WO2016192613A1 - Bivalent antibody having single-domain antigen-binding fragment fused to conventional fab fragment - Google Patents

Abstract

Provided is a bivalent antibody, comprising: (a) an antigen-binding fragment (Fab) comprising a light chain and a portion of a heavy chain of the antibody, and (b) a single-domain antigen-binding fragment (VHH) fused to a C terminal of the light chain or the portion of the heavy chain, wherein the portion of the heavy chain does not comprise a CH2 or CH3 domain.

Description

Bivalent antibody having a single domain antigen-binding fragment fused to a conventional Fab fragment

Cross-reference to related applications

The present application claims priority to US Provisional Patent Application No. US-A-62/169,088, filed on Jun. 1, 2015, and No.

Technical field

The present invention relates to a bivalent antibody, particularly a bivalent antibody having a single domain antigen-binding fragment fused to a conventional Fab fragment.

Background technique

The bispecific antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies and thus binds to two different types of antigens. For example, in cancer immunotherapy, engineered BsMAbs bind to both cytotoxic cells and targets to be killed (eg, tumor cells).

At least three types of bispecific antibodies have been proposed or tested, including trifunctional antibodies, chemically linked Fabs, and bispecific T cell adapters. In order to overcome manufacturing difficulties, a first generation BsMAb called a trifunctional antibody has been developed. It consists of two heavy chains and two light chains, each from a different antibody. These two Fab regions are directed against two antigens. The Fc region consists of two heavy chains and forms a third binding site, hence the name.

Other types of bispecific antibodies have been designed to address certain issues, such as short half-life, immunogenicity, and side effects caused by cytokine release. These include: chemically linked Fabs consisting only of Fab regions, as well as various types of bivalent and trivalent single chain variable regions (scFv) (fusion proteins mimicking two antibody variable domains). The newly developed forms are bispecific T cell adapters (BiTE) and tetrafunctional antibodies.

Despite these advances, the main challenges of bispecific antibodies remain, such as increased manufacturing efficiency, retention of immunogenicity, and maintenance of half-life.

Summary of the invention

In one embodiment, the invention provides a bivalent or trivalent antibody comprising a conventional antigen binding fragment (Fab) and one or two single domain antigen binding (VHH) fragments. Each Fab and VHH has specificity for an antigen, which may be the same or different.

In antibody production and purification, such a bivalent or trivalent antibody with a reduced molecular weight relative to conventional antibodies presents a significant advantage. Despite its small size, unexpectedly, such antibodies are still able to efficiently bind to two different antigens to achieve its intended biological function. Although these disclosed bispecific antibodies are heterodimers which are known to be detrimental to expression and production, they are also unexpectedly capable of being readily expressed in bacterial cells (eg E. coli). Up to, resulting in the production of soluble proteins. In contrast, other known bispecific antibodies produced by E. coli are almost insoluble. In addition, since the heavy and light chains of the Fab do not bind to themselves (ie, the heavy chain binds the heavy or light chain to the light chain), avoiding the formation of undesired heterodimers or At least minimize it, resulting in a high yield of the expected heterodimer.

Accordingly, one embodiment of the present invention provides a bivalent antibody comprising (a) an antigen-binding fragment (Fab) comprising a light chain and a partial heavy chain of an antibody, and (b) fused to the light A single domain antigen binding (VHH) fragment of the C-terminus of a stranded or partially heavy chain, wherein the partial heavy chain does not comprise a CH2 or CH3 domain.

In certain aspects, a VHH fragment is fused to the partial heavy chain. In certain aspects, the antibody further comprises a second VHH fragment fused to the light chain.

In some aspects, the Fab fragment and the VHH fragment each have specificity for tumor cells or immune cells. In certain aspects, the Fab fragment has specificity for a tumor antigen and the VHH fragment has specificity for immune cells. In certain aspects, the tumor antigen is selected from the group consisting of CEA, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding Protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin. In certain aspects, the tumor antigen is CEA or Her2.

In certain aspects, the VHH fragment is specific for a mammalian T cell or a mammalian NK cell. In certain aspects, the VHH fragment is specific for an antigen selected from the group consisting of CD3, CD16, CD19, CD28, and CD64. In certain aspects, the antigen is CD16 or CD3.

In certain aspects, the VHH fragment does not comprise Val, Gly, Leu, and Trp residues at Kabat positions 37, 44, 45, and 47, respectively.

In one embodiment, the invention further provides a host cell comprising one or more polynucleotides encoding an antibody of the invention. In certain aspects, the host cell is a bacterial cell or a yeast cell. In certain aspects, the host cell is E. coli.

In another embodiment, the invention provides a method of making a soluble antibody, the method comprising administering a host of the invention and harvesting an antibody expressed in the cell.

Still further, in one embodiment, the invention provides a method of treating a tumor in a patient, the method comprising administering to the patient an antibody of the invention.

DRAWINGS

Figure 1a is a bivalent antibody comprising an antigen binding fragment (Fab) and a single domain antigen binding fragment (VHH). This Fab fragment includes a partial heavy chain having a variable domain (VH) and a constant domain (CH1), and a light chain having a variable domain (VL) and a constant domain (CL). The heavy and light chains are linked by disulfide bonds. The VHH fragment was fused to the C-terminus of the heavy chain.

Figure 1b is a trivalent antibody comprising an antigen binding fragment (Fab) and two single domain antigen binding fragments (VHH and VHH'). The Fab fragment includes a partial heavy chain having a variable domain (VH) and a constant domain (CH1), and a light chain having a variable domain (VL) and a constant domain (CL). The heavy and light chains are linked by disulfide bonds. The VHH and VHH' fragments are each fused to the C-terminus of the heavy and light chains.

Figures 2a-f show the design and preparation of the bispecific antibody S-Fab. (a) Single-stranded bacterial expression construct. This structure comprises: a PelB signal sequence, a humanized anti-CD3 (UTCH1 clone) VH or VL, CH1 or CL, and an anti-CEA VHH. A Flag tag or a his8 tag is added to the C-terminus to aid in protein detection and purification. (b) Protocol by co-expression of control Fab (HCBF4/5) and S-Fab (HCB5/6). (c) and (b), SDS-PAGE Coomassie blue staining and immunoblotting were performed to detect purified proteins after immobilized Ni-NTA affinity chromatography. Above, Ni-NTA affinity chromatography (M, molecular weight ladder; SF, sucrose component; PF, periplasmic component; P, micropore sphere; FT, penetrant; W, lotion; E, wash Deliquoring; E1, 100 nM imidazole, E2, 200 nM imidazole, E3, 300 nM imidazole, E4, 400 nM imidazole). Arrows indicate purified HCBF4/5 (left) and HCBF5/6 (right); middle panel, immunoblotting to detect HCBF5 by anti-His antibody; bottom panel, immunoblotting to detect HCBF4 (left) and HCBF6 by anti-Flag antibody (right) ). (e) Purified protein after the second immobilization of Ni-NTA affinity chromatography. (f) Gel filtration chromatography showed HCBF4/5 and HCB5/6 electrophoresis to 60 kd. Above, gel-filtered protein markers.

Figures 3a-c show that purified S-Fab recognizes T cells and CEA antigens. (a) Flow cytometric analysis of HCBF5/6 (50 ug/ml) (black line) and control OKT3 antibody (red line) on CD3-positive Jurkat cells, gray areas were unstained cells, and blue lines were Only anti-Flag-FITC stained cells. (b) and (c), SPR analysis of binding of HCBF4/5 and HCB5/6 to CEA.

Figures 4a-e show that S-Fab kills tumor cells in a T cell-dependent and tumor antigen-dependent manner. Human T cells were mixed with tumor cells in a ratio of 10:1 (a) and 5:1 (b) in the presence of the indicated concentrations of S-Fab and Fab. After the cells were incubated for 48 hours, cytotoxicity was measured as described in Methods and Materials. (c) CEA immunoblot analysis in different cell lines. (d) EC50 of S-Fab HCBF5/6 in HT29 (open squares), LS174T (open triangles), DLD-1 (open diamonds) cells. (e) Binding to anti-CD3 affects S-Fab HCB5/6 activity. Cytotoxicity assays were performed using T cells and LS174T (10:1) in the presence of the indicated concentrations of HCBF5/6 and control Fab (*P<0.05, T test, 100 nM HCBF4/5 versus HCBF4/5) . All error bars represent the standard deviation of three identical experiments. The data represents one of at least three independent experiments.

Figure 5 shows that S-Fab inhibits tumor growth in vivo. NOD/SCID mice (n=5/group) were transplanted subcutaneously with LS174T and human PBMC. Subsequently, mice were given PBS (solid diamond, PBS treatment, no PBMC transplantation); only PBMC (open triangle, PBS treatment, PBMC transplantation); HCBF4/5 (open circle, HCBF4/5 treatment, PBMC transplantation); Represents the average tumor volume of 5 mice. The error bars represent the standard deviation. (*P<0.05, T test, HCBF5/6 is better than the other three groups).

Figure 6. Bacterial expression constructs of Her2-S-Fab. (a) Her2-S-Fab and control Her2-Fab purified from E. coli, each construct containing a signal sequence pelB for periplasmic expression; anti-Her2 Co-expression of -VL-CL and anti-Her2-VH-CH1 constructs to generate Her2-Fab; co-expression of anti-Her2-VL-CL and anti-Her2-VH-CH1-anti-CD16 VHH constructs to produce Her2-S-Fab; To facilitate protein detection and purification, a Flag tag or a His8 tag was ligated at the C-terminus of each construct. (b) Coomassie brilliant blue staining of Her2-S-Fab after two-step purification, M represents a molecular weight marker in kD; (c) Gel filtration chromatography showed that the size of Her2-S-Fab was about 65 kD.

Figure 7. Her2-S-Fab recognizes Her2 positive cells. Flow cytometric analysis of Her2-PE antibody (a), trastuzumab (b), control Fab (c) and Her2-S-Fab (d) on different cell lines.

Figure 8. Her2-S-Fab induces NK cell mediated cytotoxicity. (a) CHO and SKOV3 cell lines were subjected to cytotoxicity experiments with 10 μg/ml of Her2-Fab, Her2-S-Fab or trastuzumab. The data is the average of three experiments and the error bars represent the standard deviation. Black indicates CHO in the absence of NK cells; gray indicates CHO to which NK is added; thick stripes indicate SKOV3 without NK cells; and streak indicates SKOV3 to which NK is added. (b-f) Analysis of dose-dependent cytotoxicity of different cell lines: CHO (b); MDAMB435 (c); MCF7 (d); SKBR3 (e) and SKOV3 (f). The concentration of Her2-Fab, Her2-S-Fab or trastuzumab is from 0.01 ng/ml to 10 μg/ml. The data is the average of three experiments and the error bars represent the standard deviation.

Figure 9. Her2-S-Fab inhibits tumor growth in vivo. NOD/SCID mice (n=5/group, male) were subcutaneously inoculated with a mixture of SKOV3 cells (2×10 ⁶ ) and freshly isolated human PBMC (2×10 ⁷ ). Two hours later, PBS, control Her2-Fab (1 mg/kg) or Her2-S-Fab (1 mg/kg) was intraperitoneally administered as the first dose (D ₀ ). On the following 6 days (D1, D2, D3, D4, D5 and D6), mice were injected subcutaneously (ip) with PBS (dotted line); Her2-Fab (1 mg/kg, dotted line) or Her2-S-Fab (1 mg) /kg, solid line). Data represent the mean tumor volume of 5 mice. Error bars indicate standard deviation (*P<0.05, t test, Her2-S-Fab vs. other two groups).

detailed description

I. Definition

It should be noted that the term "a" entity refers to one or more such entities, for example, "a bivalent antibody" is understood to mean one or more bivalent antibodies. Likewise, the terms "a", "one or more" and "sai" are used interchangeable.

"Homology" or "identity" or "similarity" refers to sequence similarity between two polypeptide sequences or between two nucleic acid molecules. Homology is determined by comparing the positions at each sequence, and each sequence may be aligned for comparison purposes. When the positions of such comparison sequences are occupied by the same base or amino acid, then these molecules are homologous at that position. The degree of homology between sequences varies with the number of matches or homology positions shared by these sequences. "not related" Or "non-homologous" sequence means having less than 40% identity, preferably less than 25% identity to one of the sequences of the invention.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage to another sequence (eg, 60%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, "Sequence identity" of 95%, 98% or 99%) means that when aligned, the percentage bases (or amino acids) are identical when comparing the two sequences. The percentage of such alignment and homology or sequence identity can be determined using software programs known in the art.

The term "equivalent polynucleotide" refers to a nucleic acid sequence that has a degree of homology or sequence identity to a reference nucleic acid sequence or its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide that has some degree of homology or sequence identity to the amino acid sequence of a control polypeptide. In certain aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, such equivalent sequences retain the activity (eg, epitope binding) or structure (eg, salt bridge) of the control sequence.

The equivalents of each of the polypeptides or polynucleotides disclosed herein are also contemplated. In one aspect, an equivalent of a polypeptide includes a change (ie, deletion, addition or substitution) of an amino acid residue. In one aspect, a polypeptide equivalent comprises no more than two amino acid residue changes. In one aspect, an equivalent of a polypeptide includes no more than 3, 4 or 5 amino acid residues. In some aspects, such amino acid changes are located at residues that are not critical to the activity of the referenced polypeptide. Residues critical for polypeptide activity can be readily tested by site-specific mutation analysis, or even sequence alignment (because this sequence is highly conserved).

As used herein, "antibody" or "antigen binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to one or more antigens. An antibody is a whole antibody and any antigen-binding fragment or single strand thereof. Thus, the term "antibody" includes any protein or peptide that contains at least a molecule of an immunoglobulin molecule, a portion of which has biological activity for binding to an antigen. Such examples include, but are not limited to, a complementarity determining region (CDR), a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region of a heavy/light chain or a ligand binding portion thereof Or any part thereof, or at least a portion of a binding protein. The term antibody also encompasses a polypeptide or polypeptide complex that possesses antigen binding ability upon activation.

The term "antibody fragment" or "antigen-binding fragment" as used herein is part of an antibody, such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of the structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

Antibodies, antigen binding polypeptides, variants or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, human, primatized or chimeric Antibodies, single-chain antibodies, epitope-binding fragments (eg, Fab, Fab' and F(ab') ₂ , Fd, Fvs, single-chain Fvs (scFv)), single-chain antibodies, disulfide-linked Fvs (sdFv) Fragments comprising a VK or VH domain, fragments generated from a Fab expression library, and anti-idotype (anti-Id) antibodies (including, for example, the anti-Id antibodies disclosed herein to LIGHT antibodies). The immunoglobulin or antibody molecule of the invention may be immunized with any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), species (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass Globulin molecule.

The light chain is divided into kappa or lambda (,). Each heavy chain species can be combined with a kappa or lambda light chain. In general, the light and heavy chains are covalently bound to each other. When immunoglobulins are produced by hybridoma, B-cell or genetically engineered host cells, the "tail" of the two heavy chains is passed through a covalent disulfide bond or Non-covalent bonds combine with each other. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked end of the Y conformation to the C-terminus at the bottom of each strand.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that both the light chain variable domain (VK) and the heavy chain variable domain (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental fluidity, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become distant from the antigen binding site or the amino terminus of the antibody. The N-terminal portion is the variable domain and the C-terminal portion is the constant domain; the CH3 and CK domains actually comprise the carboxy terminus of the light and heavy chains, respectively.

As described above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on the antigen. That is, the VK domain and VH domain of an antibody, or a subclass of complementarity determining regions (CDRs), combine to form a variable domain that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site that is presented at the end of each arm of Y. More specifically, the antigen binding site is determined by three CDRs on each VH and VK chain (ie, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some instances, for example, certain immunoglobulin molecules derived from Camelidae or Camelidae immunoglobulin-based engineering, intact immunoglobulin molecules may consist solely of heavy chains without a light chain, see for example Hamers -Casterman et al., Nature 363:446-448 (1993).

In natural antibodies, the six "complementarity determining domains" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically localized to present a three-dimensional conformation of the antibody in an aqueous environment. The form forms an antigen binding domain. The remaining portion of the amino acid in the antigen binding domain (referred to as the "framework region") exhibits lower intermolecular variability. The framework regions are predominantly in a folded conformation, the CDRs forming a loop, and these loops are connected-folded structures, in some cases forming part of a folded structure. Thus, the framework regions are used to form scaffolds that position the CDRs in the correct orientation by non-covalent interactions between the strands. The antigen binding domain formed by the aligned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface causes the antibody to bind non-covalently to its cognate epitope. For any variable domain of a heavy or light chain, one of ordinary skill in the art can readily identify these amino acids comprising the CDRs and framework regions, respectively, since they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987), which are incorporated by reference in their entirety. In this article).

If two or more definitions are used and/or accepted in the art for a term, the definition of the term is intended to include all such meanings unless otherwise indicated. A specific example is the term "complementarity determining region" ("CDR") used to describe a non-contiguous antigen binding site found in both the variable region of a heavy chain polypeptide and a light chain polypeptide. This particular region has been described in Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. MoI. Biol. : 901-917 (1987), which is incorporated herein by reference. According to the definition of CDRs by Kabat and Chothia, it includes overlapping or subpopulations of amino acid residues when compared to each other. However, the use of any definition of a CDR for an antibody or variable region thereof is considered to be within the scope of the terms defined and used in the present invention. Suitable amino acid residues comprising the CDRs as defined in any of the above cited documents are listed in the table below for comparison. The exact number of residues comprising a particular CDR varies depending on the sequence and size of the CDRs. For the variable region amino acid sequence of an antibody, one skilled in the art can determine a residue comprising a specific CDR by a conventional method.

Kabat et al. also define a numbering system that is adaptable to the variable domain sequences of any antibody. One skilled in the art can explicitly apply the "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in: Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

"Specific binding" or "specific to" generally refers to the binding of an antibody to an epitope by its antigen binding domain, and such binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it is capable of binding to a particular epitope more rapidly via its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used to quantify the relative affinity of a particular antibody for binding to a particular epitope. For example, for a given epitope, antibody "A" is considered to be more specific than antibody "B", or antibody "A" binds to epitope "C" more specifically than its binding to the relevant The specificity of epitope "D".

The term "treating" as used herein refers to a therapeutic or prophylactic means in which a subject is prevented or slows (mitigates) an undesired pathological change or disorder, such as the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of disease states (ie, no deterioration), delay or slowing of disease progression, improvement or mitigation of disease states, and remission ( Whether local or holistic, whether or not these results are detectable or undetectable. "Treatment" also refers to prolonged survival as compared to the expected survival without treatment. Subjects in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition or those in need of prevention of the disease or condition.

"Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, in whom a diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, domesticated animals, farm animals, and zoo animals, competitive animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

The term "patient in need of treatment" or "subject in need of treatment" includes a subject, such as a mammalian subject, that can benefit from administration of an antibody or composition of the invention to achieve, for example, detection, diagnostic procedures, and/or therapeutic purposes. .

II. Bivalent and trivalent antibodies

In one embodiment, the invention provides a bivalent antibody having a single domain antigen-binding fragment (VHH) fused to one of the strands of a Fab fragment. When another strand of the Fab fragment is also fused to a single domain antigen-binding fragment (VHH'), the antibody is trivalent.

The bivalent or trivalent antibodies of the invention may be monospecific, bispecific or trispecific. When Fab and VHH have specificity for binding to the same antigen, the antibody is monospecific. If they have different binding specificities, the antibody is bispecific. If VHH, VHH' and Fab each have a different binding specificity, the antibody is trispecific.

As demonstrated in the test examples, the schematic type of bispecific antibody is an antibody that targets two different antigens, one of which is present on tumor cells or microorganisms and the other on immune cells. When administered to an individual, such bispecific antibodies specifically bind to tumor cells or microorganisms while specifically binding to immune cells (eg, cytotoxic cells). This dual binding can result in the bound tumor or microorganism being killed by the host's immune system.

A. Unexpected properties of the bispecific antibodies of the invention

Since the VHH fragment is much smaller and shorter than conventional antibodies, it has previously been suspected that when VHH is fused to a Fab fragment, large proteins inhibit the binding ability of VHH due to steric effects. Moreover, it is suspected that this chimeric protein will not be stable under physiological conditions.

However, it is unexpected that the suspected steric effect has a limited effect on the function of the bivalent antibodies of the invention. Moreover, the overall affinity of this antibody (which does not require an Fc fragment) is comparable to that of a conventional antibody. Another surprise is that VHH binds efficiently to its target, whether it is in the same orientation (ie, the N-terminus of VHH is attached to the N-terminus of the heavy or light chain of the Fab fragment) or in the opposite direction (ie The C-terminus of VHH is linked to the N-terminus of the heavy or light chain of the Fab fragment) to the Fab fragment.

The advantages of the invention are also manifested in cell expression efficiency and protein stability. Although small antibodies are generally considered to be more readily produced in cells than larger antibodies, heterodimeric antibodies pose particular challenges for antibody production. This is at least because two (or three or four) different protein chains can interact inside and outside the cell, causing interference, thereby reducing the efficiency of the host cell expression system and increasing protein instability. Thus, heterodimeric antibodies with all heavy and light chains are typically expressed in two separate cells, or a common light chain is used to reduce mismatching of the light and heavy chains, which results in the production of non-functional antibodies.

Even those bispecific antibodies that are smaller than the bispecific antibodies disclosed herein have these problems. For example, a bispecific T cell adaptor (BITE) is a fusion protein composed of two typical single chain variable fragments. Although small The bispecific antibody (about 65 kDa) disclosed herein (usually 55 KDa), but BITE cannot be expressed in soluble form in bacterial cells. Currently, BITE is expressed in mammalian cells, which is very expensive.

Unexpectedly, as demonstrated in the test examples, up to about 20% of the bispecific antibodies of the invention were soluble (based on Western blot analysis) when expressed in E. coli cells. As mentioned above, these antibodies are much larger than BITE. However, although the bacterially expressed BITE is completely insoluble, the antibody of the present invention can be easily produced by bacteria. Therefore, the results are surprising and unexpected.

Thus, the antibodies of the invention are relatively small in size compared to conventional antibodies and can be produced efficiently, particularly in large scale production, such as in yeast and bacterial hosts. The stability, solubility and half-life of these antibodies are vastly superior to those of bispecific antibodies being developed in the art.

As can be seen from the foregoing description, the antibodies of the present invention exhibit high bacterial productivity, stability, and binding affinity. Examples of such bivalent and trivalent antibodies are shown in Figures 1a and 1b, respectively.

Figure 1a shows a bivalent antibody with a Fab portion and a VHH portion. The Fab portion includes a light chain having a variable domain (VL) and a constant domain (CL). The Fab portion also includes a heavy chain having a variable domain (VH) and a constant domain (CH1). Unlike the heavy chain of a common antibody, the heavy chain does not include any one or both of the CH2 domain and the CH3 domain. A Fab fragment is a functional unit that specifically binds to an antigen. Structurally, VH and VL comprise CDRs suitable for this specificity, with CH1 and CL having appropriate amino acid residues such that they are joined to each other to form the desired Fab structure.

Figure 1a shows that the VHH fragment is fused to the C-terminus of the CH1 domain of the heavy chain of the Fab fragment. It will be readily appreciated that in some embodiments, the VHH fragment can also be fused to the light chain. When fused to a Fab fragment, the VHH and Fab can have the same N-terminal to C-terminal orientation such that the entire strand can be expressed as a single polynucleotide. In another embodiment, the C-terminus of the VHH can be linked to the C-terminus of the heavy or light chain of the Fab fragment.

Similarly, Figure 1b shows a trivalent antibody of the invention comprising a Fab portion and two VHHs (VHH and VHH'), each VHH fused to a strand of a Fab fragment.

The Fab portion of the antibody can be readily identified and prepared. For example, the sequences of the light and heavy chains can be identified from any conventional antibody comprising two identical Fab fragments. Conventional antibodies can be derived from animals or humanized and can be further modified by methods known in the art.

A "single domain antigen binding fragment" or "single domain antibody fragment" or "VHH" is an antigen binding fragment that is capable of binding to an antigen without the need for a light chain. VHH was originally isolated as a single domain antibody (sdAb) as a single antigen-binding fragment. The first known single domain antibody was isolated from camel (Hamers-Casterman et al., Nature 363:446-8 (1993)) and thereafter isolated from cartilage fish. Camels produce functional antibodies without light chains, their single N-terminal domain (VHH) binding antigen without domain pairing (see Harmsen and Haard, App Microbiol Biotechnol., 77: 13-22 (2007)). Single domain antibodies do not include the CH1 domain, and in conventional antibodies, the CH1 domain interacts with the light chain.

VHH comprises four framework regions (FR1-FR4) constituting the core structure of the immunoglobulin domain and three complementarity determining regions (CDR1-CDR3) involved in antigen binding. Compared to the human VH domain, the VHH framework area shows High sequence homology (>80%) of the VH domain. See Harmsen and Haard, 2007, which further describes: "The most characteristic feature of VHH is the amino acid substitutions at the four FR2 positions (positions 37, 44, 45 and 47; Kabat numbering), which are in the conventional VH structure. The domains are conserved and involve hydrophobic interactions with the VL domain." VHH typically has different amino acids (such as Leu11Ser, Va137Phe or Tyr, Gly44Glu, Leu45Arg or Cys, Trp47Gly) at these and other positions that are highly conserved in conventional VH.

Harmsen and Haard, 2007 also describes that the CDRs of VHH have certain known characteristic features. For example, the N-terminal portion of CDR1 is more variable than conventional antibodies. Moreover, certain VHHs have an extended CDR3 that is normally stabilized by the formation of additional disulfide bonds with cysteines in CDR1 or FR2, resulting in the CDR3 loop folding over the entire interface of the previous VL. The specific subfamily of llama VHH (VHH3) also contains an extended CDR3 that is stabilized by the formation of additional disulfide bonds with the cysteine at position 50 of FR2.

Many sdAbs are known in the art and can be readily prepared from animals such as camels. Based on these sdAbs, their VHH can be easily identified and prepared. Table 1 lists non-limiting examples of many VHHs and sdAbs. Accordingly, in some embodiments, the invention provides polypeptides comprising each such disclosed sequence or equivalent thereof, as well as polynucleotides encoding each polypeptide. In one aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having one, two or three amino acid insertions/deletions/substitutions.

Table 1. Exemplary single domain antigen binding fragments (VHH) and single domain antibodies (sdAb)

As is apparent from the above table, certain regions of these sequences are highly conserved (e.g., FR1-3), while CDRs are more variable.

B. Monospecific, bispecific and trispecific antibodies

Bivalent antibodies can be monospecific when the Fab fragment and VHH have the same binding specificity, or bispecific antibodies can be bispecific when they have different binding specificities. Similarly, trivalent antibodies can be monospecific, bispecific or trispecific.

Bispecific antibodies can be configured to target different antigens. For example, between a Fab fragment and a VHH fragment, one has specificity for the first immune cell and the other can target the second immune cell; one has specificity for the first tumor cell and the other has the second The specificity of tumor cells; one has specificity for immune cells and the other has specificity for microorganisms, infected cells, tumor cells, inflammatory cells, apoptotic cells or exogenous cells (undefined).

Similarly, trivalent antibodies can be configured as desired. In one aspect, the Fab, VHH and VHH&apos; fragments have specificity for the same antigen or epitope. In another aspect, the Fab fragment has specificity for one antigen or epitope, while the VHH and VHH&apos; fragments have specificity for another antigen or epitope. In another aspect, the Fab fragment has the same binding specificity as VHH but different from VHH'. In another aspect, each of them has a different binding specificity.

Non-limiting examples of binding structures of divalent and trivalent antibodies are listed in the table below.

Table 2. Exemplary structures of bivalent antibodies

Fragment	Binding specificity	Fragment	Binding specificity
Fab	Tumor cell	VHH	Same tumor cell
Fab	Immune Cells	VHH	Identical immune cell
Fab	Tumor cell	VHH	Another tumor cell

Fab	Immune Cells	VHH	Another immune cell
Fab	Tumor cell	VHH	Immune Cells
Fab	Immune Cells	VHH	Tumor cell
Fab	Immune Cells	VHH	microorganism
Fab	microorganism	VHH	Immune Cells

Table 3. Exemplary structures of trivalent antibodies

Fragment	Binding specificity	Fragment	Binding specificity	Fragment	Binding specificity
Fab	Immune Cells	VHH	Second immune cell	VHH’	Second immune cell
Fab	Immune Cells	VHH	Second immune cell	VHH’	Third immune cell
Fab	Tumor cell	VHH	Second tumor cell	VHH’	Second tumor cell
Fab	Tumor cell	VHH	Second tumor cell	VHH’	Third tumor cell
Fab	Immune Cells	VHH	Tumor cell	VHH’	Tumor cell
Fab	Immune Cells	VHH	Tumor cell	VHH’	Second tumor cell
Fab	Tumor cell	VHH	Immune Cells	VHH’	Immune Cells
Fab	Tumor cell	VHH	Immune Cells	VHH’	Second immune cell
Fab	Tumor cell	VHH	Immune Cells	VHH’	Second tumor cell
Fab	Immune Cells	VHH	Tumor cell	VHH’	Second immune cell

C. Combining the target

In some embodiments, the Fab or VHH (or VHH&apos;) fragment of a bivalent antibody has immunological specificity for a tumor antigen.

A "tumor antigen" is an antigenic substance produced in a tumor cell, that is, it causes an immune response of the host. Tumor antigens can be used to identify tumor cells and are potential candidates for cancer therapy. Normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and thus appear to be "exogenous" to the body. This may include proteins that are well evaded from the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only at a particular developmental stage, or proteins whose structure is modified by mutation.

Many tumor antigens are known in the art and many new tumor antigens are readily found by screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2 , VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

In some aspects, the Fab or VHH fragment has specificity for a protein that is overexpressed on tumor cells compared to the corresponding non-tumor cells. "Corresponding non-tumor cells" refers to non-tumor cells having the same cell type as cells from which tumor cells are derived. It should be noted that such proteins are not necessarily different from tumor antigens. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon cancer, rectal cancer, breast cancer, lung cancer, pancreatic cancer, and gastrointestinal cancer; Protein receptor (HER-2, neu or c-erbB-2), which is commonly overexpressed in breast, ovarian, rectal, lung, prostate, and cervical cancer; epidermal growth factor receptor, in a series of Overexpression in solid tumors (including breast cancer, head and neck cancer, non-small cell lung cancer, and prostate cancer); asialoglycoprotein receptor; transferrin receptor; serine protease inhibitor enzyme complex expressed on hepatocytes Fibroblast growth factor receptor (FGFR) overexpressed on islet ductal adenocarcinoma cells; vascular endothelial growth factor receptor (VEGFR) for anti-angiogenic gene therapy; selectively overexpressed at 90% Folate receptors for non-mucinous ovarian cancer; cell-surface glycocalyx (glycocalyx); carbohydrate receptors; and polyimmunoglobulin receptors, which are beneficial for gene transport to respiratory epithelial cells and for lung disease Treatment (eg, cystic fibrosis) is attractive.

In one aspect, the VHH fragment is specific for CEA or Her2. A representative sequence of such VHH is SEQ ID NO: 1 or 6 (Table 1). In some aspects, the VHH comprises the amino acid sequence of SEQ ID NO: 1 or 6 and has one or two or three insertions, deletions or substitutions. In one aspect, the VHH comprises the amino acid sequence of SEQ ID NO: 7 (anti-EGFR1), and optionally has one or two or three insertions, deletions or substitutions.

In some aspects, the Fab or VHH fragment is specific for a microorganism (eg, a virus or a bacterium). Non-limiting examples of microorganisms include microbial surface receptors and endotoxins. Examples of endotoxins include, but are not limited to, lipopolysaccharide (LPS) and lipooligosaccharides (LOS).

In some aspects, the VHH fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-11 (Table 1), or optionally one or two or three insertions, deletions or substitutions.

In some aspects, another fragment of the bivalent antibody is specific for the immune cell. In one aspect, the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocytic cell, a natural killer cell, an eosinophil, a basophil, and a hypertrophy. A group of cells.

In one aspect, the additional fragment specifically recognizes an antigen selected from the group consisting of CD3, CD16, CD19, CD28, and CD64. In one aspect, the second VHH specifically recognizes CD3 or CD16.

In one aspect, the additional fragment is specific for CD16 or CD3. Representative sequences of VHH fragments are SEQ ID NO: 2, 3, 4, 5, 12 and 13 (Table 1), or optionally one or two or three insertions, deletions or substitutions.

In some aspects, the Fab portion comprises: one or two amino acid sequences selected from the group consisting of SEQ ID NOs: 14-25 (Table 4), or optionally one or two or three insertions, deletions or substitutions.

Table 4. Example of Fab sequence

Table 5 below shows the amino acid sequences of one embodiment of the bispecific antibody of the present invention.

Table 5. Bispecific antibodies targeting CD3 (at Fab) and CEA (at VHH)

Any of the antibodies or polypeptides described above may further comprise additional polypeptides, for example, signal peptides that direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.

One of ordinary skill in the art will appreciate that the antibodies disclosed herein can be modified such that they differ in amino acid sequence from the native binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar, for example, having a certain percentage of identity to the starting sequence, for example it may be 60%, 70%, 75%, 80% identical to the starting sequence. , 85%, 90%, 95%, 98% or 99% consistency.

In addition, conservative substitutions, deletions or insertions of nucleotides or amino acids can be made in the "non-essential" amino acid region. For example, a polypeptide or amino acid derived from a particular protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions (eg, one, two, three, four, five, six, Seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions or deletions). In certain embodiments, the polypeptide or amino acid sequence derived from a particular protein has from one to five, one to ten, or one to twenty individual amino acid substitutions, insertions or deletions relative to the starting sequence.

In certain embodiments, an antigen binding polypeptide comprises an amino acid sequence or one or more portions that are not normally associated with an antibody. Exemplary modifications are described in detail below. For example, a fragment of the invention may comprise a flexible linker sequence or may be modified to add a functional moiety (eg, PEG, drug, toxin or marker).

An antibody, variant or derivative thereof of the invention includes a modified derivative, i.e., any type of molecule covalently linked to the antibody, so long as the covalent linkage does not prevent binding of the antibody to the epitope. For example, but not by way of limitation, antibodies may be modified, such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting or blocking groups, protease cleavage, ligation to cells Ligand or other protein. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, an antibody may comprise one or more atypical amino acids.

In other embodiments, an antigen binding polypeptide of the invention may comprise a conservative amino acid substitution.

In a "conservative amino acid substitution", one amino acid residue is substituted with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) Uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine, Proline, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine) and aromatic Family side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues in the immunoglobulin polypeptide are more suitably substituted with other amino acid residues from the same side chain family. In another embodiment, the amino acid chain can be substituted with a structurally similar but sequential or compositionally different chain of side chain family members.

The following table provides non-limiting examples of conservative amino acid substitutions. A similarity score of 0 or higher in the table indicates a conservative substitution at both amino acids.

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	I	L	F	Y	W
W	-8	-7	-6	-2	-6	-5	-7	-7	-4	-5	-3	-3	2	-6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	-2	-2	-1	-1	7	10
F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	-1	0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
I	-2	-3	-2	-1	-1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5
M	-5	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
V	-2	-1	-1	-1	0	0	-2	-2	-2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
K	-5	-2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
T	-2	0	0	1	1	3
A	-2	1	1	1	2
S	0	1	1	1
P	-3	-1	6
G	-3	5
C	12

In some embodiments, an antibody can be bound to a therapeutic formulation, prodrug, peptide, protein, enzyme, virus, lipid, biological effect modifier, pharmaceutical formulation, or PEG.

These antibodies can be bound or fused to a therapeutic preparation, which can include a detectable label (eg, a radiolabel), an immunomodulator, a hormone, an enzyme, an oligonucleotide, a phototherapeutic or diagnostic agent, a cell. Toxic agents (drugs or toxins), ultrasound enhancers, non-radioactive labels, combinations thereof, and other formulations known in the art.

By labeling the antibody with a compound that is coupled to chemiluminescence, the antibody can be detected. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of fluorescence (during the course of a chemical reaction). Extremely Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridine salt and oxalate.

III. Polynucleotide encoding an antibody and method for preparing the same

The invention also provides an isolated polynucleotide or nucleic acid molecule encoding a bispecific antibody, a variant or derivative thereof.

Polynucleotides of the invention may encode all VHH, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, a polynucleotide of the invention may encode a portion of an antibody or VHH, a variant or derivative thereof, on the same polynucleotide molecule or on a separate polynucleotide molecule.

In certain embodiments, the prepared antibody will not cause a deleterious immune response on a subject animal, such as a human. In one embodiment, the antigen binding polypeptides of the invention, variants or derivatives thereof, are modified using domain recognized techniques to reduce their immunogenicity. For example, the antibody can be humanized, primatized, deimmunized, or a chimeric antibody can be prepared.

The binding specificity of the bispecific antibodies of the invention can be determined by in vitro assays (e.g., immunoprecipitation, radioimmunoassay (RIA) or immunoenzymatic adsorption (ELISA)).

IV. Production systems and methods

The invention also provides methods and systems for producing the bispecific antibodies of the invention. Cells suitable for the production of antibodies known in the art include human cells (e.g., CHO cells), mammalian cells, and bacterial cells. The use of bacterial cells to produce bispecific antibodies presents significant challenges. However, as shown in the examples, when an antibody is expressed in a bacterial cell, even when both peptide chains are expressed in the same cell, the produced antibody is largely soluble.

Accordingly, in one embodiment, the invention provides a host cell comprising one or more polynucleotides encoding two strands of a bispecific antibody of the invention. In one aspect, a single nucleotide construct (eg, a plasmid) includes two coding sequences. In another aspect, the invention provides two different polynucleotide constructs, each encoding one of the polynucleotide strands. In one embodiment, the invention also provides a host cell comprising two polypeptide chains of a bispecific antibody of the invention.

In some aspects, the host cell is a human cell. In some aspects, the host cell is a mammalian cell. In some aspects, the host cell is a yeast cell. In some aspects, the host cell is a bacterial cell, including G+ and G-bacterial cells. Representative bacterial cells include, but are not limited to, Escherichia coli and Salmonella typhimurium.

In certain aspects, the invention also provides methods of making the bispecific antibodies of the invention. In one aspect, the method entails expressing two peptide chains of the antibody in a host cell and extracting the antibody from the cell lysate. Furthermore, the present invention also provides bispecific antibodies obtained by these methods.

V. Treatment and diagnostic methods

As noted above, the bispecific antibodies, variants or derivatives thereof of the invention are useful in certain therapeutic and diagnostic methods associated with cancer or infectious diseases.

The invention further relates to antibody-based therapies which involve administering a bispecific antibody of the invention to a patient, such as an animal, a mammal and a human, to treat one or more of the diseases or conditions described herein. Therapeutic compositions of the invention include, but are not limited to, antibodies of the invention (including variants and derivatives thereof described herein) and antibodies encoding the invention (including variants and derivatives thereof described herein) Nucleic acid or polynucleotide.

The antibodies of the invention can be used to treat, inhibit or prevent a disease, disorder, or condition, including a malignant disease, disorder, or condition associated with, for example, a disease or disorder (such as cancer) associated with increased cell survival or inhibition of apoptosis, Such cancers include, but are not limited to, follicular lymphoma, cancer with p53 mutations, and hormone-dependent tumors (including but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinal neoplasia, glioblastoma, Lung cancer, colon cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, fibroids, lymphoma, endothelial tumor, osteoblastoma, osteoclast, osteosarcoma, chondrosarcoma, adenocarcinoma, breast cancer, Prostate cancer, Kaposi sarcoma; autoimmune disorders (eg multiple sclerosis, Sjogren syndrome, Grave disease, Hashimoto thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic Lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura and rheumatoid arthritis; and viral Dye (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft vs. host disease (acute and / or chronic), acute graft rejection, and chronic graft rejection. The antigen binding polypeptides, variants or derivatives thereof of the invention are used to inhibit the development, progression and/or metastasis of cancer, particularly the cancers listed above or in subsequent paragraphs.

Other diseases or conditions that may be associated with an increase in cell viability, including, but not limited to, progression and/or metastasis of an antibody, or a variant or derivative thereof, of the invention, or a variant or derivative thereof, include: malignancy and Related diseases such as leukemia (acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia (including myeloblasts, promyelocytic leukemia, myelomonocytes, monocytes, and leukemia cells)) and chronic leukemia (such as chronic Myeloid (granulocyte) leukemia and chronic lymphocytic leukemia), true erythrocytosis, lymphoma (such as Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumor ( Including but not limited to: sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, spinal tumor, angiosarcoma, endothelial sarcoma, lymphosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelium) Tumor, Ewing tumor, leiomyomas, rhabdomyosarcoma, colon cancer, pancreatic cancer, thymic carcinoma, ovarian cancer, prostate cancer, squamous cell carcinoma, Bottom cell carcinoma, adenocarcinoma, sweat adenoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryogenic Cancer, Wilm tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, squamous cell tumor, medulloblastoma, craniopharyngioma, ependymoma, pineal gland Tumor, hemangioblastoma, acoustic neuroma, mesenchymal glioma, meningioma, melanoma, neuroblastoma, and ocular cell membrane tumor)).

The antibodies of the present invention can also be used to treat infectious diseases caused by microorganisms or to kill microorganisms by targeting microorganisms and immune cells to affect the elimination of microorganisms. In one aspect, the microorganism is a virus (including RNA and DNA viruses), a Gram-positive bacterium, a Gram-negative bacterium, a protozoa or a fungus.

The dosage and treatment regimen specific for any particular patient will depend on a variety of factors (including the specific antigen binding polypeptide used, variants or derivatives thereof, age, weight, overall health, sex, diet, time of administration, excretion of the patient) Rate, combination of drugs, and severity of the particular disease being treated). The determination of such factors by a medical professional is within the judgment of one of ordinary skill in the art. The dosage will also be based on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the composition employed, the severity of the disease, and the desired effect. The dosage employed can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

Methods of administration of bispecific antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption of the epithelial or mucosal protective layer (eg, oral mucosa, rectal and intestinal mucosa, etc.); Formulations are used together. Thus, a pharmaceutical composition comprising an antigen binding polypeptide of the invention may be administered orally, rectally, parenterally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops or dermal patches) Spray application of the mouth or nose.

The term "parenteral" as used herein refers to the manner of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injection and infusion.

Administration can be systemic or topical. Furthermore, it is contemplated that the antibodies of the invention can be introduced into the central nervous system by any suitable route, including: intraventricular and intrathecal injection; promotion by intraventricular catheters (eg, attachment to a reservoir (eg, Ommava reservoir)) Intraventricular injection. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and a formulation with an aerosol.

It may be desirable to topically administer a bispecific antibody or composition of the invention to a region in need of treatment; this may be accomplished, for example, but not limited to, by local infusion, topical administration (eg, post-operative combination) Use a wound dressing), inject, through a catheter, through a suppository, or through an implant (the implant is a porous, non-porous, non-permeable or gel-like material, including a film (eg, a silicone membrane) or fiber) . Preferably, when a protein (including an antibody) comprising the present invention is administered, care should be taken to use a material that the protein does not adsorb.

An effective amount of an antibody of the invention in the treatment, inhibition, and prevention of an inflammatory, immune or malignant disease, disorder, or condition can be determined by standard clinical techniques. In addition, in vitro assays can be optionally employed to help identify the optimal dose range. The exact dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined in accordance with the judgment of the practitioner and the condition of each patient. Effective doses can be deduced from dose-response curves derived from in vitro or animal model test systems.

As a general recommendation, the dose of the antigen-binding polypeptide of the present invention administered to a patient is usually from 0.1 mg/kg to 100 mg/kg of patient body weight, from 0.1 mg/kg to 20 mg/kg of patient body weight, or from 1 mg/kg to 10 mg/kg of patient. body weight. Total In general, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to exogenous polypeptides. Therefore, lower doses and lower doses of human antibodies are generally possible. Moreover, the frequency and dosage of administration of the antibodies of the invention can be reduced by modification (e.g., lipidation) to enhance the uptake of these antibodies and tissue penetration (e.g., into the brain).

A method for treating an infection or a malignant disease, disorder or disorder, comprising administering an antibody, variant or derivative thereof, for use in a human body, usually in vitro, and then in vivo in an acceptable animal model Test to obtain the desired therapeutic or prophylactic activity. Suitable animal models, including transgenic animals, are well known to those of ordinary skill in the art. For example, in vitro assays demonstrating the therapeutic utility of the antigen binding polypeptides described herein include the effect of antigen binding polypeptides on cell lines or patient tissue samples. The effect of an antigen binding polypeptide on a cell line and/or tissue sample can be determined using techniques known to those of skill in the art, such as those disclosed elsewhere herein. In vitro assays that can be used to determine whether a specific antigen-binding polypeptide is required for use in accordance with the present invention include in vitro cell culture assays (where patient tissue samples are grown in culture and exposed or otherwise administered to the antibody) and observed The effect of antibodies on this tissue sample.

In a further embodiment, the compositions of the invention are administered in combination with an anti-neoplastic, anti-viral, antibacterial or antibiotic or anti-fungal formulation. Any of these formulations known in the art can be administered in the compositions of the present invention.

In another embodiment, the compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, actinomycin), antiestrogens (eg, tamoxifen) ), antimetabolites (eg, fluorouracil, 5-FU, methotrexate, fluorouridine, interferon alpha-2b, glutamate, pucamycin, guanidine, and 6-mercaptoguanine), cytotoxic agents (eg, Carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, benzamidine, mitomycin, busulfan, cisplatin and vincristine Sulfate), hormones (eg medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, metotestosterone, diethylstilbestrol, ketene estradiol and testosterone) ), nitrogen mustard derivatives (phenylalanine mustard, chlorambucil, dichloromethyldiethylamine (nitrogen mustard), thiotepa), steroids and products (betamethasone sodium phosphate) and others (eg, dacarbazine, asparaginase, mitoxantrone, vincristine sulfide, vinblastine sulfide, and etoposide).

In additional embodiments, the compositions of the invention are administered in combination with a cytokine. Cytokines that can be administered with the compositions of the invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L and TNF-α.

In additional embodiments, the compositions of the invention are administered in combination with other therapeutic or prophylactic therapies, such as radiation therapy.

VI. Composition

The invention also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more specifically For people. Further, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of excipient.

The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the drug is used. Such pharmaceutical carriers can be sterile liquids such as water and oils including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous dextrose and glycerol solutions can also be employed as carriers for the liquid phase, especially for injectable solutions. Suitable pharmaceutical excipients include: starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, c Triol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents such as, for example, acetate, citrate or phosphate. It is also contemplated to add an antibacterial formulation (such as benzyl alcohol or methyl benzoate); an antioxidant (such as ascorbic acid or sodium bisulfite); a chelating agent (ethylenediaminetetraacetic acid) and a formulation for isotonicity adjustment (such as chlorine) Sodium or dextrose). These compositions may take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described by Re. E. W. Martin in Remington's Pharmaceutical Sciences (which is incorporated herein by reference). Such compositions will contain a therapeutically effective amount of the antigen binding polypeptide (preferably in a purified form) with a suitable amount of carrier to provide a suitable mode of administration for the patient. This formulation should be suitable for the mode of administration. Such parental preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. The composition may also include a solubilizing agent and a local anesthetic (e.g., lidocaine to reduce pain at the injection site), if necessary. The components are usually provided, either alone or in combination, in unit dosage form, for example, as a lyophilized powder or a water-free concentrate in a closed container, such as an ampoule or sachette indicating the amount of active agent. When the composition is administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients can be mixed prior to administration.

The compositions of the invention may be formulated in a neutral or salt form. The pharmaceutically acceptable salts include salts formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed from cations, For example, those derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, ethylhydroxyethylamine, histidine, procaine and the like.

Experimental example

Example 1. Bispecific antibody induces T-cell mediated cytotoxicity

This example provides a novel bispecific antibody format by ligating a single domain anti-CEA VHH targeting a CEA positive tumor cell to an anti-CD3 Fab that binds to T cells. Such antibodies (S-Fab) can recruit T cells to CEA-positive tumor cells. This S-Fab antibody effectively kills CEA-positive tumor cells in vitro by involving T cells. In a xenograft model, this S-Fab antibody exhibited potent tumor suppressive effects, suggesting that such strategies are used in many immunotherapies. Further, the S-Fab antibody can be efficiently and stably expressed and purified from E. coli.

This example utilizes a combination of a single domain antibody (VHH) and a Fab fragment to produce a heterodimeric antibody. In this format, an anti-CEA VHH is ligated to the C-terminus of a portion of the heavy chain of the anti-CD3 antibody (VH-CH1), and the resulting VH-CH1-VHH chain is linked to the anti-CD3 light chain (VL- CL) paired to form an S-Fab bispecific antibody. CEA was chosen as the target because it is usually overexpressed in cancer (colon cancer, pancreatic cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, uterine cancer, ovarian cancer, endometrial cancer).

This S-Fab antibody was expressed and purified from E. coli. In vitro experiments confirmed that the tumor cell killing function of S-Fab is specific to CEA-positive tumor cells and is T cell dependent. In vivo, this S-Fab antibody inhibits tumor progression. Thus, such antibody formats present a novel form of bispecific antibodies as immunotherapeutic agents.

Method and material

Fab design and protein purification

The S-Fab protein HCBF5/6 and the control Fab HCBF4/5 as shown in Figures 2a-b were constructed. The anti-CD3 (humanized UCHT1) gene was synthesized based on the published sequence (see Table 5). The camel anti-CEA VHH sequence was cloned into the C-terminus of the CH1 domain. A signal sequence was added to the N-terminus of each strand for periplasmic expression.

Plasmids of recombinant antibodies were co-transformed into BL21 (DE3) sensing cells and co-expressed in LB medium with appropriate antibiotic selection. For protein expression, 1 L culture was grown at 16 °C for 20 h to OD600 0.8-1.0 (0.1 mM IPTG induction). Bacterial cells were pelleted by centrifugation at 4400 rpm for 30 min at 4 °C. The periplasmic extract was carried in a frozen sucrose solution (20 mM Tris-HCl pH 8.0; 25% (w/v) sucrose; 1 mM EDTA; 1 mM PMSF) at a 1:4 (m/v) resuspended cell mass. This suspension was centrifuged at 8,500 g for 20 min and the supernatant was a sucrose component. The cell pellet was resuspended in a frozen periplasmic solution (5 mM MgCl2) and the suspension was centrifuged at 8,500 g for 20 min. The supernatant was a periplasmic component.

This protein was purified from the mixed components of the sucrose component and the periplasmic component by two steps of C-terminal His8-labeled immobilized metal affinity chromatography.

Gel filtration was performed using GE Superdex 200 Increase 10/300 GL as described by the manufacturer. Gel-filtered protein markers were purchased from Sigma (MWGF1000).

Cell lines and animals

All cell lines (including CEA-positive human rectal cancer cell lines HT29, DLD-1 and LS174T, CEA-negative human rectal cancer cell line SKOV3, and Jurkat T cell line) were purchased from the Chinese Academy of Sciences' Typical Culture Collection (China, Shanghai) . HT29 and SKOV3 in DMEM medium containing 10% fetal bovine serum (Gibco, Life Technologies, China) and 1% penicillin/streptomycin (Hyclone) in a 5% CO ₂ , 37 ° C humidified incubator (Gibco) , Life Technologies, China), LS174T and Jurkat RPMI-1640 medium containing 10% fetal bovine serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (Hyclone) (Gibco, Life Technologies, China) ) cultured. Nod/SCID mice were purchased from the Experimental Animal Center of Sun Yat-sen University. Human blood collection, animal care and animal experiments were approved by Sun Yat-sen University.

Isolation of T cells from PBMC

Human PBMC were prepared from healthy donors using Ficoll density centrifugation. EasySep ^TM with the human CD3 positive selection kit (STEMCELL Technologies, Inc., Vancouver, Canada) was purified T cells. Isolated T cells were cultured in complete RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin and placed in a 37 ° C, 5% CO ₂ humidified incubator.

Surface Plasmon Resonance (SPR) Test of CEA and S-Fab Interaction

The carcinoembryonic antigen CEA full-length protein (ab742, Abcam) was used for the immobilization chip (GLC chip, #33D211O1, Bio-Rad). After immobilization, interaction analysis of different samples was carried out at 25 ° C with a flow rate of 5 μl/min of PBST (10 mM Na ₃ PO ₄ , 150 mM NaCl, 0.01% Tween, pH=7.4) as a running buffer. 36 (Bio-rad) to collect data ProteOn ^TM XPR, using Protein On evaluation software (Bio-rad) to analyze the data to apply the appropriate binding model. Kinetic constants were determined from the fit of the bound and dissociated phases. KD is derived from the ratio of these constants. A positive control (CEA/CD66e (CB30) Mouse mAb (#2383, Cell Signaling) was used to optimize this process.

Flow cytometry

To examine the binding of S-Fab to CD3 cell surface markers, 1 x 10 ⁶ Jurkat cells/samples were collected by centrifugation (1000 rpm, 5 min) and then washed with 1 ml ice cold PBS + 0.1% BSA. The cell pellet was resuspended in 100 ul of ice-cold PBS + 0.1% BSA. Anti-CD3 antibodies (R&D systems, MAB100), HCBF4/5 and HCBF5/6 (S-Fab) were added to a final concentration of 50 ug/ml. The cells were incubated with primary antibodies for one hour on ice. The cells were washed twice with ice cold PBS + 0.1% BSA. Then, sheep-anti-mouse Alexa 488 or anti-Flag-FITC was added to a final concentration of 10 ug/ml. The cells were incubated for an additional hour on ice. After washing the cells twice, flow cytometry was performed.

Cytotoxicity test

SKOV3, HT29 and LS174T were used as target cells. Human PBMC or isolated, unpre-stimulated T cells are used as effector cells. 100 ul of target cells (5000 cells) were transferred to each well of a 96-well plate (in quadruplicate). After 6 hours of incubation, an equal volume of CD3 ⁺ T cells was added to each well at a ratio of E:T (10:1 or 5:1). The indicated concentrations of antibody from 0.01 ng/ml to 10 ug/ml are then added. After 48 hours of incubation, the cells were washed twice with PBS. 10 ul of CCK8 Kit was then added to each well. Measurements were made using the TECAN Infinite F50 via the "Magellan" software. The target cell survival rate (%) was calculated according to this formula: [(survival target cell (sample)-medium) / (viable target cell (control)-medium)] × 100.

In a competitive assay, LS174T was used as a target cell. The ratio of effector cells to target cells is 10:1. HCBF4/5 and HCBF5/6 were added at a ratio of 1:1, 1:10 and 1:100. Then, the cytotoxicity test was carried out as described above.

In vivo test

Non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice were housed in the Experimental Animal Center of Sun Yat-sen University under sterile and standardized environmental conditions (20-26 ° C room temperature, 40%-70% relative humidity, and 12 hours day and night) Rhythm) Mice receive autoclaved food, litter and sterile drinking water.

LS174T human colon cancer cells harvested from cell culture medium were washed once with PBS, resuspended in PBS, and then mixed with PBMC freshly isolated from healthy donors. Cell suspension was injected subcutaneously on the right, a total volume of 0.4mL / mouse mixed with 1x10 ⁶ LS174T cells and 5x10 ⁶ human PBMC. One hour after LS174T/T cell transplantation, antibody or vehicle control (PBS) was administered intraperitoneally (ip). These animals were then treated daily for the next 7 days. Tumors are generally formed approximately 8 to 10 days after transplantation. The tumor volume was measured in two perpendicular directions using a vernier caliper, and the volume was calculated using this formula (width ² x length)/2. All results are expressed as the arithmetic mean of each group.

result

Construction and purification of S-Fab bispecific antibody

The bispecific antibody S-Fab was designed by ligation of an anti-CEA single domain antibody with a conventional anti-CD3 Fab (Fig. 2a, 2b). This monovalent anti-CD3 Fab is expected to recognize T cells and recruit T cells. At the C-terminus of the VH-CH chain, the anti-CEA VHH is fused to the Fab. The expression level of S-Fab was comparable to that of the control anti-CD3 Fab. To facilitate expression and purification of S-Fab, the two strands of S-Fab were cloned into two different plasmids; periplasmic expression was used to purify this complex (Fig. 2c, 2d).

Similar to Fab, this S-Fab was purified as a heterodimer. Immunoblotting (Fig. 2c, 2d) revealed that the half antibody did fold into a complete Fab. After a second Ni-NTA affinity purification, the purity of this protein exceeded 90% (Fig. 2e). Gel filtration was then used to analyze this purified protein. The light and heavy chains are combined into a complete Fab antibody (Fig. 2e) with molecular weights (~65 KD and ~50 KD) as expected.

S-Fab recognizes antigens CD3 and CEA

To test whether this purified antibody recognizes these antigens, flow cytometry analysis was performed to characterize the binding of the CD3 antigen to S-Fab HCBF5/6. For the control antibody OKT3, positive expression on D3 positive Jurkat cells was observed (Fig. 3a). S-Fab HCBF5/6 also had positive expression on Jurkat cells (Fig. 3a), indicating that HCBF5/6 can bind to Jurkat cells. This low intensity may be due to the unit price of the Fab.

To confirm the binding of S-Fab to CEA, this example uses SPR to measure the binding of S-Fab to CEA. No binding to CEA was detected for the control FabHCBF4/5 (Fig. 3a). Strong binding of S-Fab HCBF5/6 to CEA was observed at kd ~ 12.5 nM (Fig. 3b).

HCBF5/6 S-Fab kills tumor cells that specifically express CEA

To assess whether S-Fab can target tumor cells to achieve T cell-mediated cell killing, cytotoxicity assays were performed. At the ratio of E:T 10:1 (Fig. 4a) or 5:1 (Fig. 4b), no cell line negative for CEA was observed for either S-Fab (HCBF5/6) or control CD3 Fab (HCBF4/5). Cytotoxicity of (SKOV3). For the CEA positive cell line (LS174), the control anti-CD3 Fab (HCBF4/5) had no cell killing effect; however, S-Fab (HCB5/6) induced cell killing (Fig. 4b). For the CEA positive cell line (HT29), the control CD3 Fab (HCBF4/5) itself kills tumor cells, however, S-Fab (HCBF5/6) induces higher cell killing activity than HCBF4/5 (Figures 4a and 4b). ).

Immunoblot analysis indicated that LS174T cells had higher CEA expression than HT29 cells (Fig. 4c). In a dose-response experiment, S-Fab HCBF5/6 has a lower IC50 on LS174T cells compared to HT29 cells (Fig. 4d), further confirming that CEA expression is mediated by S-Fab HCBF5/6 Cell killing is important.

T cell binding mediates the cell killing activity of S-Fab

To further confirm that T cells are essential for S-Fab HCBF5/6 induced cell killing, a competitive assay was performed. CD3+ T cells and LS174T were used at a ratio of E:T=10:1. When 1 nM HCBF5/6 was used for tumor cells and T cells, effective cell killing was induced (Fig. 5). However, when an increased amount of Fab HCBF4/5 was applied, the cell killing effect was reduced. Since FabHCBF4/5 alone has no effect on cell killing, this data indicates that S-Fab HCBF5/6 needs to bind to T cells to direct cell killing. When the binding of T cells was competitively sequestered by the control antibody HCB4/5, a lower cell killing effect was induced.

S-Fab inhibits tumor growth in vivo

This example tested whether S-Fab HCBF5/6 inhibited tumor growth in vivo. LS174T cells were mixed with human PBMC and then transplanted subcutaneously in NOD-SCID mice. Then, these mice were treated with 20 ug of S-Fab HCBF5/6, or PBS as a control group or control Fab HCBF4/5 (n=5). No tumor growth inhibition was observed for tumors treated with PBMC only or with PBMC containing control Fab HCBF4/5. Significant tumor growth inhibition was observed in mice treated with PBMC and HCBF5/6 (Figure 5). Of the mice treated with PBMC and HCBF5/6, only two-fifths of the mice developed tumors. No tumor growth was observed in the other three groups of mice. These data confirm that S-Fab HCB5/6 specifically inhibits tumor growth in transplanted tumor mice.

This example provides a novel form (S-Fab) of the bispecific antibody. Bispecific antibodies have received great interest as a powerful immunotherapeutic approach. Various forms of their antitumor activity have been proposed and studied. Among them, BITE (two single-chain Fv-fused antibodies) exhibits broad prospects because BITE induces strong anti-tumor activity by directly targeting T cells. Blinatumomab (BITE antibody targeting CD19 and CD3) has been approved for clinical use in B cell leukemia. However, single-chain Fv fusion proteins (used by BITE and other similar techniques) have a tendency to aggregate and present in bacterial cells.

A novel bispecific antibody (S-Fab) is based on a conventional Fab (Fig. 2b) in which a single domain antibody is ligated to the C-terminus of VH-CH1. Unlike BITE or other single-chain Fvs, the anti-CD3 portion of S-Fab in this study is a natural form of Fab and therefore more stable. A number of different anti-CD3 antibodies were tested, and since the anti-CD3 Fab derived from the UTCH1 clone exhibited better expression and solubility at E. coli. it was used in this study. Since this anti-CD3 Fab can be expressed and produced in E. coli, it greatly reduces the complexity of ScFv expression, which typically requires an expensive mammalian cell expression system.

S-Fab provides a new form of bispecific antibody with several advantages. In the present invention, natural human anti-CD3 Fab is used, and T cells can participate in killing tumor cells.

Example 2. Bispecific antibody induces NK-cell mediated cytotoxicity

This example provides a novel bispecific antibody form Her2-S-Fab by ligation of a single domain anti-CD16 VHH to trastuzumab (Trastuzumab,

The C-terminus of the Fab is constructed. Her2-S-Fab can be expressed and purified by bacterial cells. In in vitro cell experiments, Her2-S-Fab specifically kills Her2 overexpressing cancer cells by recruiting NK cells. Enhanced tumor cell killing effects were observed compared to trastuzumab. In vivo studies have shown that Her-2-S-Fab inhibits tumor progression.

Method and material

Fab design and protein purification

The constructs of Her2-S-Fab and control Her2-Fab are shown in Figure 6a. The standard DNA cloning technique was used to first chemically synthesize trastuzumab anti-Her2 VL-CL and VH-CH1 (see Table 5) and clone Into the pET21a vector and the pET26b vector. Subsequently, VHH-CD16 was cloned into the C-terminus of trastuzumab against Her2 VH-CH1. The signal sequence pelB was added at the N-terminus to achieve periplasmic expression. Heterodimerization of VL-CL/VH-CH1-VHH (CD16) forms Her2-S-Fab, while heterodimerization of VL-CL/VH-CH1 forms Her2-Fab.

The purification of periplasmic proteins in E. coli is briefly described below. Two plasmids encoding different polypeptides were co-transformed into BL21 (DE3) cells with appropriate antibiotic markers. Induction of protein expression and extraction of cell periplasm. Anti-Her2 Fab or Her2-S-Fab was purified from a mixture of sucrose and periplasmic components by Ni-NTA agarose affinity chromatography and anti-IgG CH1 affinity purification (Fig. 6b). Gel filtration was performed using a GE Hiload 16/600 Superdex 200 pg according to the manufacturer (GE) instructions. Gel filtered protein markers were purchased from Sigma (MWGF 1000).

Cell lines and animals

Her2-positive cancer cell lines SKBR3 (human breast cancer cells), BT474 (human breast cancer cells), SKOV3 (human ovarian cancer cells), MCF7 (human breast cancer cells), and Her-2 negative cancer cell lines MDAMB435, MDAMB468, Chinese hamsters Ovarian cells (CHO) were purchased from the Central Culture Center of the Chinese Academy of Sciences (Shanghai). SKBR3, SKOV3 and MDAMB435 cells were cultured in DMEM medium (Gibco, Life Technologies, China) supplemented with 10% HI fetal bovine serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone); BT474 And CHO cells were cultured in RPMI-1640 medium (Gibco, Life Technologies, China) supplemented with 10% HI fetal bovine serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone); MDAMB468 cells The cells were cultured in L15 (Gibco, Life Technologies, China) and placed in a CO2 incubator at a temperature of 37 ° C and a humidity of 5%.

Nod/SCID mice were purchased from the Experimental Animal Center of Sun Yat-sen University. Human blood collection, animal care and animal experiments were approved by Sun Yat-sen University.

Blood cell separation

Human peripheral blood mononuclear cells (PBMC) were prepared from healthy donors using Ficoll density centrifugation. EasySep ^TM with purified human NK cells NK cell enrichment kit (STEMCELL Technologies, Inc., Vancouver, Canada). Isolated NK cells were cultured in complete RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin and placed in a 37 ° C, 5% CO ₂ humidified incubator.

Flow cytometry

When the tissue culture cell fusion degree reached 80-90%, 0.25% trypsin digestion was added. The cells were collected at 1 × 10 ⁵ cells/sample by centrifugation at 1500 rpm for 5 minutes, and then washed twice with 3 ml of ice-cold PBS + 0.1% BSA. The cell pellet was resuspended in 500 ul of ice-cold PBS + 0.1% BSA. Trastuzumab, Her-2 Fab or Her2-S-Fab were added as primary antibodies to each tube; sheep-anti-human IgG (H+L) Alexa Fluor 488 (Invitrogen, cat#A11013) as secondary antibody . HER2/neu-PE (BD, ca #340552) served as a control. After washing the cells twice, flow cytometry was performed.

Cytotoxicity test

SKBR3, BT474, MCF7, SKOV3, MDAMB435, MDAMB468 and CHO cells were used as target (T) cells. Human PBMC cells or isolated NK cells (unstimulated) were used as effector (E) cells. 100 μl of target cells (5000 cells) were transferred to each well of a 96-well plate (in triplicate). After 12 hours of incubation, an equal volume of NK cells was added to each well at a ratio of E:T (10:1). The indicated concentrations of antibody from 0.01 ng/ml to 10 [mu]g/ml are then added. After 72 hours of incubation, cell viability was quantified using CCK8 reagent (Dojindo, CK04). The target cell survival rate (%) was calculated according to this formula: [(survival target cell (sample)-medium) / (viable target cell (control)-medium)] × 100.

In vivo tumor growth inhibition test

SKOV3 cells were harvested from the cell culture medium, washed once with PBS, resuspended in PBS, and then mixed with PBMC just isolated from healthy donors. Cell suspension was injected subcutaneously in NOD / SCID mice on the right side, the total volume of 0.2mL / mouse, 2x10 ⁶ SKOV3 mixed with 1x10 ⁷ cells and human PBMC. Two hours after SKOV3/PBMC cell transplantation, antibody or vehicle control (PBS) was administered intraperitoneally (ip). Then, these animals were treated daily for the next 6 days and the state of the mice was observed. The tumor volume was measured in two perpendicular directions using a vernier caliper, and the volume was calculated using the formula (width ² x length)/2. All results are expressed as the arithmetic mean of each group.

result

Purification of Her2-S-Fab and Her2-Fab

Purification of Her2-Fab and Her2-S-Fab was achieved by a two-step procedure using Ni-NTA-agarose first and affinity purification using anti-CH1 (Fig. 6b). Since VHH is relatively small and soluble, the addition of anti-CD16 VHH does not affect the expression level and solubility of anti-Her2 Fab. The solubility and expression level of Her2-S-Fab was comparable to that of the control Her2-Fab (0.6 mg/L). To determine if the Her2-S-Fab was correctly folded into a heterodimer, gel filtration was used to analyze the purified protein. Most of the protein forms a single peak. The light and heavy chains constitute a complete Fab antibody with a molecular weight of approximately 65 kD (Fig. 6c) and approximately 50 kD (data not shown), similar to the expected molecular weights of Her2-S-Fab and Her2-Fab, respectively, indicating most of the Her2-S -Fab is folded correctly.

Her2-S-Fab recognizes Her2 positive cells

To verify whether Her2-S-Fab can bind to Her2 positive expressing cells, flow cytometric analysis was performed using HER2-positive and HER2-negative cells. The results showed that the Her2 negative cells CHO, MDAMB435 and MDAMB 468 had very low, even no staining using the control anti-Her2 antibody; MCF7 had very low Her2 expression; whereas BT474, SKBR3 and SKOV3 cells had high Her2 expression (Fig. 7a) .

Trastuzumab can stain HER2 expressing cells (Fig. 7b). Both Her2-Fab and Her2-S-Fab bind to HER2 positive cells (Figures 7c and 7d) and show similar changes in fluorescence intensity, indicating that the control Fab and Her2-S-Fab have similar binding capabilities. Her2-Fab and Her2-S-Fab also showed the same staining pattern for trastuzumab, with lower intensity, which is consistent with the monovalent nature of Her2-Fab and Her2-S-Fab.

Her2-S-Fab induces NK cell-mediated cytotoxicity

To evaluate the cytotoxic effects of Her2-S-Fab, Her2-Fab, Her2-S-Fab, and trastuzumab were cultured with cancer cells and freshly isolated NK cells. At a concentration of 10 μg/ml, no cytotoxicity was observed in the HER2-negative cell line CHO even in the presence of NK cells (Fig. 8a). For the HER2 overexpressing cell line SKOV3, only trastuzumab reduced cell proliferation when NK cells were absent, with a survival rate of 72% (Fig. 8a). Her2-S-Fab or Her2-Fab had no cell viability. influences. However, when NK cells were present, Her2-S-Fab induced strong cytotoxicity and was more cytotoxic than trastuzumab (survival rate 10.70% vs 33.12%) (Fig. 8a). The cytotoxic effect of Her2-S-Fab is dependent on anti-CD16, since the Her2-Fab alone has no cytotoxic effect even in the presence of NK cells (Fig. 8a).

To further evaluate the effect of Her2-S-Fab on tumor cells, we determined the dose response of different antibodies on cancer cells. Her2-Fab had little or no effect on cell viability regardless of Her2 expression status (Fig. 8b-f). Trastuzumab only had a slight tumor suppressive effect on Her2 high expressing cells SKOV3 in the absence of NK cells (Fig. 8b-f). In the presence of NK cells, trastuzumab and Her2-S-Fab caused strong cytotoxic effects on SKBR3 and SKOV3 cells in a dose-dependent manner, but had no effect on the HER2-negative cell line MDAMB435 and CHO (Fig. 8b-f). A high concentration of antibodies is required for MCF7 (Her2 low expression cancer cells) to induce cytotoxicity, indicating that the activity of Her2-S-Fab is dependent on the expression of Her2. In MCF7, SKBR3 and SKOV3 cells, Her2-S-Fab showed a stronger cytotoxic effect than trastuzumab, indicating that Her2-S-Fab is more effective than trastuzumab.

Her2-S-Fab inhibits tumor growth in vivo

The adoptive transfer model was used to test whether Her2-S-Fab can inhibit tumor growth in vivo. SKOV3 cells were first mixed with human PBMC cells and subsequently transplanted subcutaneously into NOD/SCID mice. Mice were subsequently treated with PBS, Her2-Fab or Her2-S-Fab. No inhibition of tumor growth was observed in mice treated with Her2-Fab. However, for Her2-S-Fab, a strong tumor growth inhibition phenomenon was observed (Fig. 9).

Thus, Her2-S-Fab can be used to direct NK cells to Her2 overexpressing cells. Her2-S-Fab is effective in killing Her2-positive cancer cells in vivo and in vitro.

***

All of the patents and other references cited in the specification are indicative of the level of ordinary skill in the art to which the invention pertains, which are incorporated herein by reference in their entirety, including any The literature is hereby incorporated by reference in its entirety in its entirety.

Those skilled in the art will readily appreciate that the present invention can be readily adapted to obtain the objects and advantages described herein as well as those objects and advantages herein. The methods, variations, and compositions described herein are representative of the presently preferred embodiments, and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that changes may be made or used for other purposes, but are intended to be included within the scope of the invention as defined by the appended claims.

Moreover, when describing some features or aspects of the present invention in the form of a Markush group or other alternative group, those skilled in the art will appreciate that the present invention also employs any of the Markush groups or other groups. The form of a single member or a subgroup of members is described.

Moreover, unless otherwise indicated, when various numerical values are provided in some embodiments, other embodiments are described by taking any two different values as the endpoints of the range. These range values are also within the scope of the invention.

Where appropriate, the matters illustratively described herein can be implemented in the absence of any one or more of the elements, one or more limitations not specifically disclosed herein. So, for example, in each case of this article, the term Any of "including", "consisting essentially of", and "consisting of" can be replaced by the other two terms. Therefore, it is to be understood that the invention may be modified and changed by those skilled in the art, It is intended to define the scope of the invention.

Claims (16)

1. A bivalent antibody comprising (a) an antigen-binding fragment (Fab) comprising a light chain and a partial heavy chain of an antibody, and (b) a single domain antigen fused to the C-terminus of the light chain or the partial heavy chain A binding fragment (VHH), wherein the partial heavy chain does not comprise a CH2 or CH3 domain.
2. The bivalent antibody according to claim 1, wherein the VHH fragment is fused to the partial heavy chain.
3. The bivalent antibody of claim 2, wherein the bivalent antibody further comprises a second VHH fragment fused to the light chain.
4. The bivalent antibody according to any one of claims 1 to 3, wherein the Fab fragment and the VHH fragment each have specificity for tumor cells or immune cells.
5. The bivalent antibody according to claim 4, wherein the Fab fragment is specific for a tumor antigen, and the VHH fragment is specific for an immune cell.
6. The bivalent antibody according to claim 5, wherein the tumor antigen is selected from the group consisting of CEA, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX , PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.
7. The bivalent antibody according to claim 5, wherein the tumor antigen is CEA or Her2.
8. The bivalent antibody according to claim 5, wherein the VHH fragment is specific for mammalian T cells or mammalian NK cells.
9. The bivalent antibody according to claim 5, wherein the VHH fragment is specific for an antigen selected from the group consisting of CD3, CD16, CD19, CD28 and CD64.
10. The bivalent antibody according to claim 9, wherein the antigen is CD16 or CD3.
11. The bivalent antibody according to any one of claims 1 to 3 or 5 to 9, wherein the VHH fragment does not contain Val, Gly, Leu and Trp at Kabat sites 37, 44, 45, 47, respectively. Residues.
12. A host cell comprising one or more polynucleotides encoding a bivalent antibody of any of the preceding claims.
13. The host cell according to claim 12, wherein the host cell is a bacterial cell or a yeast cell.
14. The host cell according to claim 12, wherein the host cell is E. coli.
15. A method of producing a soluble antibody, the method comprising culturing the host cell of any one of claims 12-14, and harvesting an antibody expressed in the cell.
16. Use of the bivalent antibody of any one of claims 1 to 11 for the preparation of a medicament for treating cancer.

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