Bispecific t cell engagers and uses thereof

Abstract

The present disclosure provides a novel fusion protein to overcome the difficulties associated with the use of monoclonal antibodies in disease treatment and other fields of application, particularly in fields requiring ADCC, such as removal of tumor cells, virus-infected cells, or immunomodulatory cells. One preferred example of the fusion protein comprises the extracellular domain of a high affinity variant of human CD16A fused to an anti-CD3 antibody or antigen-binding fragment thereof that specifically binds to an epitope on human CD3 or a fragment thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a fusion protein, in particular to CD16A-bispecific T cell engager (BiTE) and uses thereof. BACKGROUND

[0002] Therapeutic monoclonal antibodies (mAbs) have been the fastest growing class of drugs in recent years and have been approved for various indications in the treatment of cancer, infectious diseases, and autoimmune diseases (Almagro et al., Front Immunol 8, 1751 (2017)). Most of the approved mAbs for the treatment of tumors are what is called direct targeting mAbs, such as rituximab (anti-CD20 mAb), which are designed to directly target tumor cells. This type of mAbs is usually produced in vitro and passively injected into patients, where they can combat established or residual tumors and thereby activate various Fc receptor-mediated effector pathways to kill target cells. When combined with chemotherapy, these therapeutic mAbs have achieved impressive results in hematological malignancies, with examples of anti-CD20 and anti-CD38 having clinical efficacy in follicular lymphoma (Subramanian et al., Cancer Management and Research, 9, 131-140 (2017)) and multiple myeloma (van de Donk et al., Blood, 131, 13-29 (2018)), respectively. On the other hand, approved immunomodulatory mAbs, such as anti-CTLA-4 (cytotoxic T lymphocyte antigen 4), anti-PD-1 (programmed cell death-1), and anti-PD-L1 (programmed cell death-ligand 1), are designed to block immune checkpoints to reactivate anti-tumor immune cell responses (K. Chin et al., Annals of Oncology, 28, 1658-1666 (2017)), but can also be used as direct targeting mAbs to eliminate cells (Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515-523 (2017)).

[0003] The mechanism of action of direct targeting therapeutic mAbs is derived from various natural functions of antibodies: neutralization, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (Suzuki et al., J Toxicol Pathol, 28, 133-139 (2015)). The extent of contribution of each mode of action to clinical efficacy is not yet known.

[0004] Therefore, there is a need to develop a novel method to improve the therapeutic efficacy of mAbs and to facilitate the application of mAbs in various fields. The present disclosure addresses the above and other needs. SUMMARY

[0005] The present disclosure provides a novel bispecific T cell engaging protein to overcome the difficulties associated with the current mAbs in disease treatment and other fields of application, particularly in the fields requiring ADCC, such as removal of tumor cells, virus-infected cells, or immunomodulatory cells, etc.

[0006] The present disclosure provides a fusion protein comprising

[0007] an extracellular domain of a human CD16A; and

[0008] an antibody or antigen-binding fragment thereof that can specifically bind to an epitope of human CD3 or a fragment thereof.

[0009] The present disclosure also provides a polynucleotide encoding the fusion protein as shown herein.

[0010] The present disclosure further provides a host cell comprising the polynucleotide as shown herein.

[0011] The present disclosure still further provides a pharmaceutical composition comprising a therapeutically effective amount of the fusion protein as shown herein and optionally a pharmaceutically acceptable carrier or excipient.

[0012] The present disclosure still further provides the use of the pharmaceutical composition as shown herein in the preparation of a medicament for inducing antibody-dependent cellular cytotoxicity in a subject in need thereof.

[0013] The present disclosure will be described in detail in the following paragraphs, and other features, objects and advantages of the present disclosure can be seen in the embodiments and claims. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figures 1A-1D Gene construct of haCD16A-BiTE is shown. Figure 1A Gene construct of haCD16A-CD3 bispecific T cell engager cloned in an adeno-associated virus (AAV) shuttle plasmid. H: 6x histidine tag; ITR: inverted terminal repeat of AAV; S: secretion signal; WPRE: woodchuck hepatitis B virus post-transcriptional regulatory element. Figure 1B Coding sequences of the extracellular domain of human high affinity CD16A (haCD16A) (edCD16) and a single chain antibody against human CD3 (anti-CD3 scFv) were fused in the same coding frame by gene synthesis. Figure 1C: Synthetic gene with 1341 bp of nucleotides encodes a fusion protein with 446 amino acids. Figure 1D : Schematic representation of haCD16A-BiTE.

[0015] Figures 2A-2E Binding of haCD16A-BiTE to T cells and to tumor cells coated with immunoglobulin G (IgG) antibodies is shown. Figure 2A : Analysis strategy for binding of haCD16A-BiTE to cells coated with IgG antibodies. Figure 2B : Antigen expression on tumor cells. Figure 2C : Binding of haCD16A-BiTE to tumor cells coated with IgG antibodies. Figure 2D : Analysis strategy for binding of haCD16A-BiTE to T cells. Figure 2E : Binding of haCD16A-BiTE to T cells.

[0016] Figure 3 Effect of killing CD20-expressing cell lines by treating them with anti-CD20 antibodies (rituximab, Rituxan®, MabThera®, Rixathon®, ) and haCD16A-BiTE, alone or in combination, in the presence of T cells is shown.

[0017] Figure 4 Effect of killing epidermal growth factor receptor (EGFR)-expressing cell lines by treating them with anti-EGFR antibodies (cetuximab, Erbitux®, Merck, ) and haCD16A-BiTE, alone or in combination, in the presence of T cells is shown.

[0018] Figure 5 Effect of killing human epidermal growth factor receptor 2 (HER2)-expressing cell lines by treating them with anti-HER2 antibodies (trastuzumab, Herceptin®, Genentech, ) and haCD16A-BiTE, alone or in combination, in the presence of T cells is shown.

[0019] Figure 6 Effect of plasma on the killing of CD20-expressing cell lines treated with rituximab and haCD16A-BiTE is shown.

[0020] Figure 7A and Figure 7B Comparative study of IgG antibody-mediated cell killing effect between CD16-γ9δ2 T cells and CD16+γ9δ2 T cells treated with a pulse of haCD16A-BiTE is shown.

[0021] Figure 8The effect of removing malignant B cells in T cell expansion cultures by treating the T cell expansion cultures with an anti-CD20 antibody (rituximab) and haCD16A-BiTE is shown.

[0022] Figure 9 The effect of killing EBV-infected cell lines by treating the cell lines with an anti-latent membrane protein 1 (LMP1) antibody and haCD16A-BiTE, alone or in combination, in the presence of T cells is shown.

[0023] Figure 10 The effect of killing PD-L1 -expressing cell lines by treating the cell lines with an anti-PD-L1 antibody and haCD16A-BiTE, alone or in combination, in the presence of T cells is shown.

[0024] Figure 11 The in vivo effect of combination therapy with haCD16A-BiTE and rituximab and rituximab monotherapy on reducing cancer cell growth in the presence of T cells is shown.

[0025] Figure 12 The in vivo production of haCD16A-BiTE after AAV-mediated gene transfer of haCD16A-BiTE is shown. DETAILED DESCRIPTION

[0026] The present disclosure provides a fusion protein comprising:

[0027] a Fc gamma receptor or a ligand-binding fragment thereof; and

[0028] an antibody or an antigen-binding fragment thereof that can specifically bind to an antigenic determinant of a T cell surface antigen or a fragment thereof, wherein the surface antigen can trigger antibody-dependent cell-mediated cytotoxicity and / or activation of T cells.

[0029] In particular, the fusion protein according to the present disclosure can be referred to as a bispecific T cell engager protein, which possesses bispecific affinity to two antigens / ligands, and can act as a bridge between target cells and T cells.

[0030] Preferably, the fusion protein can activate an ADCC immune response, whereby the ADCC immune response, T cells can recognize and kill target cells coated with antibodies, which express tumor or antigens derived from pathogens on their surface.

[0031] monotherapy using direct targeting therapeutic mAbs to treat cancer often produces only limited therapeutic effect without combination with other treatment modalities such as chemotherapy (Hiddemann et al., Blood, 106, 3725-32 (2005); Sehn et al., J Clin Oncol, 33, 3467-3474 (2015)). Although not wishing to be bound by theory, the present disclosure as disclosed herein is based on the Applicant's idea that amplifying ADCC is a promising approach to increase the clinical benefit of therapeutic antibodies. The major immune effector cells that mediate ADCC are natural killer (NK) cells (Wang et al., Front Immunol, 6, 368 (2015)). NK cells express Fc gamma receptors (FcγR), mainly CD16A (FcγRIIIA), which recognize and bind to the Fc portion of IgG antibodies. Upon binding of Fcγ receptors to the Fc region of IgG bound to the surface of target cells, natural killer cells release cytotoxic factors that cause target cell death (Wang et al., Front Immunol, 6, 368 (2015)). Thus, ADCC involves three components: immune effector cells, antibodies, and target cells surrounded by antibodies. Binding of FcγR expressed on the surface of NK cells to the Fc region of IgG molecules can trigger ADCC. Therefore, given that genetic variations of FcγR are known to cause different binding affinities and thus differences in the magnitude of ADCC they mediate, the affinity / number of FcγR and the quality / number of NK cells can cause differences in the magnitude of ADCC they mediate.

[0032] Examples of the Fcγ receptor include, but are not limited to, CD16A, CD16B, CD32A, CD32B, CD64A, CD64B, and CD64C.

[0033] Examples of the antibody or antigen-binding fragment thereof that can specifically bind to an epitope of a T cell surface antigen or a fragment thereof include, but are not limited to, an anti-CD3 antibody, an anti-4-1BB antibody, an anti-CD28 antibody, or an anti-OX40 antibody.

[0034] Preferably, the present disclosure provides a fusion protein comprising:

[0035] an extracellular domain of a human CD16A; and

[0036] an antibody or antigen-binding fragment thereof that can specifically bind to an epitope of human CD3 or a fragment thereof.

[0037] According to the present disclosure, the fusion protein comprises an extracellular domain of CD16A. Preferably, the CD16A is human CD16A. CD16A, also known as FcγRIIIA, is a transmembrane glycoprotein, and there are two allelic variants of CD16A with phenylalanine (F) or valine (V) residues at position 158. The CD16A-158V variant has higher affinity for IgG, but CD16A-158F is the predominant allele in the human population. Clinical analyses revealed that the therapeutic efficacy of tumor-targeting therapeutic mAbs positively correlates with their affinity for binding to CD16A. In comparison of clinical outcomes for using clinically approved anti-tumor therapeutic antibodies, such as rituximab, trastuzumab, and cetuximab, patients with homozygous CD16A valine variant (CD16A-V / V) have improved clinical outcomes after treatment with anti-tumor therapeutic antibodies than patients with lower affinity CD16A allotype heterozygous (CD16A-V / F) or homozygous (CD16A-F / F) (Cartron et al., Blood, 99, 754-758 (2002); Kim et al., Blood, 108, 2720-2725 (2006); Zhang et al., J Clin Oncol, 25, 3712-3718 (2007); Musolino et al., J Clin Oncol, 26, 1789-1796 (2008); Veeramani et al., Blood, 118, 3347-3349 (2011); Mellor et al., J Hematol Oncol, 6, 1 (2013)). However, only 10-20% of the total population have high-affinity CD16A variants. In one preferred embodiment of the present disclosure, the CD16A is a high-affinity CD16A variant.

[0038] In one preferred embodiment of the present disclosure, the extracellular domain of CD16A has an amino acid sequence as set forth in SEQ ID NO: 2 or a substantially similar sequence thereof.

[0039] The term "substantial similarity" or "substantially similar" when applied to a polypeptide means that, when a protein sequence is aligned with another (reference) protein sequence, using a program such as GAP or BESTFIT with default gap weights, to achieve optimal alignment, the sequence has at least 90%, 95%, more preferably at least 96%, 97%, 98%, or 99% sequence identity with the reference protein over the entire length of the reference protein. Preferably, the difference at the positions of non-identical amino acid residues is a conservative amino acid substitution. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain (substituting group). Generally, a conservative amino acid substitution does not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by an equal number of conservative substitutions, the percentage of sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of the substitutions. Methods for making this adjustment are well known to those of ordinary skill in the art. Examples of groups of amino acids that have chemical properties similar include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; and (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitutions are: valine - leucine - isoleucine, phenylalanine - tyrosine, lysine - arginine, alanine - valine, glutamic acid - aspartic acid, and asparagine - glutamine. Alternatively, conservative substitutions are defined in accordance with the PAM250 log-likelihood matrix of Gonnet et al. (1992) Science 256: 1443-1445, as any change that has a non-negative value.

[0040] Sequence similarity, also known as sequence identity, of a polypeptide is typically measured using sequence analysis software. Protein analysis software matches similar sequences using their specified degrees of similarity including conservative amino acid substitutions, gaps, and other modifications. For instance, the GCG software contains programs such as Gap and Bestfit that can be used to determine sequence homology or sequence identity between closely related polypeptides, for example, homologous polypeptides from different species of the same organism group or between a wild type protein and a protein formed from a mutation thereof, using default or recommended parameters. Polypeptide sequences also can be compared using the GCG Version 6.1 program FASTA, (e.g., FASTA2 and FASTA3) using either default parameters or recommended parameters, which provides alignments and percent sequence identity of the regions of the best overlap between the queried and searched sequences (Pearson (2000) supra). Another preferred algorithm is a BLAST program, particularly when comparing a sequence of the application to a database containing a large number of sequences from different organisms, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0041] According to the present disclosure, the fusion antibody comprises an antibody or antigen binding fragment thereof that can specifically bind to an epitope of human CD3 or a fragment thereof.

[0042] In an immune response, CD3 is a surface antigen that associates with the T cell receptor (TCR) to form a complex involved in antigen recognition and signal transduction. CD3 T cell co-receptors help to activate both cytotoxic T cells (CD8+ T cells) as well as T helper cells (CD4+ T cells). With the fusion protein according to the present disclosure, all cells expressing CD3 in the body, including alpha-beta T cells, gamma-delta T cells, and natural killer T cells, can be recruited via the anti-CD3 portion of the fusion protein that binds to the CD3 molecule on the T cell, thereby becoming cells that carry high affinity CD16A and are able to perform ADCC.

[0043] Many techniques are known to those skilled in the art to which this invention pertains for determining whether an antibody specifically binds to one or more amino acids within a peptide or protein. Exemplary techniques include, for example, conventional cross-linking-blocking analysis, alanine scanning mutagenesis analysis, peptide blotting analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Additionally, methods such as excision of antigenic determinants, antigenic determinant extraction, and chemical modification can also be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a peptide that specifically bind to an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves labeling the protein of interest with deuterium, followed by binding an antibody to the deuterium-labeled protein. The protein / antibody complex was then transferred to water, allowing hydrogen-deuterium exchange to occur at all amino acid sites except those protected by the antibody (which remained deuterated). After antibody dissociation, protease cleavage and mass spectrometry analysis of the target protein were performed to reveal that the deuterated amino acid sites were the specific amino acids that interacted with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0044] The antibodies disclosed herein may be full-length or may contain only one antigen-binding moiety and may be modified to affect their function.

[0045] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (such as CD3). The term "antibody" also includes immunoglobulin molecules composed of four polypeptide chains—two heavy (H) chains and two light (L) chains—interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (HCVR or V) as referred to herein. H ) and a heavy-chain constant region. The heavy-chain constant region contains three domains, C H1 C H2 and C H3Each light chain contains a light chain variable region (LCVR or V for short). L ) and a light chain constant region. The light chain constant region contains a domain (C L1 V H and V L The region can be further divided into highly variable regions called complementarity-determining regions (CDRs), and conserved regions scattered within them called framework regions (FRs). Each V H and V L Each consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In other embodiments of the invention, the FRs of the anti-CD3 antibody (or its antigen-binding portion) may be identical to human germline sequences or may be naturally or artificially modified. A common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0046] As used herein, the term "antibody" also includes the antigen-binding fragment of the entire antibody molecule. The terms "antigen-binding portion" and "antigen-binding fragment" of an antibody, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically modified polypeptide or glycoprotein that has the ability to specifically bind to an antigen to form a complex. The antigen-binding fragment of an antibody can be generated from the whole antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable or constant regions of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA databases (including, for example, phage-antibody databases), or can be synthesized. The DNA can be sequenced and manipulated using chemical methods or molecular biological techniques, such as arranging one or more variable and / or constant regions into a suitable conformation, or introducing codons, adding cysteine, modifying, adding, or deleting amino acids, etc.

[0047] Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., a complementarity determining region (CDR) such as CDR3 or a constrained FR3-CDR3-FR4 peptide). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen binding fragment" as used herein.

[0048] An antigen binding fragment of an antibody will typically comprise at least one variable region. The variable region can be of any size or have any amino acid composition and will generally comprise at least one CDR which is held in close proximity or located within a framework held in close proximity to one or more framework sequences. In some embodiments, a V L region is associated with a V H region. In some embodiments, a V H region and a V L region are associated with each other. H In some embodiments, a V H region and a V H region are associated with each other. L In some embodiments, a V L region and a V L region are associated with each other. H In some embodiments, a V L region is associated with a V H region.

[0049] In certain embodiments, an antigen binding fragment of an antibody will comprise at least one variable region covalently linked to at least one constant region. Non-limiting exemplary configurations of variable and constant regions that can be found within an antigen binding fragment of an antibody of the present application include: (i) V H1 -C H ; (ii) V H2 -C H ; (iii) V H3 -C H ; (iv) V H1 -C H2 ; (v) V H -C H1 -C H2 -C H3 ; (vi) VH -C H2 -C H3 ; (vii) V H -C L ; (viii) V L -C H1 ; (ix) V L -C H2 ; (x) V L -C H3 ; (xi) VL-C H1 -C H2 ; (xii) V L -C H1 -C H2 -C H3 ; (xiii) V L -C H2 -C H3 ; and (xiv) V L -C L In any configuration of variable and constant regions, including any of the exemplary configurations described above, the variable and constant regions can be directly connected to one another or can be connected by a full or partial hinge or linker region. A hinge region can contain at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that form a flexible or semi-flexible connection between adjacent variable and / or constant regions in a single polypeptide molecule. In addition, antigen-binding fragments of the antibodies of the present disclosure can comprise homo- or hetero-dimers (or other multimers) of any of the configurations of variable and constant regions described above, connected to one another by non-covalent linkages and / or to one or more single V H regions or V L regions (e.g., by disulfide bonds).

[0050] In a preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof is an anti-CD3 single chain variable fragment (scFv).

[0051] In another preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO: 4 or a substantially similar sequence thereof; preferably a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or a substantially similar sequence thereof.

[0052] The antibodies disclosed herein can comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the variable regions of the heavy and light chains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue of the germline sequence from which the antibody was derived, or to the corresponding residue of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline sequence residue (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can readily produce many antibodies and antigen-binding fragments thereof that comprise one or more single germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the V H and / or V L regions are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues in CDR1, CDR2 or CDR3. In other certain embodiments, one or more framework and / or CDR residues are mutated back to the corresponding residue of a different germline sequence, i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived. Moreover, antibodies of the present disclosure can contain any combination of two or more mutations in the framework and / or CDR regions, e.g., where certain residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that are different from the original germline sequence are either left unchanged or are mutated to the corresponding residue of a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced biological antagonists or agonists as the case can be, decreased immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are included within the scope of the present disclosure.

[0053] The disclosure also includes anti-CD3 antibodies comprising variants of any of the V H , V L and / or CDR amino acid sequences disclosed herein that contain one or more conservative amino acid substitutions. For example, the present disclosure includes anti-CD3 antibodies comprising V H , V Land / or CDR amino acid sequences disclosed herein. In another embodiment, the anti-CD3 antibody comprises one or more of the following CDR amino acid sequences: a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and / or a CDR3 amino acid sequence of SEQ ID NO: 3. H , V L and / or CDR amino acid sequences contain, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions.

[0054] In a preferred embodiment of the disclosure, the extracellular domain of CD16A is directly linked to the antibody or antigen-binding fragment thereof. In another embodiment of the disclosure, there is a linker between the extracellular domain of CD16A and the antibody or antigen-binding fragment thereof.

[0055] In a preferred embodiment of the disclosure, the fusion protein further comprises a secretion signal peptide. As used herein, the signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) refers to a short peptide located at the N-terminus of a protein that functions to direct the protein to the secretory pathway. In an embodiment of the disclosure, the secretion signal peptide has an amino acid sequence as set forth in SEQ ID NO: 6 or a substantially similar sequence thereof.

[0056] In a preferred embodiment of the disclosure, the fusion protein further comprises a protein purification tag.

[0057] In an embodiment of the disclosure, the fusion protein has an amino acid sequence as set forth in SEQ ID NO: 8 or a substantially similar sequence thereof.

[0058] In another preferred embodiment of the disclosure, a novel bispecific T cell engaging protein (haCD16A-BiTE; haCD16A-CD3) is composed of the extracellular domain of a high affinity CD16A variant and a single chain anti-CD3 antibody. Figure 1AThe haCD16A-BiTE has several advantages for such applications: (1) overcoming the limited number of CD16A-expressing natural killer cells in vivo, because with the haCD16A-BiTE, all CD3-expressing cells in vivo, including a-beta T cells, gamma-delta T cells, and natural killer T cells, can be recruited via the anti-CD3 moiety of the haCD16A-BiTE that binds to CD3 molecules on T cells to become cells carrying high-affinity CD16A and capable of performing ADCC; (2) by administering the haCD16A-BiTE to any individual, and thus endowing a large number of CD3+ T cells in each individual with high-affinity CD16A via the anti-CD3 moiety of the haCD16A-BiTE that binds to CD3 molecules on T cells, the problem that only 10-20% of the total population have high-affinity CD16A variants that express CD16A capable of enhancing the therapeutic efficacy of mAbs is solved; (3) in the design of this haCD16A-BiTE, the downregulation of CD16A number that naturally occurs on activated NK cells is avoided by using the extracellular domain of high-affinity CD16A; (4) by endowing T cells with ADCC activity with this haCD16A-BiTE, it becomes possible to use ADCC to eliminate undesirable cells in the preparation of T cell expansion cultures for immunotherapy; (5) a potential method is provided to generate a virus vaccine with the ability to induce ADCC by antibody through the combined use of a vaccine and this haCD16A-BiTE, and to improve the therapeutic efficacy of antibody therapy for viral infectious diseases; (6) transforming T cells that are no longer inhibited in patients receiving immune checkpoint inhibitors into T cells with ADCC activity, thereby increasing the therapeutic potential of similar avelumab anti-PD-L1 mAbs.

[0059] The present disclosure also provides a polynucleotide encoding the fusion protein as shown herein.

[0060] Preferably, the polynucleotide comprises a fragment encoding the extracellular domain of high-affinity CD16A, and has a nucleic acid sequence as shown in SEQ ID NO: 1 or a substantially identical sequence thereof.

[0061] Preferably, the polynucleotide comprises a fragment encoding the antibody or antigen binding fragment thereof, and has a nucleic acid sequence as shown in SEQ ID NO: 3 or a substantially identical sequence thereof.

[0062] Preferably, the polynucleotide further comprises a fragment encoding the secretion signal peptide, and has a nucleic acid sequence as shown in SEQ ID NO: 5 or a substantially identical sequence thereof.

[0063] Preferably, the polynucleotide further comprises a fragment encoding the protein purification tag.

[0064] Most preferably, the polynucleotide has a nucleic acid sequence as set forth in SEQ ID NO: 7 or a substantially identical sequence thereof.

[0065] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof, in optimal alignment with another (reference) nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, measures the nucleotide base identity between the nucleic acid and the reference nucleic acid sequence as determined by any of the known sequence identity algorithms such as FASTA, BLAST or Gap, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% nucleotide base identity. In certain instances, a nucleic acid molecule that is substantially identical to a reference nucleic acid molecule encodes a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0066] In a preferred embodiment of the disclosure, the fusion protein can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. Numerous such systems are widely available from commercial suppliers. In a specific embodiment, the fusion protein can be expressed using a vector in which the polynucleotide encoding the fusion protein is operably linked to a promoter sequence. In a specific embodiment, the promoter is a constitutive promoter. In another specific embodiment, the promoter is an inducible promoter.

[0067] In one embodiment, the polynucleotide or vector is contained in a virus. In another embodiment, the virus is selected from the group consisting of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus. In a preferred embodiment of the disclosure, the polynucleotide or vector is contained in an adeno-associated virus shuttle plasmid.

[0068] The disclosure further provides a host cell comprising a polynucleotide as shown herein. In a specific embodiment, the host cell is a prokaryotic cell. In another specific embodiment, the host cell is a eukaryotic cell. In another specific embodiment, the host cell is a mammalian cell. In a preferred specific embodiment, the host cell is a human cell.

[0069] Preferably, the host cell comprises an adeno-associated virus vector comprising a polynucleotide of the disclosure.

[0070] The disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein as shown herein and optionally a pharmaceutically acceptable carrier or excipient.

[0071] The present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of a host cell according to the present disclosure. Bi-specific T cell engager proteins generally have very short serum half-lives, on the order of 2 hours. This makes administration of BiTEs require continuous intravenous infusion, a procedure that is inconvenient for human use. The preferred way to overcome this drawback is to use a virus to mediate the continuous production of BiTEs in vivo.

[0072] The pharmaceutical composition is formulated with a suitable carrier, excipient, and other agents that are appropriate for the type of composition and the desired effect of the composition. A wide variety of appropriate formulations can be found in the Handbook of Pharmaceutical Excipients, 4thEdition, Rowe et al., Eds., American Pharmaceutical Association, Washington, D.C. (2003). These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, liposomes (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water or water-in-oil emulsions, emulsions in which the particles are dispersed in a semi- solid hydrocarbon matrix, semi-solid gels, and semi-solid mixtures with water-soluble polymers. See also Powell et al., Compendium of excipients for parenteral formulations, PDA (1998) J Pharm Sci Technol 52:238-311.

[0073] The dosage of the fusion protein administered to a patient can vary depending on factors such as the age and size of the patient to be treated, the target disease, the condition, the route of administration, etc. The preferred dosage is usually calculated on the basis of body weight or body surface area. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective doses and schedules for administration of the fusion protein can be determined empirically; for example, progress of the patient's disease can be monitored by periodic assessment and the dosage adjusted accordingly. Also, interspecies scaling of dosages can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0074] A variety of drug delivery systems are known which can be used to administer the pharmaceutical compositions of this application, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant virus and of undergoing receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., the linings of the mouth, rectum, and intestine, etc.), and can be compounded, for example, with other biologically active agents. Administration can be systemic or local.

[0075] The pharmaceutical compositions of this application can be administered in a further amount effective to deliver a further amount of the mutant virus to the tumor, subcutaneously, or intravenously, using a standard needle and syringe.

[0076] In certain instances, the pharmaceutical composition can be administered using a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be employed; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity to the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0077] In certain states, the pharmaceutical composition can be delivered in a formulation for injection. The formulation for injection can include intratumoral, intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, and the like. These formulations for injection can be prepared by well-known methods. For example, the above-mentioned antibody or salt thereof can be dissolved, suspended, or emulsified in a sterile aqueous medium, or an oily medium generally used for injection, in this way, the formulation for injection can be prepared. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, and a suitable solubilizing agent such as an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant such as polysorbate 80, HCO-50 (a 50-mole polyoxyethylene adduct of hydrogenated castor oil), and the like can be used in combination. As the oily medium, sesame oil, soybean oil, and the like can be used, and a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like can be used in combination. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0078] Preferably, the above-mentioned pharmaceutical composition for oral or injection can be prepared in a unit-dose form to contain a certain dose of the active ingredient. Such a unit-dose form includes, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, and the like.

[0079] The present disclosure further provides the use of a pharmaceutical composition as shown herein for the preparation of a medicament for inducing antibody-dependent cellular cytotoxicity in a subject in need thereof.

[0080] Preferably, the present disclosure provides the use of a pharmaceutical composition for the preparation of a medicament for treating cancer, an infectious disease, an autoimmune disease, a graft-versus-host disease, or a post-transplant lymphoproliferative disease in a subject in need thereof.

[0081] As used herein, the term "treating" or "treatment" refers to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an undesirable condition, disorder, or disease, in order to effect a reduction in severity and / or frequency of symptoms, eliminate symptoms, and / or their underlying cause, and / or to assist in healing or repair. The term "preventing" or "prevention" refers to the administration of an agent or composition to a clinically asymptomatic individual who is predisposed to a particular undesirable condition, disorder, or disease, and thus is directed to preventing the appearance of symptoms and / or their underlying cause. As understood by those skilled in the art, prevention need not be absolute (complete) blocking or avoidance of the condition. Rather, prevention can achieve substantial (e.g., more than about 50%) reduction or avoidance of the disease or condition to be prevented. Unless otherwise clearly indicated or implied by context, the term "treatment" or "treating" as used herein is intended to encompass prevention, unless otherwise indicated or clearly implied by context.

[0082] "Cancer", "tumor", "transformation" and like terms include pre-cancerous, neoplastic, transformed and cancerous cells, and can refer to solid tumors, or non-solid cancers (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign and malignant neoplasms (abnormal growths). "Transformation" refers to spontaneous or induced phenotypic changes, e.g., immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition and anchorage dependence, and / or malignant disease (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Although transformation can be caused by infection with a transforming virus and incorporation of new genomic DNA or uptake of foreign DNA, it can also occur spontaneously or as a result of exposure to a carcinogen.

[0083] In one preferred embodiment of the disclosure, the pharmaceutical composition further comprises one or more antibodies. More preferably, the antibody is an IgG antibody. In another aspect, the antibody is a monoclonal antibody or a polyclonal antibody. Examples of antibodies include, but are not limited to, an anti-CD20 antibody, an anti-EGFR antibody, an anti-HER2 antibody, an anti-latent membrane protein 1 (LMP1) antibody, or an anti-PD-L1 antibody.

[0084] The development of cancer is characterized by the evasion of immune responses, including tumor evasion mediated through immune checkpoint pathways (Pardoll Nat Rev Cancer, 12, 252-264 (2012)). By overexpressing PD-L1, tumor cells exploit the PD-1 / PD-L1 pathway to promote an immunosuppressive environment and allow tumor growth (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012)). Restoring T cell anti-tumor activity by blocking PD-L1 inhibitory signals represents a key therapeutic strategy (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012); Postow et al., J Clin Oncol, 33, 1974-1982 (2015)). Avelumab is an approved human IgG anti-PD-L1 mAb that is believed to specifically bind to PD-L1, preventing the interaction between PD-L1 and the T cell inhibitory receptor PD-1. Blockade of PD-L1 abrogates the inhibition of T cell activity, resulting in T cell-mediated anti-tumor immune responses (Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515-523 (2017)). In addition, unlike other approved anti-PD-L1 antibodies, avelumab has a wild-type IgG Fc region that enables avelumab to engage FcyRs on NK cells and induce tumor-directed ADCC (Boyerinas et al., Cancer Immunol Res, 3, 1148-1157 (2015); Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515-523 (2017)). Thus, avelumab has the potential to re-activate T cell-mediated anti-tumor immune responses and mediate ADCC to eradicate tumor cells. The pharmaceutical compositions according to the present disclosure enable such T cells that have been de- immunosuppressed to have ADCC activity, and significantly increase the number of cells with ADCC activity and the therapeutic potential of the mAb.

[0085] In one embodiment of the present disclosure, the fusion protein is used to remove unwanted cells. For example, to reduce graft versus host disease and prevent post-transplant lymphoproliferative disease, such as Epstein-Barr virus (EBV)-induced post-transplant lymphoproliferative disease.

[0086] In one embodiment of the disclosure, the fusion proteins are applied to treat infectious diseases. Preferred examples of infectious diseases according to the disclosure include viral infections, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). Without being limited by theory, it is generally believed that ADCC is an important protective mechanism in HIV vaccines against patients (Haynes et al., N Engl J Med, 366, 1275-1286 (2012); Parsons et al., Retrovirology, 15, 58 (2018)). Furthermore, elimination of latently infected cells harboring viral reservoirs is an important work in HIV treatment. In recent years, a number of potent neutralizing antibodies against multiple HIV-1 isolates, called broadly neutralizing antibodies (Mujib et al., J Virol, 91, e00634-17 (2017)), have been isolated. Currently, passive transfer of the broadly neutralizing antibody-VRC01 is being evaluated in the clinic for its potential to eliminate latently infected cells (NCT02716675 and NCT02568215), and there is substantial evidence that NK cell-mediated ADCC can have the potential to eliminate latently infected cells (Madhavi et al., J Virol, 91, e00700-17 (2017)). Nonetheless, chronic HIV-1 infection has been shown to alter the phenotype, function, and subset distribution of NK cells. Novel methods of enhancing ADCC in HIV-infected patients, such as the methods disclosed herein, can contribute to the achievement of HIV cure in a critical extent. Similar strategies can also be applied to vaccine design and treatment of other viral infections, such as HBV, EBV, and CMV (Gao et al., Human Vaccines Immunotherapeutics, 13, 1768-1773 (2017); Coghill et al., Clin Cancer Res, 22, 3451-3457 (2016); McVoy et al., Int J Mol Sci, 19, 3982 (2018)).

[0087] The following examples are provided to assist a person of ordinary skill in the art with practicing the present disclosure.

[0088] Example

[0089] Example 1

[0090] haCD16A-BiTE constructs

[0091] Genetic construct of haCD16A-BiTE is shown in Figures 1A-1D . Figure 1AGenetic construct of CD16A-CD3 bispecific T cell engager protein cloned into adeno-associated virus (AAV) shuttle plasmid pAAV-CD16CD3 and driven by cytomegalovirus promoter (CMV) to express a fusion protein of 446 amino acids. H: 6x histidine tag; ITR: inverted terminal repeat of AAV; S: secretion signal; WPRE: woodchuck hepatitis B virus post-transcriptional regulatory element. Figure 1B Coding sequences of extracellular domain of human high affinity CD16A (edCD16) and single chain antibody against human CD3 (anti-CD3 scFv) were fused in the same coding frame by gene synthesis. Figure 1C Synthetic gene of 1341 bp nucleotides encodes a fusion protein of 446 amino acids. Figure 1D Schematic diagram of haCD16A-BiTE.

[0092] In vitro binding analysis of haCD16A-BiTE.

[0093] haCD16A-BiTE has the ability to bind human IgG antibodies and T cells. This binding ability of haCD16A-BiTE was first demonstrated herein. haCD16A-BiTE with a histidine tag was produced and purified from the supernatant of haCD16A-BiTE gene transfected HEK 293 cells by affinity chromatography. To show the binding of haCD16A-BiTE to IgG antibodies, the analysis strategy was outlined in Figure 2A Raji (CD20 + Burkitt's lymphoma cells) and A431 (EGFR + epidermoid carcinoma cells) cells were incubated with 1 μg of rituximab and cetuximab (both IgG antibodies) respectively for 10 minutes, followed by incubation with 50 ng of haCD16A-BiTE in 100 μl PBS for 10 minutes at 4°C. Next, the cells were pelleted by centrifugation at 400 x g for 5 minutes, washed once with PBS, and incubated with 2 μl of PE-labelled anti-6x-histidine tag antibody (Miltenyi Biotech) in 100 μl PBS for 1 hour at 4°C. After washing once with PBS, the cells were subjected to flow cytometry analysis and the data were presented as histograms of counts versus fluorescence. CD20 and EGFR expression of Raji and A431 cells were also checked by flow cytometry.

[0094] As Figure 2BRaji and A431 cells express CD20 and EGFR, respectively. In the presence of haCD16A-BiTE, over 90% of Raji or A431 cells treated with rituximab or anti-EGFR antibody stained positive with anti-His antibody, but cells without rituximab or anti-EGFR antibody treatment were negative (Figure 1C), indicating that haCD16A-BiTE has the ability to bind IgG antibody. These experimental data show that haCD16A-BiTE is able to bind IgG antibody-coated tumor cells. Figure 2C

[0095] To show the binding of haCD16A-BiTE to T cells, Figure 2D The analysis strategy is shown in Figure 2A, and Figure 2E CD16-negative T cells (over 95% of which express CD3 molecules) can be detected by anti-CD16 antibody in the presence of haCD16A-BiTE, but not in the absence of haCD16A-BiTE. These results indicate that haCD16A-BiTE binds to T cells. The experimental methods used are as described above, except that CD16 - T cells were used and IgG antibody was omitted.

[0096] Example 2

[0097] In vitro activity analysis of haCD16A-BiTE

[0098] The ability of haCD16A-BiTE to mediate killing of CD20-expressing tumor cells in the presence of an approved anti-CD20 IgG antibody (rituximab) and T cells

[0099] Cytotoxicity experiments were performed according to the method disclosed in Sheehy et al. (J Immunol Methods, 249, 99-110 (2001)). In this experiment, Raji, VAL, and Toledo hematological tumor cell lines expressing CD20 were used as target cells, with the VAL cell line being an acute lymphoblastic leukemia (ALL) cell and the Toledo cell line being a diffuse large B-cell lymphoma cell. The RS4 cell line is a CD20 - ALL cell. Raji, VAL, Toeldo, and RS4 cells were independently stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and seeded in wells of a culture dish (5 x 105 4 cells / well). haCD16A-BiTE (80 ng / well), rituximab (10 μg / well), and T cells (5 x 105 5 ​cells / well) were added independently or together to each well containing different tumor cells. After 6 hours of incubation of the cultures, the number of viable cells was determined by counting CFSE + T cells were obtained from peripheral blood mononuclear cell (PBMC) cultures expanded with CD3 / CD28 beads, IL-7 and IL-15 according to the method described by Chen et al. (Clinical Immunology, 104, 58-66, (2002)).

[0100] As shown in Figure 3 , the results demonstrate that, in the presence of T cells, haCD16A-BiTE shows a synergistic effect in killing CD20-expressing hematological tumor cells (Raji, VAL and Toeldo) when combined with rituximab compared to haCD16A-BiTE or rituximab alone. *: p<0.001 compared to haCD16A-BiTE or rituximab alone.

[0101] Ability of haCD16A-BiTE to mediate killing of EGFR-expressing tumor cells in the presence of an approved anti-EGFR IgG antibody (cetuximab) and T cells

[0102] The experiment was performed exactly as described in Figure 3 except that the EGFR-expressing tumor cell line (A431) and cetuximab (2.5 μg / well) were used. In this experiment, A431 cells expressing EGFR molecules were used as target cells. The MCF-7 cell line is an EGFR low breast tumor cell.

[0103] As shown in Figure 4 , the results demonstrate that, in the presence of T cells, haCD16A-BiTE shows a synergistic effect in killing EGFR-expressing cells (A431) when combined with cetuximab compared to haCD16A-BiTE or cetuximab alone. *: p<0.001 compared to haCD16A-BiTE or cetuximab alone.

[0104] Ability of haCD16A-BiTE to mediate killing of HER2-expressing tumor cells in the presence of an approved anti-HER2 IgG antibody (trastuzumab) and T cells

[0105] The experiment was performed exactly as described in Figure 3The procedure was performed except that the HER2-expressing tumor cell line and trastuzumab (2.5 pg / well) were used. In this experiment, the BT474 tumor cell line, which is more than 80% of cells express HER2 molecules, was used as target cells, where BT474 is a breast ductal carcinoma cell. The T47D cell line is a HER2 low breast ductal carcinoma cell, where 10% of cells express HER2 at a low density.

[0106] As shown in Figure 5 Figure 6, the results demonstrate that haCD16A-BiTE exhibits synergistic effect in killing HER2 high cells (BT474 cells) when combined with trastuzumab in the presence of T cells, but not in killing HER2 low cells (T47D cells) when compared to haCD16A-BiTE or trastuzumab alone. *: p<0.01 when compared to haCD16A-BiTE or trastuzumab alone.

[0107] Overall, the experimental data as shown in Example 2 demonstrate that haCD16A-BiTE is able to recruit T cells to mediate killing of tumor cells bound by IgG antibodies. Importantly, these results also show that haCD16A-BiTE is able to kill tumor cells in combination with various FDA-approved IgG therapeutic antibodies.

[0108] Example 3

[0109] Effect of plasma on haCD16A-BiTE activity

[0110] The IgG content in normal plasma can compete with therapeutic mAbs of the same IgG isotype to bind haCD16A-BiTE, resulting in loss of haCD16A-BiTE activity. To assess such competition, whole blood from a healthy individual was centrifuged at 400 x g for 5 minutes, and the supernatant was taken as plasma. The plasma was added to the serum-free medium for the cytotoxicity assay experiment at different volume ratios, as shown in Figure 3 Figure 6, to analyze the effect of plasma on rituximab cytotoxicity in the presence of haCD16A-BiTE and T cells. Plasma also mediates complement-dependent cytotoxicity of rituximab, so the data are presented as rituximab / plasma / T cell cytotoxicity minus complement-dependent cytotoxicity.

[0111] Figure 6 As shown in Figure 6, plasma (up to 50%) did not significantly reduce rituximab cytotoxicity against Raji cells in the presence of haCD16A-BiTE and T cells.

[0112] Example 4

[0113] CD16 pulsed with haCD16A-BiTE- Comparison of IgG antibody-mediated cell killing between γδ9 T cells and CD16 + Comparison of IgG antibody-mediated cell killing between γδ9 T cells and CD16

[0114] To assess the equivalence of haCD16A-BiTE with the high affinity CD16A expressed on T cells, we compared CD16 - Antibody-mediated cytotoxicity of γδ9 T cells with CD16 + Antibody-mediated cytotoxicity of γδ9 T cells.

[0115] γδ9 T cells were generated as described below. PBMC from different donors (2 x 10 6 cells / ml) were stimulated with recombinant human IL2 (25 ng / ml; Prospec) and zoledronate (1 μΜ; Sigma) in RPMI-1640 medium containing 10% heat-inactivated FBS, penicillin (100 IU / ml) and streptomycin (100 μg / ml) at 37°C in a humidified incubator with 5% C02 for 14 days. Subsequently, CD16 expression of γδ9 T cell cultures was analyzed and those cultures positive for CD16 staining were subjected to a polymerase chain response specific for CD16 high affinity variant to select CD16 + high affinity variant cultures. CD16 + high affinity variant cultures. CD16 Figure 7A ) expressing CD16 - cell source of γδ9 T cells.

[0116] Antibody-mediated cell killing was analyzed according to the method described in Example 2 using Rituximab and Raji cells as target cells. To investigate antibody-mediated killing of CD16 + high affinity variant cultures. CD16 4 high affinity variant cultures. CD16 + high affinity variant cultures. CD16 5 high affinity variant cultures. CD16 -Antibody-mediated cell killing by γ9δ2 T cells, Raji cells treated with CFSE (5×10⁻⁶). 4 Staining was performed using haCD16A-BiTE (80 ng / well), rituximab (20 μg / well), and CD ...20 μg / well). - -γ9δ2 T cells (5×10) 5 CFSEs were added to each well (one cell per well). After incubating the culture for 6 hours, CFSEs were counted using flow cytometry. + Cellular analysis was used to determine the survival rate of Raji cells.

[0117] Figure 7B The results shown indicate that CD16 combined with haCD16A-BiTE - Rituximab-mediated Raji cell killing of γ9δ2 T cells and CD16 + The activity of haCD16A-BiTE is equivalent to that of the high-affinity CD16 variant expressed on γ9δ2 T cells, indicating that its functional activity is comparable to that of the high-affinity CD16 variant expressed on γ9δ2 T cells. *: Comparable to CD16 - Compared with haCD16A-BiTE or rituximab alone for -γ9δ2 T cells, p < 0.001. *: compared with CD16 + Compared with haCD16A-BiTE alone, -γ9δ2 T cells showed p<0.001.

[0118] Example 5

[0119] Unwanted cells were removed from T cell expansion cultures using haCD16A-BiTE.

[0120] This example aims to demonstrate the usefulness of haCD16A-BiTE in removing unwanted cells (such as tumor cells) from T cell expansion cultures.

[0121] Written informed consent was obtained from a patient with chronic lymphocytic leukemia (CLL), followed by peripheral blood samples. PBMCs were isolated from 5 ml of venous blood using density gradient centrifugation with Ficoll-Paque PLUS (Sigma) according to the manufacturer's instructions. To expand γ9δ2 T cells, PBMCs (2 × 10⁶ cells / ml) were stimulated with recombinant human IL-2 (25 ng / ml; Prospec) and zoledronic acid (1 μM; Sigma) in RPMI-1640 medium containing 10% heat-deactivated FBS, penicillin (100 IU / ml), and streptomycin (100 μg / ml) at 37°C in a humidified incubator with 5% CO₂. 6T cells were expanded in the presence of anti-EBV IgG antibodies and T cells for 14 days. To deplete malignant B cells from these T cell expansion cultures, haCD16A-BiTE (80 ng / well) and rituximab (10 pg / well) were added to the cultures on day 10. Three days later, the extent of malignant B cell death in the cultures was analyzed by flow cytometry analysis of CD19 marker (marker for malignant B cells) and propidium iodide (PI) staining.

[0122] Figure 8 It is shown that CD19 positive cells are reduced after treatment with haCD16A-BiTE and rituximab, indicating that haCD16A-BiTE in combination with rituximab has the ability to deplete malignant B cells from T cell expansion cultures. *: p<0.001 compared to haCD16A-BiTE or rituximab alone.

[0123] Example 6

[0124] Ability of haCD16A-BiTE to mediate killing of EBV-infected B cells in the presence of anti-EBV IgG antibodies and T cells

[0125] To determine the usefulness of haCD16A-BiTE in the treatment of viral diseases, an EBV-infected B cell line was used as a model virus-infected cell to test the ability of haCD16A-BiTE to mediate killing of EBV-infected B cells in the presence of anti-EBV IgG antibodies and T cells.

[0126] EBV is a gamma herpes virus that infects 90% of the population. EBV establishes a lifelong latency in memory B cells and oral epithelial cells. In immunocompetent hosts, cytotoxic T lymphocytes specific for EBV maintain EBV-infected B cells at levels less than 1% of the total B cell population. However, in immunocompromised hosts, uncontrolled proliferation of these EBV-infected B cells can lead to lymphoproliferative disorders, such as post-transplant lymphoproliferative disease (PTLD) after solid organ or hematopoietic stem cell transplantation. Furthermore, a large proportion of Hodgkin lymphoma, non-Hodgkin lymphoma, and nasopharyngeal carcinoma are associated with EBV infection. Current treatment options for EBV-associated malignancies are limited to standard chemotherapy and radiation. One promising therapy is the use of T cell therapy specific for EBV, which has shown efficacy against PTLD (success rate up to 70%). A more desirable therapeutic agent is an antibody that can specifically recognize one of the more broadly expressed antigens: latent membrane protein-1 (LMP1) or LMP2 (Ahmed et al. JCI Insight, 3, e97805, (2018)).

[0127] In this study, EBV-infected, LMP1-expressing B cells were used as target cells, while jurkat T cells were used as negative control cells. The experiment was performed exactly as described in Example 2, except that human anti-LMP1 IgG antibody (10 μg / well) was used instead.

[0128] Figure 9 This demonstrates that, in the presence of T cells, haCD16A-BiTE, when combined with anti-LMP1 IgG antibody, exhibits a synergistic effect in killing EBV-infected B cells compared to haCD16A-BiTE or anti-LMP1 antibody alone. *: p < 0.001 compared to haCD16A-BiTE or anti-LMP1 antibody alone.

[0129] Example 7

[0130] In vitro activity analysis of haCD16A-BiTE-mediated killing of PD-L1-expressing tumor cells

[0131] By overexpressing PD-L1, tumor cells utilize the PD-1 / PD-L1 pathway to promote an immunosuppressive environment and allow tumor growth (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012)). Therefore, PD-L1 itself is also a tumor-associated antigen. Blocking PD-L1 inhibitory signaling with anti-PD-L1 IgG mAb can not only restore T cell anti-tumor activity but also provide an opportunity to eradicate tumor cells using ADCC.

[0132] To examine the potential of haCD16A-BiTE to confer ADCC activity on T cells, thereby mediating the elimination of PD-L1-expressing tumor cells by anti-PD-L1 IgG mAb, we screened PD-L1-expressing tumor cells and performed experiments exactly as described in Example 2, except that we used a PD-L1-expressing tumor cell line (A431) and human anti-PD-L1 IgG antibody (10 μg / well) (Invivogen). In this experiment, A431 cells were used as target cells. The MCF-7 cell line is a low-PD-L1 breast cancer cell line.

[0133] like Figure 10 As shown, the results demonstrate that, in the presence of T cells, haCD16A-BiTE, when combined with anti-PD-L1 IgG antibody, exhibits a synergistic effect in killing PD-L1-expressing cells (A431) compared to haCD16A-BiTE or anti-PD-L1 antibody alone. *: p < 0.001 compared to haCD16A-BiTE or anti-PD-L1 antibody alone.

[0134] Example 8

[0135] In vivo analysis of the activity of haCD16A-BiTE in killing CD20-expressing tumor cells in an immunodeficient NOD mouse model

[0136] To effectively enhance the efficacy of therapeutic antibodies, our invention requires the combination of four components together to form a complex. These components include tumor cells, antibodies, haCD16A-BiTE, and T cells. We performed in vivo proof-of-concept studies to demonstrate not only the in vivo activity of haCD16A-BiTE, but also that the formation of this complex actually occurs in vivo.

[0137] These experiments were performed in immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl were performed in YckNarl (RMRC 13288) mice. First, the genes of luciferase and green fluorescent protein were transduced into Raji hematological tumor cells using a lentiviral vector (Zhou et al., Blood, 120, 4334-4342 (2012)). Raji cells expressing luciferase and green fluorescent protein (1 x 10 6 were injected into NOD mice via the tail vein. On day 7 after Raji cell implantation, T cells obtained as described in Example 2 were implanted into NOD mice via the tail vein. The amount of T cells implanted per injection was 10 times the amount of Raji cells. T cells were implanted every 4 days, for a total of 2 implantations. In addition, on day 7 after Raji cell implantation, haCD16A-BiTE was infused via the tail vein for 9 consecutive days at a daily dose of 800 ng, which was delivered by a single bolus injection. On days 7 and 11 after Raji cell implantation, rituximab (10 mg / kg) was administered via the tail vein twice. Control mice received only T cell implantation.

[0138] Bioluminescence imaging (BLI) was performed at different time points to monitor the clearance of Raji cells. At the end of the experiment, the body weight of the treatment groups was comparable to that of the control group. Figure 11 The results in Figure 9 show the BLI difference at the end of the experiment. haCD16A-BiTE significantly improved Raji cell clearance by two-fold compared to mice treated with rituximab and T cells (without haCD16A-BiTE). Thus, tumor cells, antibodies, haCD16A-BiTE, and T cells can be combined together as needed for in vivo activity. More importantly, these data demonstrate the in vivo activity of haCD16A-BiTE. *: p < 0.01 compared to the haCD16A-BiTE, rituximab, and T cell combination.

[0139] Example 9

[0140] AAV-haCD16A-BiTE mediates peripheral effects of haCD16A-BiTE in mice

[0141] To overcome the need for continuous infusion of BiTE in a therapeutic setting due to its short in vivo half-life, we explored an AAV-mediated gene transfer method.

[0142] Mice (B6, male, 8 weeks old; n=5) were intraperitoneally injected with the viral vector AAV-haCD16A-BiTE. Figure 1A 10 9 VGC (vgc / animal; vgc: viral genome copy). Serum samples were collected via tail vein before (day 0) and after (days 2, 7, and 14) viral injection and subjected to Western blot analysis to assess the protein content of haCD16A-BiTE. HRP-conjugated polyclonal antibodies were used to detect the 6× histidine tag at the C-terminus of haCD16A-BiTE. The detected protein content was measured by optical density analysis and expressed as optical density per milliliter of serum.

[0143] Figure 12 The results showed that haCD16A-BiTE was detected in the peripheral blood of mice 7 days after injection of AAV-haCD16A-BiTE, and its presence persisted for at least 7 days. These results indicate that continuous in vivo production of haCD16A-BiTE can be achieved through in vivo virus-mediated haCD16A-BiTE gene transfer.

[0144] This invention is illustrated in conjunction with the specific embodiments described above, and substitutions, adjustments, and variations thereof are as will be understood by those skilled in the art. These substitutions, adjustments, and variations still fall within the scope of this invention. sequence list <110> Baitiwei Biotechnology Co., Ltd. <120> Bispecific T cell adaptor proteins and their uses <130> none <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 531 <212> DNA <213> people <220> <221> CDS <222> (1)..(531) <400> 1 agg aca gag gac ctg cca aag gcc gtg gtg ttt ctg gag ccc cag tgg 48 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 tac cgc gtg ctg gag aag gac tcc gtg aca ctg aag tgc cag ggc gcc 96 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 tat agc cct gag gat aac tcc acc cag tgg ttc cac aat gag agc ctg 144 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 atc agc tcc cag gcc tct agc tac ttt atc gac gca gca acc gtg gac 192 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 gat tcc gga gag tat cgg tgc cag acc aac ctg agc aca ctg tcc gat 240 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 cca gtg cag ctg gag gtg cac atc gga tgg ctg ctg ctg cag gca cct 288 Pro Val Gin Leu Glu Val His He Gly Trp Leu Leu Leu Gin Ala Pro 85 90 95 aga tgg gtg ttc aag gag gag gac ccc atc cac ctg cgc tgt cac agc 336 Arg Trp Val Phe Lys Glu Glu Asp Pro He His Leu Arg Cys His Ser 100 105 110 tgg aag aat acc gcc ctg cac aag gtg aca tac ctg cag aac ggc aag 384 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gin Asn Gly Lys 115 120 125 ggc cgg aag tac ttc cac cac aat tct gac ttt tat atc ccc aag gcc 432 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr He Pro Lys Ala 130 135 140 aca ctg aag gat agc ggc tcc tat ttt tgc aga ggc ctg gtg ggc agc 480 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser 145 150 155 160 aag aac gtg tcc tct gag acc gtg aat atc acc atc aca cag gga ctg 528 Lys Asn Val Ser Ser Glu Thr Val Asn He Thr He Thr Gin Gly Leu 165 170 175 gca 531 Ala <210> 2 <211> 177 <212> PRT <213> Human <400> 2 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro 85 90 95 Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser 100 105 110 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys 115 120 125 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala 130 135 140 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser 145 150 155 160 Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu 165 170 175 Ala <210> 3 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> scFv <220> <221> CDS <222> (1)..(732) <400> 3 cag gtg cag ctg cag cag agc gga gca gag ctg gca agg cct gga gcc 48 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 tcc gtg aag atg tct tgt aag gcc agc ggc tac acc ttc aca cgg tat 96 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 aca atg cac tgg gtg aag cag aga cca gga cag gga ctg gag tgg atc 144 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga tac atc aac cct tcc cgc ggc tac acc aac tat aat cag aag ttt 192 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag gac aag gcc acc ctg acc aca gat aag agc tcc tct aca gcc tac 240 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg cag ctg agc tcc ctg acc tct gag gac agc gcc gtg tac tat tgc 288 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 gcc aga tac tat gac gat cac tac tgt ctg gat tat tgg ggc cag ggc 336 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 acc aca ctg aca gtg tct agc gtg gag gga ggc tcc gga ggc tct gga 384 Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly 115 120 125 ggc agc ggc ggc tcc gga gga gtg gac cag atc gtg ctg acc cag tcc 432 Gly Ser Gly Gly Ser Gly Gly Val Asp Gin He Val Leu Thr Gin Ser 130 135 140 cca gca atc atg tct gcc agc cct gga gag aag gtg acc atg aca tgc 480 Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 145 150 155 160 tct gcc tcc tct agc gtg agc tac atg aat tgg tat cag cag aag tct 528 Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser 165 170 175 ggc aca agc cca aag cgg tgg atc tac gac acc tcc aag ctg gca tct 576 Gly Thr Ser Pro Lys Arg Trp He Tyr Asp Thr Ser Lys Leu Ala Ser 180 185 190 gga gtg cca gca cac ttc aga ggc tct ggc agc ggc acc tcc tat tct 624 Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser 195 200 205 ctg aca atc tcc gga atg gag gca gag gat gca gca acc tac tat tgt 672 Leu Thr He Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220 cag cag tgg tcc tct aac ccc ttc acc ttt ggc tct ggc aca aag ctg 720 Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 225 230 235 240 gag atc aat aga 732 Glu Ile Asn Arg <210> 4 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly 115 120 125 Gly Ser Gly Gly Ser Gly Gly Val Asp Gin He Val Leu Thr Gin Ser 130 135 140 Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 145 150 155 160 Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gin Gin Lys Ser 165 170 175 Gly Thr Ser Pro Lys Arg Trp He Tyr Asp Thr Ser Lys Leu Ala Ser 180 185 190 Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser 195 200 205 Leu Thr He Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220 Gln Gin Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 225 230 235 240 Glu He Asn Arg <210> 5 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construction <220> <221> CDS <222> (1)..(57) <400> 5 atg gag tgc agc tgc gtg atg ctg ttc ctg ctg tcc gga acc gca ggc 48 Met Glu Cys Ser Cys Val Met Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 gtg ctg tct 57 Val Leu Ser <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construction <400> 6 Met Glu Cys Ser Cys Val Met Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val Leu Ser <210> 7 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construction <220> <221> CDS <222> (1)..(1338) <400> 7 atg gag tgc agc tgc gtg atg ctg ttc ctg ctg tcc gga acc gca ggc 48 Met Glu Cys Ser Cys Val Met Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 gtg ctg tct agg aca gag gac ctg cca aag gcc gtg gtg ttt ctg gag 96 Val Leu Ser Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu 20 25 30 ccc cag tgg tac cgc gtg ctg gag aag gac tcc gtg aca ctg aag tgc 144 Pro Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys 35 40 45 cag ggc gcc tat agc cct gag gat aac tcc acc cag tgg ttc cac aat 192 Gln Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn 50 55 60 gag agc ctg atc agc tcc cag gcc tct agc tac ttt atc gac gca gca 240 Glu Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala 65 70 75 80 acc gtg gac gat tcc gga gag tat cgg tgc cag acc aac ctg agc aca 288 Thr Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr 85 90 95 ctg tcc gat cca gtg cag ctg gag gtg cac atc gga tgg ctg ctg ctg 336 Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu 100 105 110 cag gca cct aga tgg gtg ttc aag gag gag gac ccc atc cac ctg cgc 384 Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg 115 120 125 tgt cac agc tgg aag aat acc gcc ctg cac aag gtg aca tac ctg cag 432 Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln 130 135 140 aac ggc aag ggc cgg aag tac ttc cac cac aat tct gac ttt tat atc 480 Asn Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile 145 150 155 160 ccc aag gcc aca ctg aag gat agc ggc tcc tat ttt tgc aga ggc ctg 528 Pro Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu 165 170 175 gtg ggc agc aag aac gtg tcc tct gag acc gtg aat atc acc atc aca 576 Val Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr 180 185 190 cag gga ctg gca cag gtg cag ctg cag cag agc gga gca gag ctg gca 624 Gln Gly Leu Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala 195 200 205 agg cct gga gcc tcc gtg aag atg tct tgt aag gcc agc ggc tac acc 672 Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr 210 215 220 ttc aca cgg tat aca atg cac tgg gtg aag cag aga cca gga cag gga 720 Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly 225 230 235 240 ctg gag tgg atc gga tac atc aac cct tcc cgc ggc tac acc aac tat 768 Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr 245 250 255 aat cag aag ttt aag gac aag gcc acc ctg acc aca gat aag agc tcc 816 Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser 260 265 270 tct aca gcc tac atg cag ctg agc tcc ctg acc tct gag gac agc gcc 864 Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala 275 280 285 gtg tac tat tgc gcc aga tac tat gac gat cac tac tgt ctg gat tat 912 Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 290 295 300 tgg ggc cag ggc acc aca ctg aca gtg tct agc gtg gag gga ggc tcc 960 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser 305 310 315 320 gga ggc tct gga ggc agc ggc ggc tcc gga gga gtg gac cag atc gtg 1008 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Gln Ile Val 325 330 335 ctg acc cag tcc cca gca atc atg tct gcc agc cct gga gag aag gtg 1056 Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val 340 345 350 acc atg aca tgc tct gcc tcc tct agc gtg agc tac atg aat tgg tat 1104 Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr 355 360 365 cag cag aag tct ggc aca agc cca aag cgg tgg atc tac gac acc tcc 1152 Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser 370 375 380 aag ctg gca tct gga gtg cca gca cac ttc aga ggc tct ggc agc ggc 1200 Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly 385 390 395 400 acc tcc tat tct ctg aca atc tcc gga atg gag gca gag gat gca gca 1248 Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala 405 410 415 acc tac tat tgt cag cag tgg tcc tct aac ccc ttc acc ttt ggc tct 1296 Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser 420 425 430 ggc aca aag ctg gag atc aat aga cat cac cac cac cac cac tga 1341 Gly Thr Lys Leu Glu Ile Asn Arg His His His His His His 435 440 445 <210> 8 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Met Glu Cys Ser Cys Val Met Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val Leu Ser Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu 20 25 30 Pro Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys 35 40 45 Gln Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn 50 55 60 Glu Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala 65 70 75 80 Thr Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr 85 90 95 Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu 100 105 110 Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg 115 120 125 Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln 130 135 140 Asn Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile 145 150 155 160 Pro Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu 165 170 175 Val Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr 180 185 190 Gln Gly Leu Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala 195 200 205 Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr 210 215 220 Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly 225 230 235 240 Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr 245 250 255 Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser 260 265 270 Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala 275 280 285 Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 290 295 300 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser 305 310 315 320 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Gln Ile Val 325 330 335 Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val 340 345 350 Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr 355 360 365 Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser 370 375 380 Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly 385 390 395 400 Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala 405 410 415 Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser 420 425 430 Gly Thr Lys Leu Glu Ile Asn Arg His His His His His His 435 440 445

Claims

1. A fusion protein comprising an extracellular domain of human CD16A and an anti-human CD3 single-stranded variable fragment, wherein the extracellular domain of human CD16A comprises the amino acid sequence shown in SEQ ID NO: 2; and The anti-human CD3 single-chain variable fragment specifically binds to human CD3, and the anti-human CD3 single-chain variable fragment is composed of the amino acid sequence shown in SEQ ID NO: 4; and the C-terminal amino acid of the extracellular domain of human CD16A is directly linked to the N-terminal amino acid of the anti-human CD3 single-chain variable fragment.

2. A polynucleotide encoding the fusion protein as described in claim 1.

3. The polynucleotide of claim 2, wherein the fragment encoding the human CD16A extracellular domain is composed of the nucleotide sequence shown in SEQ ID NO: 1; and wherein the single-stranded variable fragment encoding the anti-human CD3 is composed of the nucleotide sequence shown in SEQ ID NO:

3.

4. The polynucleotide of claim 2, further comprising a fragment encoding a secretion signal peptide and consisting of the nucleotide sequence shown in SEQ ID NO:

5.

5. The polynucleotide of claim 2, wherein it comprises the nucleotide sequence shown in SEQ ID NO:

7.

6. The polynucleotide of claim 2, wherein it is contained in an adeno-associated virus vector.

7. A host cell comprising the polynucleotide as described in claim 2.

8. Use of a pharmaceutical composition for the preparation of a medicament for treating individuals in need of treatment for Burkert lymphoma, epidermoid carcinoma, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, or Epstein-Barr virus infection, said pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of claim 1 and, if necessary, a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition further comprises an IgG antibody having an Fc region that can specifically bind to tumor cell surface antigens CD20, EGFR, HER2, or PD-L1, or to Epstein-Barr virus-infected cell surface antigen LMP1.

Images