,ADA27940.1,ADA27930.1,ADA27920.1,ADA27925.1,ADA27941. 1,ADA27929.1,ADA27924.1,ADA27939.1,AAE82528.1,AAE82524.1,ADA27942.1,ADA27915.1,ADA27917.1,ADA27934.1,ADA27938.1,ADA27936.1,ADA27935.1, ADA27933.1, ADA27937.1, AAE82526.1, AAE82521.1, AAE82529.1. In addition, exemplary functional FLT3 ligands with amino acid changes and methods for their screening and characterization are also disclosed in US5554512, US6291661, US7294331, US7361330, and US9486519. For the purpose of the present invention, the above documents and accession numbers are all incorporated herein by reference.

In some preferred embodiments, the present invention provides a FLT3 ligand fusion protein, wherein the fusion protein comprises a truncated FLT3 ligand polypeptide having an amino acid sequence from the extracellular region of a human FLT3 ligand protein, preferably , comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, and wherein, according to SEQ ID NO: The amino acid residue numbering of 1, the truncated FLT3 ligand polypeptide lacks the sequence corresponding to the sequence after amino acid residue 160 of SEQ ID NO: 2.

In further preferred embodiments, the present invention provides a FLT3 ligand fusion protein, wherein the fusion protein comprises a truncated FLT3 ligand polypeptide that is the amino acid sequence of SEQ ID NO: 2 lacking the C-terminal amino acid sequence DSSTLPPPWSPRPLEATAPTAP ; or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it; or differs by no more than 15 , for example, an amino acid sequence of 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, Wherein the amino acid change is preferably an amino acid substitution, especially a conservative amino acid substitution.

(b) linker

In the fusion protein of the present invention, the FLT3 ligand is operably linked to the Fc region of the antibody through a linker.

In one embodiment, the linker is a short flexible amino acid sequence. Such peptide linkers are typically rich in glycine as well as serine or threonine. For example, glycine and/or serine residues can be used alone or in combination. However, other flexible peptide linkers may also be used in the present invention.

In some embodiments, the amino acid residues of the constituent linkers can be selected from the twenty natural amino acids. In certain other embodiments, the one or more amino acids are selected from the group consisting of glycine, serine, threonine, alanine, proline, asparagine, glutamine, and lysine. In a preferred embodiment, the one or more amino acids are selected from Gly, Ser, Thr, Lys, Pro, and Glu.

In some embodiments, the linker is about 1-30 amino acids in length, preferably about 5 to about 25 amino acids, more preferably about 15 to about 20 amino acids, or about 10 to about 20 amino acids in length, Or about 10 to 15 amino acids, or any intermediate amino acid length. In a preferred embodiment, the linker is 10-20 amino acid residues in length. In some embodiments, the length of the linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25 or more amino acids.

Examples of peptide linkers that can be used in the present invention include: glycine polymer (G)n; glycine-serine polymer ( _G1-5S1-5 )n, wherein n is at least ₁ , ₂ , 3, 4 or an integer of 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. It will be understood by those skilled in the art that, in some embodiments, the linker may consist entirely of the flexible linker peptide, or the linker may consist of the flexible linker peptide moiety and one or more moieties that impart a less flexible structure.

In one embodiment, the peptide linker is a Gly/Ser linker peptide. In one embodiment, the peptide linker is a (GxS)n linker, where G=glycine, S=serine, (x=3, n=1, 2, 3 or 4) or (x=4 and n=1 , 2, 3 or 4), in one embodiment x=4 and n=2 or 3. In some embodiments, the linker can include the amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, eg, n is an integer from 1-7. In one embodiment, the linker used to link the Fc region and the FLT3 ligand has the sequence (GGGGS)n, wherein n is an integer of 1, 2, 3, 4 or 5, preferably n is 2 or 3. In one embodiment, the linker is (G4S)2. In one embodiment, the linker is (G4S)3. In one embodiment, the linker is (G4S)4. Preferably, the linker has the sequence GGGGSGGGGS. Still preferably, the linker has the sequence GGGGSGGGGSGGGGS.

(c) Antibody Fc region

As understood by those of skill in the art, suitable Fc regions for use in fusion proteins of the present invention may be any antibody Fc region, eg, an Fc region from an IgG antibody. In some embodiments, the Fc region is an Fc region of an IgGl, IgG2, or IgG4 subtype. In some embodiments, the Fc region is a native sequence Fc region. As used herein, "native sequence Fc region" refers to antibody Fc region sequences found in nature, including various native allelic sequences. In other embodiments, the Fc region is a variant Fc region comprising amino acid modifications to the native sequence Fc region, including, but not limited to, amino acid insertions, deletions and/or substitutions, preferably amino acid substitutions.

In one embodiment, the Fc region may be modified with one or more properties selected from the group consisting of receptor binding properties of the Fc region, effector function, and complement activation function of the Fc region. In some embodiments, the antibody Fc region of a fusion protein according to the invention comprises a mutation that reduces or eliminates binding of the Fc region to one or more Fc receptors. In a preferred embodiment, the mutation reduces or eliminates the binding of the Fc region to FcyRs, especially FcyRIII. Preferably, the mutations comprise L234A and L235A mutations.

In yet another embodiment, the antibody Fc region of the fusion protein according to the invention has effector function or complement activation function that has been reduced or eliminated relative to the native sequence Fc region of the same isotype. As known to those of skill in the art, effector function can be reduced or eliminated by a method selected from the group consisting of reducing glycosylation of the Fc region, using Fc isotypes that naturally have reduced or eliminated effector function, and Fc region modification .

In one embodiment, effector function is reduced or eliminated by reducing glycosylation of the Fc region. In one embodiment, glycosylation of the Fc region is reduced by modifying a method such that wild-type glycosylation does not occur, such as including a mutation at position 297 of the Fc region such that the wild-type asparagine residue at this position is replaced by another An amino acid substitution that interferes with glycosylation at this position, such as the N297A mutation.

In one embodiment, binding to one or more FcR receptors and/or one or more effector functions is reduced or eliminated by at least one Fc region modification. In one embodiment, the at least one Fc region modification may be selected from Fc region point mutations that impair binding to one or more Fc receptors at any one or more of the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439.

In one embodiment, the invention provides FLT3L fusion proteins comprising a FLT3L polypeptide of the invention fused to an Fc region of an antibody, wherein the Fc region of the antibody comprises a mutation that reduces or eliminates Fcγ receptor binding. In some preferred embodiments, the Fc region has a L234A/L235A mutation or L234A/L235E/G237A that reduces binding to Fcγ receptors.

In some preferred embodiments, the Fc region fused to the FLT3L polypeptide of the invention is a human IgG Fc, eg, human IgGl Fc, human IgG2 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith, the amino acid sequence of SEQ ID NO:7 % or 99% identical amino acid sequences.

Exemplary fusion protein molecules

Any suitable truncated FLT3L polypeptide, linker, and antibody Fc can be included in the FLT3L-Fc fusion proteins provided herein.

In some preferred embodiments of the FLT3L-Fc fusion proteins described herein, the fusion proteins of the invention may comprise the amino acid sequence of SEQ ID NO:3, or comprise at least 80% sequence identity to SEQ ID NO:3 (e.g. , at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity). In certain embodiments, fusion proteins of the invention comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 3.

In further preferred embodiments, the fusion protein of the present invention comprises the amino acid sequence of SEQ ID NO:4, or comprises at least 80% (eg, at least 80%, at least 81%, at least 82%, at least 80% with SEQ ID NO:4) 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%) amino acid sequences of sequence identity. In certain embodiments, fusion proteins of the invention comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO:4.

In addition to the above components, in further embodiments, the present invention provides FLT3 ligand fusion proteins, wherein the fusion proteins further comprise a signal peptide to facilitate secretory expression. Truncated FLT3 ligand polypeptide. The signal peptide can be that of a native FLT3 ligand, or a heterologous signal peptide, eg, a signal peptide that is functional in the host cell used to express the fusion protein of the invention.

2. Polynucleotides, Vectors and Hosts

The present invention provides isolated nucleic acid molecules encoding any of the above FLT3L fusion proteins. The polynucleotide sequences encoding the fusion proteins of the invention can be generated by de novo solid phase DNA synthesis or by PCR reactions using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention may comprise a segment encoding a secretion signal peptide operably linked to a segment encoding a fusion protein of the present invention, thereby directing secretory expression of the fusion protein of the present invention.

The present invention also provides vectors comprising the nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

The present invention also provides host cells comprising the nucleic acid or the vector. Host cells suitable for replication and to support expression of the fusion proteins of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large numbers of vector-containing cells can be grown for seeding large scale fermentors to obtain sufficient quantities of fusions for in vitro or in vivo applications. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells. Examples of useful mammalian host cell lines include, but are not limited to, the monkey kidney CV1 line (COS-7) transformed by SV40; the human embryonic kidney line (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 ( 1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells, as described for example in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse Breast tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., ProcNatlAcadSciUSA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63 and Sp2/0. In one embodiment, the host cells are eukaryotic cells, preferably mammalian cells such as Chinese hamster ovary (CHO) cells or human embryonic kidney (293) cells.

3. Preparation method

In yet another aspect, the present invention provides a method for preparing a FLT3L fusion protein of the present invention, wherein the method comprises, under conditions suitable for expression of the FLT3L fusion protein, culturing a host cell comprising a nucleic acid encoding the protein, as provided above, and optionally recovering the protein from the host cell (or host cell culture medium). After recovery of the protein, purification can be performed by protein A and/or various chromatographic techniques, such as size exclusion chromatography. Preferably, in some embodiments, the fusion protein of the present invention reaches a purity of more than 95% after two-step purification by protein A affinity chromatography and size exclusion chromatography.

4. Assay

FLT3L fusion proteins provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

The FLT3L fusion protein of the present invention can be tested for its binding activity to the FLT3 receptor. For example, binding to the human FLT3 receptor can be determined by methods known in the art, such as ELISA, Western blotting, etc., or by the exemplary methods disclosed in the Examples herein. For example, the KD value of the binding affinity of a FLT3L fusion protein to the FLT3 receptor can be determined using a ForteBio assay at room temperature or about 25°C. Advantageously, a native non-membrane bound FTL3 ligand can be included as a control in the assay.

In order to investigate the druggability of the FLT3L fusion protein of the present invention, various analytical methods known in the art can be used, including but not limited to hydrophobic-charge separation chromatography (CIC), size exclusion chromatography (Zenix), hydrophobic interaction Chromatography (HIC) and melting temperature Tm value determination. Preferably, in some embodiments, the fusion protein of the present invention is detected by zenix chromatography and exhibits good molecular colloid stability. In still other embodiments, the fusion proteins of the invention are analyzed for their hydrophobic properties and their homogeneity using HIC chromatography.

Glycoform analysis methods known in the art, including but not limited to CE-SDS capillary electrophoresis methods and mass spectrometry methods, can be used to detect changes in the molecular weight of fusion proteins before and after cleavage of polysaccharides with N-glycosidase.

The stability of the fusion protein of the present invention can be characterized by various stability tests known in the art, such as the 40°C accelerated stability test. In an accelerated stability test, samples stored for a period of time at a specified temperature can be tested for appearance and/or purity and compared to samples before storage. In this regard, methods of purity detection are known in the art, including, but not limited to, SEC-HPLC and CE-SDS, among others. In a preferred embodiment, the fusion protein of the present invention exhibits a still colorless and clear appearance, and at least 95% or more (eg, 96%, 97%, or 98%) in an accelerated test after storage at 40°C for 2 weeks above) SEC-HPLC purity. In other embodiments, the stability of the fusion protein after storage for a period of time can also be characterized by measuring the biological activity of the fusion protein molecule. Such test methods include, but are not limited to, in vitro cell proliferation assays. For example, the CCK8 color development test in the Examples.

In still other embodiments, FLT3L fusion proteins can be characterized by examining signaling and/or immune activating effects that occur downstream of receptor binding. Said assays include, but are not limited to, in vitro DC expansion and/or phenotyping. Hematopoietic stem cells, which are DC cell precursors, can be incubated with the fusion proteins of the invention and, optionally, a positive control (such as a natural FLT3 ligand) and a negative control for a defined period of time, after which the cells are harvested and the number of DC cells counted, And/or staining for specific surface markers of DC cells, such as HLA-DR+, CD1a+ and CD11c+, to analyze DC phenotype.

In some embodiments, the in vivo blood concentration of the fusion protein of the invention can be measured in animals to determine its in vivo half-life. In still other embodiments, the effect of the fusion proteins of the invention on tumor growth and survival can also be assessed in a variety of animal tumor models known in the art. For example, xenografts of cancer cell lines can be implanted in experimental mice and treated with the fusions of the invention alone or in combination with other anti-tumor drugs such as anti-PD-1 antibodies. Thereafter, the in vivo anti-tumor effect of the fusions of the invention can be determined based on tumor inhibition rates (eg, calculated relative to an isotype control antibody).

5. Pharmaceutical compositions and drug combination products

In yet another aspect, the present invention provides a pharmaceutical composition or a pharmaceutical combination product comprising the fusion protein, nucleic acid, vector or host cell of the present invention.

In some embodiments, the present invention provides pharmaceutical compositions comprising fusion proteins of the present invention. As is clear in the art, the pharmaceutical composition may also optionally contain suitable pharmaceutical excipients, pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers. In addition, as is clear in the art, the pharmaceutical compositions may also contain other therapeutic agents that are beneficial for the particular disease to be treated.

In yet another embodiment, the present invention also provides pharmaceutical combinations comprising the fusion proteins of the present invention and further comprising other therapeutic agents beneficial for the particular disease to be treated.

In some embodiments, in the pharmaceutical composition or drug combination of the present invention, the FLT3L-Fc fusion protein of the present invention can be combined with drugs for T cell activation or inhibition, such as immune checkpoint inhibitors, such as PD-1, CD47 , CTLA-4, LAG3, TIM3, TIGIT, OX40 and other monoclonal antibody or dual antibody combination to improve the anti-tumor effect. The FLT3L-Fc fusion protein of the present invention can also be used in combination with drugs that promote DC maturation and antigen presentation, such as CD40, 4-1BB monoclonal antibody or double antibody, to improve therapeutic effects, such as anti-tumor effects. In addition, the FLT3L-Fc fusion protein of the present invention can also be used in combination with radiotherapy, chemotherapy, tumor vaccine and the like.

Accordingly, in one aspect, the present invention provides pharmaceutical combinations and pharmaceutical compositions comprising a truncated FLT3L-Fc fusion protein of the present invention, and further comprising additional therapeutic agents selected from the group consisting of:

(1) T-cell activating drugs or inhibitory drugs, such as immune checkpoint inhibitors, especially anti-PD1 antibodies;

(2) Drugs that promote DC maturation and antigen presentation, such as anti-CD40, anti-4-1BB monoclonal antibody or double antibody;

(3) Radiotherapy agents, chemotherapeutic agents, or tumor vaccines.

In some embodiments of pharmaceutical compositions and drug combinations, the fusion proteins of the present invention may be included in the same or a different composition as the other therapeutic agent. The fusion proteins of the present invention can be administered simultaneously, sequentially, or in any order and in any dosing regimen, with the other therapeutic agents.

6. USE AND TREATMENT

The term "FLT3-related disease" herein refers to non-physiological conditions associated with FLT3 receptor-mediated signaling activity, including but not limited to tumors, cancers, and hematopoietic stem cell transplantation. In some embodiments, the disease will benefit from activation of FLT3 receptor-mediated signaling.

The fusion proteins of the present invention are suitable for the treatment of FLT3-related diseases including, but not limited to, various tumors and cancers, including hematological tumors and solid tumors, such as the treatment of advanced solid tumors. In the therapeutic application, the fusion protein of the present invention can be advantageously used in combination with various anti-tumor drugs, including but not limited to radiotherapy, chemotherapy, targeted drugs, immune checkpoint inhibitors, RNA vaccines, and various cytokines. Tumors/cancers suitable for the treatment of the present invention include, but are not limited to, leukemia, colon cancer, fibrosarcoma, melanoma, breast cancer, lung cancer. In a preferred embodiment, the cancer is colon cancer.

As used herein, the term "subject" or "patient" or "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. Preferably, the subject of the present invention is a human.

As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to fusion proteins of the present invention that, when administered to cells, tissues or subjects alone or in combination with other therapeutic agents, are effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition. A therapeutically effective dose also refers to an amount sufficient to cause amelioration of symptoms, eg, to treat, cure, prevent or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention or amelioration of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers to that ingredient only. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

As used herein, "treatment" includes 1) therapeutic measures that cure, slow, alleviate the symptoms and/or halt the progression of a diagnosed pathological condition or disorder; and 2) prophylactically or preventive measures, which prevent and/or slow the development of a pathological condition or disorder. Thus, treaters include individuals already suffering from the disorder, individuals susceptible to the disorder, and individuals for whom the disorder is to be prevented. In some embodiments, the invention relates to the treatment of a disease or condition; in other embodiments, the invention relates to the prevention of a disease or condition.

In some embodiments according to the invention, "treating" of a disease or condition refers to ameliorating the disease or condition (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient. In other embodiments, "treating" refers to modulating a disease or condition physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.

In yet other embodiments according to the invention, "prevention" of a disease or condition includes the inhibition of the occurrence or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

In certain embodiments, an individual with a tumor "treated" by the methods of the present invention exhibits inhibition of tumor growth. The expression "inhibiting tumor growth" includes any mechanism by which tumor cell growth can be inhibited. In certain embodiments, tumor cell growth is inhibited by retarding tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by stopping tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by killing tumor cells. In certain embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis.

Accordingly, in some aspects, the present invention provides the following uses of the fusion proteins of the present invention:

(a) Activation of cellular FLT3 receptor signaling through FLT3 receptors;

(b) promoting the proliferation and differentiation of hematopoietic stem cells and precursor cells into dendritic cell (DC) cells;

(c) promoting proliferation and differentiation of DCs, optionally in combination with stem cell stimulating factors;

(d) improving the ability of DC cells to present tumor antigens;

(e) enhancing antigen-specific T cell immune responses;

(f) inhibiting tumor growth, optionally in combination with an anti-tumor drug (eg, an anti-PD1 antibody).

The above applications may be in vitro or in vivo. In preferred embodiments, the application is a disease therapeutic or prophylactic application in vivo. In the described methods of in vivo application, the fusion protein of the present invention is administered in a therapeutically effective amount, preferably in combination with other therapeutic agents, including, but not limited to, the aforementioned therapeutic agents. In a preferred embodiment, the therapeutic agent is an anti-PD-1 antibody.

As will be appreciated by those skilled in the art, the fusion proteins of the present invention (and pharmaceutical compositions or drug combinations comprising the same) can be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, Also, intralesional administration if required for local treatment. Preferably, parenteral infusion is carried out, including, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. For the purposes of the present invention, various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of the fusion protein of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease , administration for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in a single treatment or over a series of treatments.

In yet another aspect, the present invention also provides the use of a fusion protein, nucleic acid, vector or host cell of the present invention in the manufacture of a medicament for use in the aforementioned methods (eg, in therapy).

The following examples are further given below in order to describe and assist the understanding of the present invention. It should be understood, however, that these examples are not intended and should not be construed in any way to limit the scope of protection of the present invention.

Example

Example 1 Gene design and synthesis of recombinant FLT3 ligand-human antibody Fc fusion protein

The molecular design of the recombinant FLT3 ligand-human antibody Fc fusion protein was derived from the natural human FLT3 ligand amino acid sequence (NCBI Accession No. NP_001191432).

After preliminary research on the molecular structure, 3 recombinant FLT3 ligand-human antibody Fc fusion protein candidate molecules were designed: (1) FLT3L-D-G4S2-Fc, the amino acid sequence is shown in Seq ID No.3, and FLT3 was removed The C-terminal polypeptide sequence of the extracellular region of the ligand, the introduction of a 10 amino acid (G4S)2 linker; (2) FLT3L-D-G4S3-Fc, the amino acid sequence is shown in Seq ID No.4, and the extracellular region of the ligand is removed. The C-terminal polypeptide sequence was introduced with a (G4S)3 linker of 15 amino acids; (3) FLT3L-IgG1LALA-Fc, the amino acid sequence was shown in Seq No.5, the C-terminal polypeptide sequence of the extracellular region of the FLT3 ligand was retained, and the Introduce new linkers. In addition, referring to the amino acid sequence of the clinical trial molecule CDX301, a Benchmark reference molecule FLT3L-His was designed. The amino acid sequence is shown in Seq No. 6, and a His6-tagged polypeptide was added at the C-terminus to facilitate protein purification and detection.

Table 1: Amino acid sequence information of 3 candidate molecules and Benchmarks

The nucleotide sequences of candidate molecules and Benchmark molecules were constructed into pcDNA3.1 plasmid (purchased from Invitrogen) vector, and the corresponding plasmid was extracted. Culture HEK293F cells to a density of 3.0×10 ⁶ cells/mL; mix the plasmid and transfection reagent PEI at a mass ratio of 1:3 and let stand for 20 min, then slowly add them to HEK293F cells, shake at 37°C, 8% CO2 Culture in bed incubator for 6-7 days; collect cell supernatant. For Benchmark molecules, Ni affinity columns were used for purification. For candidate molecules, protein A affinity column and molecular sieve chromatography column were used for purification. Briefly, the collected cell supernatants were bound to Protein A purification resin (GE Healthcare) for 1 hr at room temperature; the Protein A purification resin was washed with phosphate buffered saline (PBS) pH 7.0 to remove contaminants, and then washed with pH 3.0 The protein was eluted with 0.1 M citric acid, concentrated by ultrafiltration into PBS. The purified samples were further purified by size-exclusion chromatography (SEC), the main peaks were collected, and the samples were stored in a -80°C refrigerator after packaging. The protein purity was detected by size exclusion high performance liquid chromatography (SEC-HPLC), and after purification, the purity of both candidate molecules and Benchmark molecules reached more than 95%.

Example 2 Affinity determination of recombinant FLT3 ligand-human antibody Fc fusion protein

Biolayer interferometry (BLI) was used to determine the binding affinity of candidate molecules and control molecules to FLT3. The experimental steps were as follows: the sensor was equilibrated in the assay buffer for 30 min off-line, then the baseline was established for 60 s on-line detection, and the FLT3 extracellular region recombinant protein antigen was loaded onto the AHC sensor (ForteBio, 18-5010) on-line. The loaded sensor was then exposed to 100 nM solution of candidate molecule and control molecule for 100 s, respectively, and then the sensor was transferred to assay buffer for dissociation for 120 s for dissociation rate measurement. Binding kinetics were fitted and analyzed using a 1:1 binding model, and equilibrium dissociation constant (KD) values were calculated. The experimental results are shown in Table 2. The affinity of the three candidate molecules is similar, and the KD value is reduced by about 2-3 times compared with the control molecule.

Table 2: Binding affinity data of 3 candidate molecules to control molecules

样品sample	反应值response value	KD(M)KD(M)	Kon(1/Ms)Kon(1/Ms)	Kdis(1/s)Kdis(1/s)
FLT3L-D-G4S2-FcFLT3L-D-G4S2-Fc	0.33990.3399	1.51E-081.51E-08	3.04E+053.04E+05	4.59E-034.59E-03
FLT3L-D-G4S3-FcFLT3L-D-G4S3-Fc	0.35030.3503	1.53E-081.53E-08	2.94E+052.94E+05	4.49E-034.49E-03
FLT3L-IgG1LALA-FcFLT3L-IgG1LALA-Fc	0.34170.3417	1.46E-081.46E-08	2.89E+052.89E+05	4.20E-034.20E-03
FLT3L-HisFLT3L-His	0.06550.0655	4.01E-084.01E-08	3.85E+053.85E+05	1.54E-021.54E-02

Example 3 Determination of druggability of recombinant FLT3 ligand-human antibody Fc fusion protein

Using SMAC (single molecule adsorption chromatography) and HIC (hydrophobic interaction chromatography), the druggability of the three candidate molecules and Benchmark was further studied.

The colloidal stability of antibodies was investigated by SMAC (Standup monolayer adsorption chromatography). The Zenix SEC-300 column (213300-4630, Sephax Technologies) used in this experimental method has strong heterogeneity, and the bonding surface of the packing material consists of a hydrophobic monolayer and terminal hydrophilic groups. The retention time of the sample in the Zenix column showed a significant negative correlation with its colloidal stability. The Zenix chromatographic column was inserted into the high performance liquid chromatography, and PBS (10010-023, Gibco) buffer was used as the mobile phase to fully equilibrate the liquid phase system. The system flow rate was set to 0.35ml/min, and the detection wavelength was 280/214nm. After the sample to be tested was diluted to 1 mg/ml, 20 μl were injected in sequence, and the running time of each sample was 20 min. After the experiment, the collected data were analyzed to obtain the peak time of the samples. The samples adalimumab (IBI303) and ipilimumab (IBI310) were known controls, with shorter and longer retention times, respectively. . The earlier the peak time of the sample, the better the colloidal stability, and vice versa, the poorer the colloidal stability.

The SMAC results are shown in Figure 2. The retention times of the three FLT3L-Fc fusion proteins were between 7.792 and 8.143 minutes, which were similar to the IBI303 control and much smaller than that of IBI310, indicating that the candidate molecules had good colloidal stability.

The hydrophobicity of the protein was determined by HIC (Hydophobic interaction chromatography). Ammonium sulfate buffer reduces the solvation of sample solutes, exposing the hydrophobic regions on the surface of protein molecules, promoting the adsorption of the hydrophobic regions to the hydrophobic packing material. The HIC column is bound with hydrophobic compounds, and the hydrophobicity of the detected protein sample is proportional to its retention time on the HIC column. The MAbPac HIC-10 column (088480, Thermal Scientific) was connected to high performance liquid chromatography, using buffer A [1.8M (NH4)2SO4, 100mM NaH2PO4 2H2O] and buffer B [100mM NaH2PO4 2H2O:Isopropanol 90 :10(v/v)] as the mobile phase. The system flow rate was set to 1ml/min, and the detection wavelength was 280nm. After diluting the sample to be tested to 1 mg/ml, inject 20 μl in sequence. The mobile phase gradient was 0-20min 100%A-100%B, 20-25min 100%B, 25-30min 100%A, 30-35min 100%B, each sample run time 35min. After the experiment, the collected data were analyzed to obtain the peak time of the samples. The samples IBI303 and IBI310 were known reference substances with shorter and longer retention times, respectively. The earlier the peak time of the sample, the weaker the hydrophobicity, and vice versa, the stronger the hydrophobicity of the colloid.

The HIC results are shown in Figure 3. The retention times of the three FLT3L-Fc fusion proteins ranged from 9.790 to 10.088 minutes, which were similar to the IBI303 control and much smaller than that of IBI310, indicating that the candidate molecules had low hydrophobicity. In addition, FLT3L-D-G4S2-Fc and FLT3L-D-G4S3-Fc showed a homogeneous peak, but the peak of FLT3L-IgG1LALA-Fc with C-terminal sequence and the control molecule (FLT3L-His) was broader and existed The tailing phenomenon indicates that the homogeneity of the hydrophobic properties of the molecules is relatively poor.

The purity of the protein was determined by CE-SDS (capillary electrophoresis) method. This experiment uses Caliper labchip GX Touch HT equipment for detection. The reagents are from HT Protein Express Reagent Kit (CLSP60008, Perkinelmer), which contains Sample buffer, Dye, Ladder, Lower Marker, Gel, Wash Buffer required for the experiment. Dilute the FLT3L-Fc fusion protein to 1mg/ml, take 2ul sample, add 14ul non-reducing buffer (700ul sample buffer+31.3ul 250mM NEM (E3876, Sigma), add 28ul water, mix well, put it in Heated in a metal bath at 70°C for 10 minutes, cooled and centrifuged briefly, and then transferred to a 96-well plate. Take 12ul of the Ladder and return to room temperature, heat it in a metal bath at 70°C for 10 minutes, add 120ul of ultrapure water after cooling, and transfer it to a ladder tube after mixing. (C-3258-1, Genemate). After rinsing the Protein Express Assay LabChip (760499, Perkinelmer) chip with ultrapure water, an appropriate amount of Gel-Dye (26:1, v/v) solution and decolorizing solution were added respectively. , Lower Marker, put the chip into the instrument for detection.

The experimental results found that the purities of FLT3L-D-G4S2-Fc, FLT3L-D-G4S3-Fc and FLT3L-IgG1LALA-Fc under non-reducing conditions were 95.8%, 99.3% and 99.8%, respectively, which were basically consistent with the purity data determined by SEC. The molecular weights of the three FLT3L-Fc fusion proteins and Benchmark after N-glycan excision were further determined by mass spectrometry. After the candidate molecules of FLT3L-D-G4S2-Fc and FLT3L-D-G4S3-Fc were excised by N sugar, the molecular weights identified were consistent with the theoretical molecular weights, indicating that the amino acid sequences of the fusion proteins were consistent with the theory. The FLT3L-IgG1LALA-Fc candidate molecule and Benchmark molecule were not completely removed from the N-glycan, and the molecular weight could not be accurately determined. The reason may be due to the non-specific O-glycosyl modification of the Ser- and Pro-rich polypeptides at the C-terminus of the extracellular region of FLT3L.

The above results show that the quality of the two candidate molecules, FLT3L-D-G4S2-Fc and FLT3L-D-G4S3-Fc, is better than that of FLT3L-IgG1LALA-Fc in terms of druggability.

Example 4 Accelerated stability study of recombinant FLT3 ligand-human antibody Fc fusion protein

The purified protein was concentrated to 10 mg/ml using a centrifugal ultrafiltration tube (purchased from Millipore) and replaced with preparation buffer (recipe: 1.55 mg/ml histidine, 50 mg/ml sorbitol, 0.5 mg/ml PS80, pH 5.5 ), placed in a 40-degree constant temperature and humidity box for 1 week or 2 weeks, and sampled for appearance, purity and biological activity.

The results of appearance observation are shown in Table 3. FLT3L-D-G4S3-Fc and FLT3L-D-G4S2-Fc had good thermal stability, and their appearance did not change after being placed at 40°C for 2 weeks. The stability of FLT3L-IgG1LALA-Fc was poor, and opalescence appeared after being placed at 40°C for 2 weeks; the stability of the FLT3L-His control molecule was the worst, with a large number of particles and severe opalescence.

Table 3: Appearance test results in accelerated stability test at 40°C

样品sample	0h0h	40℃ 1w40℃ 1w	40℃ 2w40℃ 2w
FLT3L-IgG1LALA-FcFLT3L-IgG1LALA-Fc	无色澄明Colorless and clear	微乳光micro-opalescence	乳光opalescence
FLT3L-D-G4S2-FcFLT3L-D-G4S2-Fc	无色澄明Colorless and clear	无色澄明Colorless and clear	无色澄明Colorless and clear
FLT3L-D-G4S3-FcFLT3L-D-G4S3-Fc	无色澄明Colorless and clear	无色澄明Colorless and clear	无色澄明Colorless and clear
FLT3L-HisFLT3L-His	无色澄明Colorless and clear	乳光严重severe opalescence	大量颗粒、乳光严重A large number of particles, severe opalescence

Purity was detected by SEC-HPLC, and the experimental results were shown in Figure 4. FLT3L-D-G4S3-Fc, FLT3L-D-G4S2-Fc and FLT3L-His had good stability. After being placed at 40 degrees for 2 weeks, the purity of the main peak was basically unchanged compared with that before placement. The stability of FLT3L-IgG1LALA-Fc was poor, and the proportion of the main peak decreased to 40.96% after being placed at 40 degrees for 1 week, and the proportion of the main peak decreased to 9.6% after being placed for 2 weeks, resulting in a large number of degraded fragments.

The biological activity of the samples was determined by the CCK8 staining method. The experimental steps are as follows: BaF3/msFLT3 cells are plated in 96-well plates at 10 ⁴ cells/well, and the proteins of different concentrations are diluted and added to the medium, 100ul per well, and placed in a 37-degree CO2 incubator for 72 hours. 20ul of CCK8 reagent (DOJINDO), continue to culture for 4 hours, and measure the OD450-OD620 light absorption value. The results are shown in Figure 5. After the candidate molecules of FLT3L-D-G4S3-Fc and FLT3L-D-G4S2-Fc were placed at 40 degrees for 2 weeks, compared with before placement, the ability to stimulate the proliferation of BaF3/msFLT3 cells did not change. Similar EC50 values. The ability of FLT3L-D-G4S3-Fc and FLT3L-D-G4S2-Fc candidate molecules to stimulate the proliferation of BaF3/msFLT3 cells was also similar compared to untreated FLT3L-His after 2 weeks at 40 degrees.

These results show that the two candidate molecules FLT3L-D-G4S3-Fc and FLT3L-D-G4S2-Fc have good druggability and thermal stability; while the FLT3L-IgG1LALA-Fc molecule has poor thermal stability, poor appearance and purity Comply with the drug requirements. Taking FLT3L-D-G4S3-Fc as an example in subsequent experiments, the biological activity of the truncated FLT3 ligand fusion protein of the present invention was further studied and compared with FLT3L-His.

Example 5 Recombinant FLT3 ligand-human antibody Fc fusion protein stimulates DC cell proliferation

CD34+ hematopoietic stem cells were isolated from the peripheral blood of healthy people, passaged into 24-well plates at 10 ⁵ , Final concentration: 100ng/ml) was added. After 7 days of culture, the cells were collected and resuspended in PBS buffer and counted using a hemocytometer to calculate the proliferation efficiency of DC cells. The cells were then divided into 3 tubes, HLA-DR+, CD1a+ and CD11c+ antibodies (Invitrogen, 1:200) were added to each tube, stained at 4°C in the dark for 30 minutes, washed twice with PBS, and then used on a FACSCELESTA flow cytometer (BD). Company) on the machine to detect the phenotype of DC cells.

The results of DC cell proliferation efficiency are shown in Figure 6A. Compared with the untreated control group, the DC proliferation efficiency in the FLT3L-D-G4S3-Fc group was increased by 2.2 times; the DC proliferation efficiency in the FLT3L-His group was increased by 1.9 times. The proliferation efficiency of FLT3L-D-G4S3-Fc and SCF combined group increased by 9.5 times; FLT3L-His and SCF combined group increased by 9.0 times.

The results of DC cell typing are shown in Figure 6B. Compared with the untreated group, the numbers of HLA-DR+, CD1a+ and CD11c+ positive DC cells in the FLT3L-D-G4S3-Fc-treated group increased by 5.9-fold, 6.0-fold and 2.9-fold, respectively; 5.4 times, 5.5 times and 2.8 times; FLT3L-D-G4S3-Fc and SCF combined group increased by 27.3 times, 27.1 times and 9.5 times respectively; FLT3L-His and SCF combined group increased by 24.9 times, 25.0 times and 8.6 times.

The above results show that FLT3L-D-G4S3-Fc can effectively expand CD34+ stem cells in vitro and promote the differentiation into DC cells. Under the condition of co-treatment with SCF, the effect of expanding DC cells is better.

Example 6 Recombinant FLT3 ligand-human antibody Fc fusion protein inhibits the growth of MC38 tumor in mice

single-dose experiment

The C57 mice were divided into 7 groups with 7 mice in each group, and each mouse was subcutaneously injected with 0.75× ^{10 6} MC38 mouse colon cancer cells. On the day of cell injection (day0), PBS, h-IgG (isotype-independent control antibody), FLT3L-His, FLT3L-D-G4S3-Fc and anti-mouse PD-1 mAb (clone No. 11430, See WO2017133540A1, Antibody C, which is hereby incorporated by reference). Among them, the dose of FLT3L-His was 15ug/mouse, and the dose of FLT3L-D-G4S3-Fc was 35ug/mouse, both were single doses; the dose of 11430 mAb was 1mg/kg, Dosing 2 times a week for a total of 4 times. The tumor size was measured twice a week, and the maximum long axis (L) and the maximum wide axis (W) of the tumor were measured with vernier calipers, and the tumor volume was calculated according to the following formula: V=L×W ² /2. At the end of the experiment, the relative tumor inhibition rate (TGI) was calculated according to the following formula:

Tumor inhibition rate TGI(%)=100%×(Tvol _control –Tvol _treated )/(Tvol _control –Tvol _predose )

Tvol _control – Tvol _treated : the terminal volume of the tumor after administration in the control group – the final volume of the tumor in the administration group after administration;

Tvol _control – _{Tvol predose} : the terminal tumor volume after administration of the control group – the tumor volume of the control group before administration.

The experimental results are shown in Figure 7A. The FLT3L-D-G4S3-Fc single-use group had a better tumor inhibition effect, with a TGI of 61%; the FLT3L-His control single-use group also had a certain tumor inhibition effect, with a TGI of 43%. The combination of FLT3L-D-G4S3-Fc and 11430 mAb significantly improved the tumor inhibition effect, and TGI increased to 94%; the TGI of FLT3-His combined with 11430 mAb increased to 84%. The above results show that under the condition of single administration, FLT3L-D-G4S3-Fc alone or in combination with anti-PD-1 monoclonal antibody has better anti-tumor effect than FLT3L-His control.

Multiple Dosing Experiment

MC38 mouse colon cancer cells were injected subcutaneously into Balb/C mice to examine the effect of dosing frequency on the in vivo efficacy of candidate molecules and Benchmark molecules. Mice were randomly divided into 10 groups. After 6-7 days after cell injection, when the tumor grew to about 50mm ³ , the corresponding doses of h-IgG, FLT3L-His, FLT3L-D-G4S3-Fc, anti-mouse PD-1 mAb (11430) were subcutaneously injected respectively. , or the combination of FLT3L and mAb. Among them, the dose of FLT3L-His was 0.5 mg/kg, once a day, a total of 10 times; the dose of FLT3L-D-G4S3-Fc was 1.15 mg/kg, with an interval of 3 days, a total of 2 times; 11430 mAbs The dosage was 1 mg/kg, 2 times a week for a total of 4 times. Tumor volumes were measured twice a week. The experimental results are shown in Figure 7B. The results showed that the antitumor effect of FLT3L-D-G4S3-Fc administered twice was similar to that of FLT3L-His administered 10 times in a row, indicating that FLT3L-D-G4S3-Fc prolongs the half-life in mice in vivo and reduces the frequency of administration. Under certain conditions, similar in vivo efficacy of Benchmark can be achieved for multiple consecutive administrations.

Example 7 Recombinant FLT3 ligand-human antibody Fc fusion protein stimulates the proliferation of DC cells in mouse tumors

The MC38 tumor-bearing C57 mice were divided into 3 groups of 3-4 mice each. The first group was injected with 2mg/kg h-IgG negative control, the second group was injected with 10ug/one FLT3L-His control, and the third group was injected with 23ug/one FLT3L-D-G4S3-Fc, all of which were single administration. Mice were sacrificed and tumor tissues were isolated on day 6, and intratumoral immune cells were analyzed by FACS. The results are shown in FIG. 8 . A single administration of FLT3L-D-G4S3-Fc significantly stimulated the number of DC cells in the tumor, and the effect was better than that of the FLT3L-His control group.

Example 8 In vivo PK study of recombinant FLT3 ligand-human antibody Fc fusion protein in mice

18 female Balb/C mice were divided into 2 groups, 9 mice in each group, were injected with a single dose of 10 mg/kg of FLT3L-D-G4S3-Fc candidate molecule or FLT3L-His control molecule, and then collected at different time points. blood, and the serum drug concentration was determined by ELISA method. The PK curve is shown in Figure 9. The half-life (t1/2) of FLT3L-D-G4S3-Fc in mice reached 45h, and the half-life of FLT3L-His was only 6 hours. The AUC _0-t of FLT3L-D-G4S3-Fc reached 4415ug*hr/ml, and the control group was only 151ug*hr/ml. Compared with the control group, FLT3L-D-G4S3-Fc increased about 29 times . Analysis of the PK curve showed that 24 hours after administration, the plasma concentration of FLT3L-D-G4S3-Fc remained about 100ug/ml, but the plasma concentration of the FLT3L-His control group was lower than 1ug/ml.

sequence listing

Human FLT-3 ligand full-length protein: SEQ ID NO: 1

Among them, the underline indicates the signal peptide, the italics indicates the extracellular region, and the bold indicates the transmembrane region and the cytoplasmic region.

Non-membrane-bound human FLT-3 ligand (without signal peptide): SEQ ID NO: 2

Wherein, italics indicate C-terminal membrane-proximal sequences

FLT3L-D-G4S2-Fc fusion protein: SEQ ID NO: 3

FLT3L-D-G4S3-Fc fusion protein: SEQ ID NO: 4

FLT3L-IgG1LALA-Fc fusion protein: SEQ ID NO:5

FLT3L-His: SEQ ID NO: 6

IgG1 Fc region amino acid sequence: SEQ ID NO:7

Claims

A FLT3 ligand fusion protein, wherein the fusion protein comprises a C-terminal truncated non-membrane-bound FLT3 ligand and an antibody Fc region,

wherein the truncated FLT3 ligand is a FLT3 ligand polypeptide in which the juxtamembrane region amino acid sequence of the C-terminus of the extracellular region of the FLT3 ligand is deleted, and

wherein the truncated FLT3 ligand is fused to the N-terminus of the Fc region of the antibody through a linker.
The fusion protein of claim 1, wherein said FLT3 ligand extracellular region is from the extracellular region of human FLT3 ligand protein, preferably, comprising at least 90%, 95%, 96%, 97% with SEQ ID NO:2 , 98% or 99% identity,

Wherein, according to the amino acid residue numbering of SEQ ID NO: 1, the truncated FLT3 ligand is a FLT3 that has deleted the near-membrane region amino acid sequence after the 160th amino acid residue of the extracellular region of the FLT3 ligand. Ligand polypeptide.
The fusion protein of claims 1-2, wherein the truncated FLT3 ligand is the amino acid sequence of SEQ ID NO: 2 lacking the C-terminal amino acid sequence DSSTLPPPWSPRPLEATAPTAP, or has at least 90%, 95%, 96%, 97% therewith %, 98% or 99% identical amino acid sequences, or amino acid sequences differing therefrom by no more than 1-10 or 1-5 amino acid changes, wherein the amino acid changes are preferably amino acid substitutions, especially conservative amino acid substitutions .
Fusion protein of claims 1-3, wherein the linker is 5-25 amino acids in length, preferably 10-20 or 10-15 amino acids, and more preferably 10 or 15 amino acids.
The fusion protein of claims 1-4, wherein the linker is a (GxS)n linker, wherein G=glycine, S=serine, and wherein x=3, n=1, 2, 3, or 4; or wherein x =4, n=1, 2, 3 or 4,

Preferably, the linker is a (G4S)2 linker or a (G4S)3 linker.
The fusion protein of claims 1-5, wherein the antibody Fc region is an IgG Fc region, preferably an IgG1, IgG2 or IgG4 Fc region, more preferably a human IgG1 Fc region,

Preferably, the antibody Fc region comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to it.
The fusion protein of claims 1-6, wherein the antibody Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to one or more Fc receptors, optionally, the mutation reduces the binding of the Fc region to FcγRs or eliminated, preferably the mutations comprise L234A and L235A mutations.
The fusion protein of claims 1-7, wherein the fusion protein comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3 or 4.
The fusion protein of claims 1-8, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 3 or 4.
An isolated nucleic acid molecule encoding the FLT3 ligand fusion protein of any one of claims 1-9.
10. A vector comprising the nucleic acid molecule of claim 10.
A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 10.
A method for producing a FLT3 ligand fusion protein, comprising: in the case of being suitable for the expression of the fusion protein of any one of claims 1-9, culturing a host cell comprising a nucleic acid encoding the protein, preferably, the host The cells are mammalian cells such as 293 cells or CHO cells.
A pharmaceutical composition or pharmaceutical combination comprising the fusion protein of any one of claims 1-9, the nucleic acid molecule of claim 10, the vector of claim 11, or the host cell of claim 12.
The pharmaceutical composition or drug combination of claim 14, further comprising an anti-tumor drug, such as an anti-PD-1 antibody.
The purposes of the fusion protein of any one of claims 1-9 in the preparation of medicine, wherein the medicine is used for the purposes selected from the following:

(a) Activation of cellular FLT3 receptor signaling through FLT3 receptors;

(b) promoting the proliferation and differentiation of hematopoietic stem cells and precursor cells into dendritic cell (DC) cells;

(c) promoting proliferation and differentiation of DCs, optionally in combination with stem cell stimulating factors;

(d) improving the ability of DC cells to present tumor antigens;

(e) enhancing antigen-specific T cell immune responses;

(f) inhibiting tumor growth, optionally in combination with an anti-tumor drug (eg, an anti-PD1 antibody).
A method of preventing or treating FLT3L-related diseases, comprising administering the fusion protein of claim 1-9, or the nucleic acid molecule of claim 10, the carrier of claim 11, or the host cell of claim 12, or claim 14 or 15 The pharmaceutical composition or drug combination.
18. The method of claim 17, wherein the disease is a hematological tumor or a solid tumor, such as colon cancer.
19. The method of claim 18, wherein the method further comprises administering other anti-tumor drugs, such as anti-PD-1 antibodies.