Bispecific T cell derivatives and uses thereof

Abstract

To provide a novel fusion protein to overcome the current difficulties related to application of monoclonal antibodies in the fields requiring ADCC, e.g. for depletion of tumor cells, virally-infected cells, or immune-modulating cells.SOLUTION: One example of the fusion protein preferably comprises an extracellular domain of a high-affinity variant of human CD 16 A fused to an anti-CD3 antibody or its antigen-binding fragment that specifically binds to an epitope on human CD3 or its fragment.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE

[0001] This disclosure relates to fusion proteins, in particular CD16A-bispecific T cell engagers (BiTEs), and uses thereof. [Background technology]

[0002] Therapeutic monoclonal antibodies (mAbs) have recently become one of the fastest-growing classes of drugs, approved for the treatment of a wide range of indications, from cancer and infectious diseases to autoimmune diseases (Almagro et al., Front Immunol 8, 1751 (2017)). Most mAbs approved for use in oncology are so-called direct-targeting mAbs, such as rituximab (anti-CD20 mAb), which are designed to directly target tumor cells. This type of mAb is typically generated ex vivo and passively infused into patients, where it acts against established or residual tumors, activating various Fc receptor-mediated effector pathways to kill target cells. When combined with chemotherapy, these therapeutic mAbs have produced excellent results in hematological malignancies, with examples of 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)) using anti-CD20 and anti-CD38, respectively. Alternatively, 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 death-ligand 1) are designed to block immune checkpoints to reactivate antitumor immune responses (K. Chin et al., Annals of Oncology, 28, 1658-1666 (2017)). These immunomodulatory mAbs can also function as direct targeting mAbs to eliminate cells (Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515–523 (2017)).

[0003]

[0003] The mechanisms of action of direct-targeting therapeutic mAbs are attributed to various natural functions of antibodies: neutralization, antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (Suzuki et al., J Toxicol Pathol, 28, 133-139 (2015)). The extent to which each mechanism contributes to clinical efficacy is unclear.

[0004]

[0004] Therefore, there is a need for the development of new methods to improve the therapeutic effectiveness of mAbs and to facilitate the application of mAbs in various fields. The present disclosure has addressed these and other needs. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] The present disclosure provides novel bispecific T cell derivatives to overcome current problems associated with mAb application in disease treatment and other fields, particularly in fields requiring ADCC, such as removal of tumor cells, virus-infected cells, or immunoregulatory cells. [Means for solving the problem]

[0006]

[0006] The present disclosure relates to The extracellular domain of human CD16A; and Antibodies or antigen-binding fragments thereof that specifically bind to epitopes or fragments thereof of human CD3 The present invention provides a fusion protein comprising:

[0007]

[0007] The present disclosure also provides polynucleotides encoding the fusion proteins disclosed herein.

[0008]

[0008] The present disclosure also provides a host cell comprising the polynucleotide disclosed herein.

[0009]

[0009] The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of the fusion proteins disclosed herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0010]

[0010] The present disclosure also provides the use of a pharmaceutical composition disclosed herein in the manufacture of a medicament for inducing antibody-dependent cellular cytotoxicity in a subject in need thereof.

[0011]

[0011] The present disclosure is described in detail in the following sections. Other features, objects, and advantages of the present disclosure can be found in the detailed description and claims. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1 shows the genetic construction of haCD16A-BiTE. Figure 2 shows the genetic construction of the haCD16A-CD3 bispecific T cell derivative cloned into an adeno-associated virus (AAV) shuttle plasmid. H: 6x histidine tag; ITR: AAV inverted terminal repeats; S: secretion signal; WPRE: woodchuck hepatitis B virus post-transcriptional regulatory element. [Figure 1B] Figure 1 shows the gene construction of haCD16A-BiTE, in which the coding sequences for the extracellular domain (edCD16) of human high-affinity CD16A (haCD16A) and a single-chain antibody against human CD3 (anti-CD3 scFv) were fused in the same coding frame by gene synthesis. [Figure 1C]

[0023] Figure 1 shows the genetic construction of haCD16A-BiTE. A 1341 bp nucleotide synthetic gene encodes a 446 amino acid fusion protein. [Figure 1D-1] 1 shows the genetic construction of haCD16A-BiTE. 2 shows the scheme of haCD16A-BiTE. [Figure 1D-2] 1 shows the genetic construction of haCD16A-BiTE. 2 shows the scheme of haCD16A-BiTE. [Figure 1D-3]1 shows the genetic construction of haCD16A-BiTE. 2 shows the scheme of haCD16A-BiTE. [Figure 1D-4] 1 shows the genetic construction of haCD16A-BiTE. 2 shows the scheme of haCD16A-BiTE. [Figure 2A] Figure 1 shows binding of haCD16A-BiTE to T cells and immunoglobulin G (IgG) antibody-coated tumor cells. Figure 2 shows a destructive strategy for haCD16A-BiTE binding to IgG antibody-coated cells. [Figure 2B] Figure 1 shows binding of haCD16A-BiTE to T cells and immunoglobulin G (IgG) antibody-coated tumor cells. Figure 2 shows antigen expression on tumor cells. [Figure 2C] Figure 1 shows binding of haCD16A-BiTE to T cells and immunoglobulin G (IgG) antibody-coated tumor cells. Figure 2 shows binding of haCD16A-BiTE to IgG antibody-coated tumor cells. [Figure 2D] Figure 1 shows binding of haCD16A-BiTE to T cells and immunoglobulin G (IgG) antibody-coated tumor cells. Figure 2 shows a destructive strategy for haCD16A-BiTE binding to T cells. [Figure 2E] Figure 1 shows binding of haCD16A-BiTE to T cells and immunoglobulin G (IgG) antibody-coated tumor cells. [Figure 3] FIG. 1 shows the effect of treatment with anti-CD20 antibody (rituximab, Rituxin®) and haCD16A-BiTE, alone or in combination, in the presence of T cells on killing of CD20-expressing cell lines. [Figure 4] FIG. 1 shows the effect of treatment with anti-EGFR antibodies (cetuximab, Erbitux®) and haCD16A-BiTE, alone or in combination, in the presence of T cells on killing of epidermal growth factor receptor (EGFR)-expressing cell lines. [Figure 5]FIG. 1 shows the effect of treatment with anti-HER2 antibodies (trastuzumab, Herceptin®) and haCD16A-BiTE alone or in combination in the presence of T cells on killing of human epidermal growth factor receptor 2 (HER2)-expressing cell lines. [Figure 6] FIG. 1 shows the effect of plasma on killing of CD20-expressing cell lines treated with rituximab and haCD16A-BiTE. [Figure 7A] FIG. 10 shows a comparative study of IgG antibody-mediated cell killing between CD16 − γ9δ2 T cells and CD16 + γ9δ2 T cells pulsed with haCD16A-BiTE. [Figure 7B] FIG. 10 shows a comparative study of IgG antibody-mediated cell killing between CD16 − γ9δ2 T cells and CD16 + γ9δ2 T cells pulsed with haCD16A-BiTE. [Figure 8] FIG. 1 shows the effect of treating T cell expansion cultures with an anti-CD20 antibody (rituximab) and haCD16A-BiTE on elimination of malignant B cells from T cell expansion cultures. [Figure 9] FIG. 1 shows the effect of treating EBV-infected cell lines with anti-latent membrane protein 1 (LMP1) antibody and haCD16A-BiTE, alone or in combination, in the presence of T cells on killing of EBV-infected cell lines. [Figure 10] Figure 10 shows the effect of treating PD-L1-expressing cell lines with anti-PD-L1 antibodies and haCD16A-BiTE, alone or in combination, in the presence of T cells on killing of PD-L1-expressing cell lines. [Figure 11] FIG. 1 shows the in vivo effects of haCD16A-BiTE and rituximab combination therapy, and rituximab monotherapy, on reducing cancer cell proliferation in the presence of T cells. [Figure 12] FIG. 1 shows in vivo production of haCD16A-BiTE following AAV-mediated haCD16A-BiTE gene transfer. DETAILED DESCRIPTION OF THE INVENTION

[0013]

[0024] The present disclosure provides: Fc gamma receptor or a ligand-binding fragment thereof; and An antibody or antigen-binding fragment thereof that specifically binds to an epitope or a fragment thereof of a surface antigen of a T cell, wherein the surface antigen is capable of inducing antibody-dependent cell-mediated cytotoxicity and / or activating a T cell. The present invention provides a fusion protein comprising:

[0014]

[0025] In particular, the fusion proteins according to the present disclosure are known as bispecific T cell derivatives that provide dual specific affinity for two antigens / ligands, acting as a bridge between the target cell and the T cell.

[0015]

[0026] Preferably, the fusion protein activates an ADCC immune response, which enables T cells to recognize and kill antibody-coated target cells that express tumor- or pathogen-derived antigens on their surface.

[0016]

[0027] Monotherapy using direct targeting of therapeutic mAbs to treat cancer without combining it with other therapeutic modalities, such as chemotherapy, generally has limited therapeutic efficacy (Hiddemann et al., Blood, 106, 3725-32 (2005); Sehn et al., J Clin Oncol, 33, 3467-3474 (2015)). Without being limited by theory, the invention disclosed herein is based on the applicant's belief that increasing ADCC is a promising approach to enhance the clinical efficacy of therapeutic antibodies. The primary immune effector cells mediating ADCC are natural killer (NK) cells (Wang et al., Front Immunol, 6, 368 (2015)). NK cells express Fc gamma receptors (FcγR), primarily CD16A (FcγRIIIA), and recognize and bind the Fc portion of IgG antibodies. When Fcγ receptors bind 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)). Therefore, ADCC involves three components: immune effector cells, antibodies, and antibody-opsonized target cells. ADCC is triggered when FcγRs expressed on the surface of NK cells bind to the Fc region of IgG molecules. Given that genetic variations in FcγRs resulting in different binding affinities are known to contribute to differences in the magnitude of ADCC, it follows that the affinity / quantity of FcγRs and the quality / quantity of NK cells may contribute to differences in the magnitude of ADCC.

[0017]

[0028] Examples of Fc gamma receptors include, but are not limited to, CD16A, CD16B, CD32A, CD32B, CD64A, CD64B, and CD64C.

[0018]

[0029] Examples of antibodies or antigen-binding fragments thereof that specifically bind to an epitope or fragment thereof of a T cell surface antigen include, but are not limited to, anti-CD3 antibodies, anti-4-1BB antibodies, anti-CD28 antibodies, or anti-OX40 antibodies.

[0019]

[0030] Preferably, the present disclosure provides The extracellular domain of human CD16A; and Antibodies or antigen-binding fragments thereof that specifically bind to epitopes or fragments thereof of human CD3 The present invention provides a fusion protein comprising:

[0020]

[0031] According to the present disclosure, the fusion protein comprises the 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, each with either a phenylalanine (F) or valine (V) residue at position 158. The CD16A-158V variant has high affinity for IgG, while CD16A-158F is the dominant allele in the human population. Clinical analysis has revealed a positive correlation between the therapeutic efficacy of tumor-targeting therapeutic mAbs and their CD16A binding affinity. Patients homozygous for the CD16A valine variant (CD16A-V / V) have improved clinical outcomes after treatment with antitumor therapeutic antibodies compared with patients either heterozygous for the low-affinity CD16A isoform (CD16A-V / F) or homozygous for the low-affinity CD16A isoform (CD16A-F / F) in response to clinically approved therapeutic antibodies, such as rituximab, trastuzumab, and cetuximab (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 general population has high-affinity CD16A variants. In a preferred embodiment of the present disclosure, CD16A is a high-affinity CD16A variant.

[0021]

[0032] In a more preferred embodiment of the present disclosure, the extracellular domain of CD16A has the amino acid sequence of SEQ ID NO: 2 or a sequence substantially similar thereto.

[0022]

[0033] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that a protein sequence shares at least 90%, at least 95%, and even more preferably at least 96%, 97%, 98%, or 99% amino acid residue sequence identity with the entire sequence of another (reference) protein sequence when optimally aligned with the reference protein sequence, e.g., using the Gap or BESTFIT programs with default gap weighting. Non-identical residue positions preferably differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Examples of groups of amino acids having side chains with similar chemical properties 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; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0023]

[0034] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, for example, between homologous polypeptides from different species, or between a wild-type protein and its variants. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the best regions of overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, 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.

[0024]

[0035] According to the present disclosure, the fusion protein comprises an antibody or antigen-binding fragment thereof that specifically binds to an epitope of human CD3 or a fragment thereof.

[0025]

[0036] In the immune response, CD3 is a surface antigen associated with the T cell receptor (TCR), forming a complex involved in antigen recognition and signal transduction. The CD3 T cell co-receptor mediates the expression of cytotoxic T cells (CD8 + T cells) and helper T cells (CD4 +When using the fusion proteins of the present disclosure, all CD3-expressing cells in the body, including alpha-beta T cells, gamma-delta T cells, and natural killer T cells, can potentially be recruited to become cells that bear high-affinity CD16A and are capable of carrying out ADCC, by binding the anti-CD3 portion of the fusion protein to the CD3 molecule on the T cells.

[0026]

[0037] Various techniques known to those skilled in the art can be used to determine whether an antibody "specifically binds to one or more amino acids" within a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. Additionally, methods such as epitope removal, epitope extraction, and chemical modification of antigens can also be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide to which an antibody specifically binds is hydrogen / deuterium exchange, detected by mass spectrometry. In general terms, hydrogen / deuterium exchange involves deuterium-labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0027]

[0038] Antibodies according to the present disclosure may be full-length or may comprise only the antigen-binding portion, optionally modified to affect functionality.

[0028]

[0039] The term "antibody" as used herein means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD3). The term "antibody" encompasses immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is made up of three domains: C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region contains one domain (C L1 ) included. V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0029]

[0040] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like encompass any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived, for example, from intact antibody molecules using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to align one or more variable and / or constant domains into the appropriate shape, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0030]

[0041] Non-limiting examples of antigen-binding fragments of antibodies 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 amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region such as a CDR3 peptide), 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 molecule modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0031]

[0042] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a V domain, H and V L The domains can be arranged in any suitable configuration relative to each other. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0032]

[0043] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H1 ;(ii)V H -C H2 ;(iii)V H -C H3 ;(iv)V H -C H1 -C H2 ;(v)V H -C H1 -C H2 -C H3 ;(vi)V H -C H2 -C H3 ;(vii)V H -CL;(viii)V L -C H1 ;(ix)V L -C H2 ;(x)V L -C H3 ;(xi)V L -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 domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monovalent VH or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, with the domains non-covalently associated (e.g., by one or more disulfide bonds).

[0033]

[0044] In one preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is an anti-CD3 single chain variable fragment (scFv).

[0034]

[0045] In another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 4 or a substantially similar sequence thereof; preferably having at least 90%, at least 95%, at least 98% or at least 99% sequence identity or a substantially similar sequence thereof.

[0035]

[0046] The antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present 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(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, VH and / or V L All of the framework and / or CDR residues within a domain are mutated back to the residue 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 mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general means are encompassed by the present disclosure.

[0036]

[0047] The present disclosure also provides a V nucleotide sequence disclosed herein with one or more conservative substitutions. H , V L and / or CDR amino acid sequences. For example, the present disclosure provides anti-CD3 antibodies comprising any variant of the V and / or CDR amino acid sequences disclosed herein. H , V L and / or V containing conservative amino acid substitutions, e.g., 10 or less, 8 or less, 6 or less, 4 or less, to any of the CDR amino acid sequences. H , V Land / or an anti-CD3 antibody having the CDR amino acid sequence.

[0037]

[0048] In one preferred embodiment of the present disclosure, the extracellular domain of CD16 is directly linked to the antibody or antigen-binding fragment thereof. In another embodiment of the present disclosure, a linker is present between the extracellular domain of CD16 and the antibody or antigen-binding fragment thereof.

[0038]

[0049] In a preferred embodiment of the present disclosure, the fusion protein further comprises a secretory signal peptide. As used herein, a signal peptide (which may also be referred to as a 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 destined for the secretory pathway. In one embodiment of the present disclosure, the secretory signal peptide has the amino acid sequence of SEQ ID NO: 6 or a sequence substantially similar thereto.

[0039]

[0050] In a preferred embodiment of the present disclosure, the fusion protein further comprises a protein purification tag.

[0040]

[0051] In one preferred embodiment of the present disclosure, the fusion protein has the amino acid sequence of SEQ ID NO: 8 or a sequence substantially similar thereto.

[0041]

[0052] In another preferred embodiment of the present disclosure, a novel bispecific T cell derivative consisting of the extracellular domain of a high-affinity CD16A variant and a single-chain anti-CD3 antibody (haCD16A-BiTE; Figure 1A) leverages mAb ADCC in a variety of applications. Such use of haCD16A-BiTEs offers several advantages: (1) it overcomes the limited availability of CD16A-expressing natural killer cells in the body, because with haCD16A-BiTEs, all CD3-expressing cells in the body, including alpha-beta T cells, gamma-delta T cells, and natural killer T cells, can be mobilized to become cells that bear high-affinity CD16A and are capable of carrying out ADCC, via binding of the anti-CD3 portion of haCD16A-BiTEs to CD3 molecules on T cells; and (2) it solves the problem of only 10-20% of the general population expressing high-affinity CD16A variants, because haCD16A-BiTEs can be applied to any individual, and each individual can be mobilized to a large number of CD3 cells that have high-affinity CD16A through binding of the anti-CD3 portion of haCD16A-BiTEs to CD3 molecules on T cells. + (3) The design of this haCD16A-BiTE employs the high-affinity CD16A extracellular domain, thereby avoiding downregulation of CD16A on activated NK cells; (4) The use of this haCD16A-BiTE to confer ADCC activity to T cells enables ADCC-mediated elimination of unwanted cells from T cell expansion cultures prepared for immunotherapy; (5) The combined use of this haCD16A-BiTE with vaccines generates viral vaccines with antibody-induced ADCC capabilities, providing a promising method for enhancing the therapeutic efficacy of antibody therapy for viral infections; and (6) the conversion of suppression-removed T cells from patients receiving immune checkpoint inhibitors into T cells with ADCC activity, enhancing the therapeutic potential of anti-PD-L1 mAbs such as avelumab.

[0042]

[0053] The present disclosure also provides polynucleotides encoding the fusion proteins disclosed herein.

[0043]

[0054] Preferably, the polynucleotide comprises a fragment encoding the extracellular domain of high affinity CD16A and has the nucleic acid sequence of SEQ ID NO: 1 or a sequence substantially identical thereto.

[0044]

[0055] Preferably, the polynucleotide comprises a fragment that encodes an antibody or antigen-binding fragment thereof and has the nucleic acid sequence of SEQ ID NO: 3 or a sequence substantially identical thereto.

[0045]

[0056] Preferably, the polynucleotide further comprises a fragment encoding a secretory signal peptide and has the nucleic acid sequence of SEQ ID NO: 5 or a sequence substantially identical thereto.

[0046]

[0057] Preferably, the polynucleotide further comprises a fragment encoding a protein purification tag.

[0047]

[0058] More preferably, the polynucleotide has the nucleic acid sequence of SEQ ID NO: 7 or a sequence substantially identical thereto.

[0048]

[0059] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another (reference) nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, there is nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98%, or 99%, of the nucleotide bases to the entire sequence of said reference nucleic acid sequence, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0049]

[0060] In a preferred embodiment of the present disclosure, the fusion protein can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. Many such systems are widely available commercially from commercial sources. In one embodiment, the fusion protein can be expressed using a vector, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter sequence. In one embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter.

[0050]

[0061] In one embodiment, the polynucleotide or vector is comprised in a virus. In another embodiment, the virus is selected from the group consisting of retrovirus, lentivirus, adenovirus, and adeno-associated virus. In a preferred embodiment of the present disclosure, the polynucleotide or vector is comprised in an adeno-associated virus shuttle plasmid.

[0051]

[0062] The present disclosure also provides a host cell comprising the polynucleotide disclosed herein. In one embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is a mammalian cell. In a preferred embodiment, the host cell is a human cell.

[0052]

[0063] Preferably, the host cell contains an adeno-associated virus vector comprising a polynucleotide disclosed herein.

[0053]

[0064] The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of a fusion protein disclosed herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0054]

[0065] The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of host cells according to the present disclosure. Bispecific T cell derivatives generally have a very short serum half-life of approximately 2 hours. This necessitates continuous intravenous infusion for BiTE application, which is inconvenient for use in humans. To overcome this drawback, it is preferable to use a virus to mediate continuous in vivo production of BiTEs.

[0055]

[0066] The pharmaceutical compositions of the present disclosure are formulated with appropriate carriers, excipients, and other agents to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of Excipients for Parenteral Formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0056]

[0067] The dose of the fusion protein administered to a patient can vary depending on various factors, including the patient's age and size, the target disease, condition, route of administration, etc. The preferred dose is typically calculated based on body weight or body surface area. The frequency and duration of treatment can be adjusted depending on the severity of the condition. The effective dosage and schedule for administering the fusion protein can be empirically determined. For example, the patient's progress can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0057]

[0068] Various delivery systems, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present disclosure (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0058]

[0069] In one embodiment of the present invention, the pharmaceutical compositions of the present disclosure can be delivered intratumorally, subcutaneously, or intravenously using a standard needle and syringe.

[0059]

[0070] In certain circumstances, pharmaceutical compositions can be delivered in sustained-release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Fla. In yet another embodiment, a sustained-release system can be placed proximal to the target of the composition, thereby 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 sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0060]

[0071] In certain circumstances, pharmaceutical compositions can be delivered in injectable preparations. Injectable preparations can include dosage forms for intratumoral, intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the pharmaceutical composition in a sterile aqueous or oily medium commonly used for injections. Examples of aqueous media for injection include saline, isotonic solutions containing glucose and other auxiliary agents, and the like. These aqueous media can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)), and the like. Examples of oily media include sesame oil and soybean oil, and these oily media can be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injection thus prepared is preferably filled into an appropriate ampule.

[0061]

[0072] The above-mentioned pharmaceutical compositions for oral or parenteral use are advantageously prepared in a dosage form of a unit dose suitable for the dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0062]

[0073] The present disclosure also provides the use of a pharmaceutical composition disclosed herein in the manufacture of a medicament for inducing antibody-dependent cellular cytotoxicity in a subject in need thereof.

[0063]

[0074] Preferably, the present disclosure provides the use of the pharmaceutical composition in the manufacture of a medicament for treating cancer, an infectious disease, an autoimmune disease, graft-versus-host disease, or post-transplant lymphoproliferative disorder in a subject in need thereof.

[0064]

[0075] As used herein, the terms "treat" and "treatment" refer to the administration of an agent or formulation to a clinically symptomatic individual suffering from an adverse condition, disorder, or disease to achieve a reduction in the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, and / or promote the amelioration or repair of damage. The terms "prevent" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is predisposed to a particular adverse condition, disorder, or disease, and thus relate to the prevention of the occurrence of symptoms and / or their underlying causes. As will be understood by those skilled in the art, prevention or preventing need not achieve absolute (complete) arrest or avoidance of a condition. Rather, prevention may achieve a substantial (e.g., greater than about 50%) reduction or avoidance of the disease or condition sought to be prevented. Unless otherwise indicated explicitly or connotatively herein, when the term "treatment" (or "treating") is used without reference to the possibility of prevention, prevention is intended to be encompassed as well.

[0065]

[0076] Terms such as "cancer," "tumor," and "transformed" encompass precancerous, 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 encompasses both benign and malignant neoplasms (abnormal growths). "Malignant transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell proliferation, contact inhibition and loss of anchorage, and / or malignant tumors (see, Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)). Malignant transformation can result from infection with a transforming virus and integration of new genomic DNA, or from uptake of exogenous DNA, but can also occur spontaneously or after exposure to carcinogens.

[0066]

[0077] In a preferred embodiment of the present 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 or 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.

[0067]

[0078] Cancer development and progression are characterized by immune evasion, including tumor evasion mediated by immune checkpoint pathways (Pardoll Nat Rev Cancer, 12, 252-264 (2012)). Overexpression of PD-L1 allows tumor cells to exploit the PD-1 / PD-L1 pathway to promote an immunosuppressive environment, promoting tumor growth (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012)). Blocking PD-L1 inhibitory signaling can restore the antitumor activity of T cells, thereby providing an important therapeutic strategy (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012); Postow et al., J Clin Oncol, 33, 1974-1982 (2015)). Avelumab, an approved human IgG anti-PD-L1 mAb, specifically binds to PD-L1 and is thought to block the interaction between PD-L1 and the inhibitory T cell receptor PD-1. PD-L1 blockade relieves suppression of T cell activity, resulting in a T cell-mediated antitumor immune response (Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515-523 (2017)). Furthermore, unlike other approved anti-PD-L1 antibodies, avelumab possesses a wild-type IgG Fc region, which enables it to engage FcγR on NK cells and induce tumor-targeted ADCC (Boyerinas et al., Cancer Immunol Res, 3, 1148-1157 (2015); Hamilton and Rath Expert Opinion on Biological Therapy, 17, 515-523 (2017)). As a result, avelumab has the potential to reactivate T cell-mediated antitumor immune responses and mediate tumor cell eradication by ADCC. The pharmaceutical compositions disclosed herein confer ADCC activity to these de-inhibited T cells, significantly increasing the number of cells with ADCC activity and the therapeutic potential of the mAb.

[0068]

[0079] In one embodiment of the present disclosure, the fusion protein is applied to eliminate unwanted cells, for example, to reduce graft-versus-host disease and to prevent post-transplant lymphoproliferative disorders, such as post-transplant Epstein-Barr virus (EBV)-induced lymphoproliferative disorders.

[0069]

[0080] In one embodiment of the present disclosure, the fusion protein is used to treat infectious diseases. According to the present disclosure, the infectious disease is preferably a viral infection, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), or cytomegalovirus (CMV). Without being limited by theory, ADCC is thought to be a potentially important defense mechanism in HIV vaccines (Haynes et al., N Engl J Med, 366, 1275-1286 (2012); Parsons et al., Retrovirology, 15, 58 (2018)). Furthermore, eliminating latently infected cells that provide viral reservoirs is a major challenge in HIV treatment. Recently, many highly potent neutralizing antibodies, broadly referred to as neutralizing antibodies, have been isolated that neutralize a broad array of HIV-1 isolates (Mujib et al., J Virol, 91, e00634-17 (2017)). Passive transfer of the broadly neutralizing antibody VRC01 is currently undergoing clinical evaluation for its potential to eliminate latently infected cells (NCT02716675 and NCT02568215), and there is a growing body of evidence suggesting that NK cell-mediated ADCC has the potential to eliminate latently infected cells (Madhavi et al., J Virol, 91, e00700-17 (2017)). Nevertheless, chronic HIV-1 infection has been demonstrated to alter the phenotype, functionality, and subset distribution of NK cells. Novel methods for enhancing ADCC in HIV-infected patients, such as those disclosed herein, may contribute significantly to achieving a cure for HIV. Similar strategies can 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)).

[0070]

[0081] The following examples are provided to aid those of ordinary skill in the art in practicing the present disclosure. [Example]

[0071] Example 1 haCD16A-BiTE construct

[0082] The gene construct for haCD16A-BiTE is shown in Figure 1A-1D. Figure 1A: The gene construct for the CD16A-CD3 bispecific T cell derivative was cloned into the adeno-associated virus (AAV) shuttle plasmid pAAV-CD16CD3 and driven by the cytomegalovirus promoter (CMV) to express a 446-amino acid fusion protein. H: 6x histidine tag; ITR: AAV inverted terminal repeat; S: secretion signal; WPRE: woodchuck hepatitis B virus posttranscriptional regulatory element. Figure 1B: The coding sequences for the extracellular domain of human high-affinity CD16A (edCD16) and a single-chain antibody against human CD3 (anti-CD3 scFv) were fused in the same coding frame by gene synthesis. Figure 1C: The synthetic gene of 1,341 bp nucleotides encodes the 446-amino acid fusion protein. Figure 1D: Scheme of haCD16A-BiTE.

[0072] In vitro binding assay of haCD16A-BiTE.

[0073]

[0083] haCD16A-BiTE has the ability to bind to human IgG antibodies and T cells. We first demonstrated this binding ability of haCD16A-BiTE. A histidine-tagged haCD16A-BiTE was generated and purified by affinity chromatography from the supernatant of HEK293 cells transfected with the haCD16A-BiTE gene. To demonstrate the binding of haCD16A-BiTE to IgG antibodies, a quantitative strategy is outlined in Figure 2A. Raji (CD20 + Burkett lymphoma cells) and A431 (EGFR +Cells from a squamous cell carcinoma (RAC) strain were first incubated with 1 μg each of rituximab and cetuximab (both IgG antibodies) for 10 minutes, followed by incubation with 50 ng of haCD16A-BiTE in 100 μl of PBS for 10 minutes at 4°C. The cells were then pelleted by centrifugation at 400 × g for 5 minutes, washed once with PBS, and incubated with 2 μL of PE-labeled anti-6x histidine tag antibody (Miltenyi Biotech) in 100 μL of PBS for 1 hour at 4°C. After washing once with PBS, the cells were subjected to flow cytometry analysis, and the data were displayed as a histogram of counts versus fluorescence. Raji and A431 cells were also examined by flow cytometry for CD20 and EGFR expression.

[0074]

[0084] As shown in Figure 2B, Raji cells and A431 cells expressed CD20 and EGFR, respectively. In the presence of haCD16A-BiTE, more than 90% of Raji or A431 cells treated with rituximab or anti-EGFR antibody stained positive with anti-6x histidine tag (anti-His) antibody, whereas untreated cells did not (Figure 2C), suggesting that haCD16A-BiTE has the ability to bind to IgG antibodies. These experimental data demonstrate that haCD16A-BiTE can bind to IgG antibody-coated tumor cells.

[0075]

[0085] To demonstrate the binding of haCD16A-BiTE to T cells, Figure 2D illustrates a degradative strategy, and Figure 2E shows that CD16-negative T cells, of which >95% express CD3 molecules, were detected by anti-CD16 antibodies in the presence of haCD16A-BiTE but not in the absence of haCD16A-BiTE. These results demonstrate that haCD16A-BiTE binds to T cells. The experimental method used was to detect CD16 - As above, except that T cells were used and IgG antibodies were omitted.

[0076] Example 2 In vitro activity assay of haCD16A-BiTE. 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.

[0086] Cytotoxicity experiments were performed according to the method disclosed by Sheehy et al. (J Immunol Methods, 249, 99-110 (2001)). In this experiment, the Raji, VAL, and Toeldo hematologic tumor cell lines, which express CD20, were used as target cells. The VAL cell line is an acute lymphocytic leukemia (ALL) cell line, and the Toeldo cell line is a diffuse large B-cell lymphoma cell line. The RS4 cell line expresses CD20. - ALL cells. Raji, VAL, Toeldo, and RS4 cells were individually stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and seeded into wells of a culture plate (5 × 10 4 / well). haCD16A-BiTE (80 ng / well), rituximab (10 μg / well), and T cells (5 × 10 5 Cells / well) were added individually or together to each well containing different tumor cells. After incubating the cultures for 6 hours, cell viability was assessed by flow cytometry using CFSE. + The T cells were obtained from peripheral blood mononuclear cell (PBMC) cultures grown with CD3 / CD28 beads, IL-7, and IL-15 according to the method described by Chen et al. (Clinical Immunology, 104, 58-66, (2002)).

[0077]

[0087] The results shown in Figure 3 demonstrate that in the presence of T cells, haCD16A-BiTE, when combined with rituximab, exhibits synergistic effects in killing CD20-expressing hematologic tumor cells (Raji, VAL, and Toeldo) compared to the use of haCD16A-BiTE or rituximab alone. *: p<0.001 compared to haCD16A-BiTE or rituximab alone.

[0078] 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.

[0079]

[0088] The experiment was performed exactly as described in Figure 3, except that an EGFR-expressing tumor cell line (A431) and cetuximab (2.5 μg / well) were used instead. In this experiment, A431 cells expressing EGFR molecules were used as target cells. MCF-7 cell line was an EGFR-low breast tumor cell line.

[0080]

[0089] The results shown in Figure 4 demonstrate that in the presence of T cells, haCD16A-BiTE, when combined with cetuximab, exhibits synergistic effects in killing EGFR-expressing cells (A431) compared to either haCD16A-BiTE or cetuximab alone. *: p<0.001 compared to haCD16A-BiTE or rituximab alone.

[0081] 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.

[0082]

[0090] The experiment was performed exactly as described in Figure 3, except that a HER2-expressing tumor cell line and trastuzumab (2.5 μg / well) were used instead. In this experiment, the BT474 tumor cell line, which expresses HER2 molecules in more than 80% of cells, was used as the target cell. BT474 is a breast ductal carcinoma cell line. The T47D cell line is a low-HER2 breast ductal carcinoma cell line, in which 10% of cells express HER2 at low density.

[0083]

[0091] The results, shown in Figure 5, demonstrate that in the presence of T cells, haCD16A-BiTE, when combined with trastuzumab, exhibits synergistic effects in killing HER2-high cells (BT474 cells) but not HER2-low cells (T47D cells) compared to using haCD16A-BiTE or trastuzumab alone. *: p<0.01 compared to haCD16A-BiTE or trastuzumab alone.

[0084]

[0092] Collectively, the experimental data presented in Example 2 demonstrate that haCD16A-BiTEs can recruit T cells and mediate the killing of IgG antibody-bound tumor cells. Importantly, these results also demonstrate that haCD16A-BiTEs can be combined with various FDA-approved IgG therapeutic antibodies to kill tumor cells.

[0085] Example 3 Effect of plasma on the activity of haCD16A-BiTE

[0093] Normal plasma levels of IgG may compete with therapeutic mAbs of the IgG isotype for binding to haCD16A-BiTE, potentially resulting in the loss of haCD16A-BiTE activity. To assess this competition, whole blood from a healthy individual was centrifuged at 400 × g for 5 minutes, and the supernatant was collected as plasma. Plasma was added at various volume ratios to the serum-free medium used in the cytotoxicity assay experiments shown in Figure 3, and the effect of plasma on rituximab cytotoxicity in the presence of haCD16A-BiTE and T cells was analyzed. Because plasma also mediates rituximab complement-dependent cytotoxicity, data were expressed by subtracting the rituximab / plasma / T cell cytotoxicity data.

[0086]

[0094] FIG. 6 shows that plasma did not significantly reduce the cytotoxicity of rituximab against Raji cells by up to 50% in the presence of haCD16A-BiTE and T cells.

[0087] Example 4 CD16 +γ9δ2 T cells and CD16 pulsed with haGD16A-BiTE - Comparative study of IgG antibody-mediated cell killing among .GAMMA.9.DELTA.2 T cells

[0095] To assess the equivalence of haCD16A-BiTE to high-affinity CD16A expressed on T cells, we measured the CD16A expressed on T cells pulsed with haCD16A-BiTE. - Antibody-mediated cytotoxicity of γ9δ2 T cells was suppressed by CD16 with a high-affinity CD16 variant. + This was compared with antibody-mediated cytotoxicity of γ9δ2 T cells.

[0088]

[0096] γ9δ2 T cells were generated as described below. PBMCs (2 × 10 6 γ9δ2 T cell cultures (100 cells / ml) were stimulated with recombinant human IL2 (25 ng / ml; Prospec) and zoledronate (1 μM; Sigma) in RPMI-1640 medium containing 10% heat-inactivated FBS, penicillin (100 IU / ml), and streptomycin (100 μg / ml) for 14 days at 37°C in a humidified incubator with 5% CO. γ9δ2 T cell cultures were subsequently analyzed for CD16 expression, and for cultures that stained positive for CD16, polymerase chain reaction specific for the CD16 high-affinity variant was performed to detect CD16. + High-affinity variant cultures of γ9δ2 T cells were selected. CD16 + γ9δ2 T cells were purified using the Miltenyi Biotech anti-PE purification kit, which conjugates with a PE-conjugated anti-CD16 antibody according to the manufacturer's instructions, and more than 95% of these purified γ9δ2 T cells express CD16 (Figure 7A). Cultures that stained negative for CD16 expression were CD16 - were used as a cell source for γ9δ2 T cells.

[0089]

[0097] Antibody-mediated cell killing was analyzed using rituximab and Raji cells as target cells according to the method described in Example 2. +To study antibody-mediated cell killing of γ9δ2 T cells, Raji were stained with CFSE and treated with rituximab (20 μg / well) and CD16. + γ9δ2 T cells (5 × 10 5 Culture plates (5 x 10 cells / well) 4 CD16 in combination with haCD16A-BiTE was seeded into wells (1000 x 1000 cells / well). - To assess antibody-mediated cell killing of γ9δ2 T cells, CFSE-stained Raji cells (5 × 10 4 / well), haCD16A-BiTE (80 ng / well), rituximab (20 μg / well), and CD16 - γ9δ2 T cells (5 × 10 5 The Raji cells were incubated for 6 hours, and then the viability of the Raji cells was assessed by flow cytometry using CFSE. + Determined by counting cells.

[0090]

[0098] The results shown in Figure 7B show that CD16 in combination with haCD16A-BiTE - Rituximab-mediated Raji cell killing of γ9δ2 T cells is mediated by CD16 + This corresponds to the killing of γ9δ2 T cells, suggesting that the functional activity of haCD16A-BiTE is equivalent to that of the high-affinity CD16 variant expressed in γ9δ2 T cells. *: p<0.001, CD16 - Comparison with haCD16A-BiTE or rituximab alone for γ9δ2 T cells. *: p<0.001, CD16 + Comparison with haCD16A-BiTE or rituximab alone for γ9δ2 T cells.

[0091] Example 5 Use of haCD16A-BiTE to remove unwanted cells from T cell expansion cultures.

[0099] This example demonstrates the utility of haCD16A-BiTE in removing unwanted cells, such as tumor cells, from T cell expansion cultures.

[0092]

[0100] Peripheral blood samples were collected from chronic lymphocytic leukemia (CLL) patients after written informed consent was obtained. PBMCs were isolated from 5 ml of venous blood by density gradient centrifugation using Ficoll-Paque PLUS (Sigma) according to the manufacturer's instructions. To expand γ9δ2 T cells, PBMCs (2 × 10 6 T cell-expanded cultures (1000 cells / ml) were stimulated with recombinant human IL2 (25 ng / ml; Prospec) and zoledronate (1 μM; Sigma) in RPMI-1640 medium containing 10% heat-inactivated FBS, penicillin (100 IU / ml), and streptomycin (100 μg / ml) in a humidified incubator at 37°C with 5% CO2 for 14 days. To eliminate malignant B cells from these T cell-expanded cultures, haCD16A-BiTE (80 ng / well) and rituximab (10 μg / well) were added to the cultures on day 10. After 3 days, cultures were analyzed for malignant B cell killing by flow cytometry analysis of both the CD19 marker (a marker for malignant B cells) and propidium iodide (PI) staining.

[0093]

[0101] Figure 8 shows that CD19-positive cells are reduced following treatment with haCD16A-BiTE and rituximab, demonstrating the ability of haCD16A-BiTE in combination with rituximab to eliminate malignant B cells from T cell expansion cultures. *: p<0.001 compared to haCD16A-BiTE or rituximab alone.

[0094] Example 6 Ability of haCD16A-BiTE to mediate killing of EBV-infected B cells in the presence of anti-EBV IgG antibodies and T cells.

[0102] To establish the utility of haCD16A-BiTE in treating viral diseases, we used an EBV-infected B cell line as a model virus-infected cell line and tested the ability of haCD16A-BiTE to mediate killing of EBV-infected B cells in the presence of anti-EBV IgG antibodies and T cells.

[0095]

[0103] EBV is a gammaherpesvirus that infects 90% of the population. EBV establishes lifelong latency in memory B cells and oral epithelial cells. In immunocompetent hosts, circulating EBV-specific cytotoxic T lymphocytes maintain EBV-infected B cells at levels below 1% of the B cell pool. However, in immunosuppressed hosts, uncontrolled proliferation of EBV-infected B cells contributes to lymphoproliferative disorders after solid organ transplantation or hematopoietic stem cell transplantation, such as post-transplant lymphoproliferative disease (PTLD). Furthermore, a significant proportion of Hodgkin's lymphoma, non-Hodgkin's lymphoma, and nasopharyngeal carcinoma are associated with EBV infection. Treatment options for EBV-associated malignancies beyond standard chemotherapy and radiation therapy are limited. One promising therapy is adoptive cell therapy using EBV-specific T cells, which has shown efficacy against PTLD (success in 70% of cases). A more ideal therapeutic agent would be an antibody that can specifically recognize one of the more widely expressed antigens: latent membrane protein-1 (LMP1) or LMP2 (Ahmed et al., JCI Insight, 3, e97805, (2018)).

[0096]

[0104] Immortalized EBV-infected B cells expressing LMP1 were used as target cells, and 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) (Creative Biolab) was used instead.

[0097]

[0105] Figure 9 demonstrates that in the presence of T cells, haCD16A-BiTE, when combined with anti-LMP1 IgG antibody, exhibits synergistic effects in killing EBV-infected B cells compared to using haCD16A-BiTE or anti-LMP1 antibody alone. *: p<0.001 compared to haCD16A-BiTE or anti-LMP1 antibody alone.

[0098] Example 7 In vitro activity assay of haCD16A-BiTEs in mediating killing of PD-L1-expressing tumor cells.

[0106] Overexpression of PD-L1 allows tumor cells to exploit the PD-1 / PD-L1 pathway, promoting an immunosuppressive environment and promoting tumor growth (Topalian et al., Curr Opin Immunol, 24, 207-212 (2012)). Therefore, PD-L1 is also a tumor-associated antigen. Blockade of PD-L1 inhibitory signals by anti-PD-L1 IgG mAb not only restores the antitumor activity of T cells but also provides an opportunity to eradicate tumor cells via ADCC.

[0099]

[0107] To test the potential of haCD16A-BiTE to confer ADCC activity to T cells that mediates eradication of PD-L1-expressing tumor cells by anti-PD-L1 IgG mAb, we screened tumor cells for PD-L1 expression and performed the experiment exactly as described in Example 2, except for using a PD-L1-expressing tumor cell line (A431) and a human anti-PD-L1 IgG antibody (10 μg / well) (Invivogen) instead. In this experiment, A431 cells were used as target cells. The MCF-7 cell line was a PD-L1-low breast cancer cell line.

[0100]

[0108] The results shown in Figure 10 demonstrate that in the presence of T cells, haCD16A-BiTE in combination with anti-PD-L1 IgG antibody exhibited synergistic effects in killing PD-L1-expressing cells (A431) compared to either haCD16A-BiTE or anti-PD-L1 antibody alone. *: p<0.001 compared to haCD16A-BiTE or anti-PD-L1 antibody alone.

[0101] Example 8 In vivo assay of the activity of haCD16A-BiTE in killing CD20-expressing tumor cells in an immunodeficient NOD mouse model.

[0109] Our invention, which is effective in enhancing the efficacy of therapeutic antibodies, requires the formation of a complex consisting of four components: tumor cells, antibodies, haCD16A-BiTEs, and T cells. In addition to demonstrating the in vivo activity of haCD16A-BiTEs, we performed in vivo proof-of-concept studies to demonstrate that this complex formation occurs in vivo.

[0102]

[0110] These experiments were performed using T cell-deficient, immunodeficient NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl This study was performed in rhesus macaque / YckNarl (RMRC 13288) mice. First, luciferase and green fluorescent protein genes were transduced into Raji hematopoietic tumor cells using lentiviral vectors (Zhou et al., Blood, 120, 4334-4342 (2012)). Raji cells (1 x 10) expressing luciferase and green fluorescent protein were cultured in vitro. 6 (cells) were transferred into NOD mice via tail vein injection. Seven days after Raji cell transfer, T cells obtained as described in Example 2 were transferred into NOD mice via tail vein injection. The amount of T cells transferred per injection was 10 times the amount of Raji cells. T cells were transferred once every four days for a total of two transfers. Additionally, seven days after Raji cell transfer, haCD16A-BiTE was infused via the tail vein at a daily dose of 800 ng for nine consecutive days, delivered by a single bolus injection. Rituximab (10 mg / kg) was administered twice via the tail vein on days 7 and 11 after Raji cell transfer. Mice in the control group received only T cell transfer.

[0103]

[0111] Bioluminescence imaging (BLI) was performed at various time points to monitor the clearance of Raji cells. At the end of the experiment, the body weights of the treatment groups were comparable to those of the controls. The results in Figure 11 show the difference in BLI at the end of the experiment. Compared to mice treated with rituximab and T cells (without haCD16A-BiTE), haCD16A-BiTE significantly enhanced Raji cell clearance by 2-fold. Thus, tumor cells, antibodies, haCD16A-BiTE, and T cells can be combined together when activity is required in vivo. More importantly, these data demonstrate the in vivo activity of haCD16A-BiTE. *: p<0.01 compared to the combination of haCD16A-BiTE, rituximab, and T cells.

[0104] Example 9 AAV-haCD16A-BiTE mediates peripheral expression of haCD16A-BiTE in mice.

[0112] To overcome the short in vivo half-life that necessitates continuous infusion of BiTEs in a therapeutic setting, we investigated AAV-mediated gene transfer methods.

[0105]

[0113] Mice (B6 strain, male, 8 weeks old; n = 5) were intraperitoneally injected with the AAV-haCD16A-BiTE viral vector (Figure 1A, 10 9 (vgc / animal; vgc: viral genome copies). Serum samples were collected from the tail vein before (day 0) and after (days 2, 7, and 14) virus injection and subjected to Western blot analysis to assess haCD16A-BiTE protein levels. An HRP-conjugated polyclonal antibody was used to probe the c-terminal 6x histidine tag of haCD16A-BiTE. The levels of detected protein were measured by densitometry analysis and expressed as optical density values ​​per ml of serum.

[0106]

[0114] The results in Figure 12 show that 7 days after injection of AAV-haCD16A-BiTE, haCD16A-BiTE was detected in the peripheral blood of mice, and its presence persisted for at least 7 days. These results demonstrate that sustained production of haCD16A-BiTE in vivo can be achieved by in vivo viral-mediated haCD16A-BiTE gene transfer.

[0107]

[0115] While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations thereto will be apparent to those skilled in the art. All such alternatives, modifications, and variations are intended to be within the scope of the present invention.

Claims

1. The extracellular domain of human CD16A having the amino acid sequence of SEQ ID NO:2; and An anti-human CD3 single-chain variable fragment that specifically binds to human CD3 and activates human T cells, the anti-human CD3 single-chain variable fragment having the amino acid sequence of SEQ ID NO:

4. wherein the C-terminal residue of SEQ ID NO:2 is fused directly to the N-terminal residue of SEQ ID NO:

4.

2. 2. The fusion protein of claim 1, further comprising a protein purification tag, wherein the protein purification tag is a 6x histidine tag.

3. A polynucleotide encoding the fusion protein of claim 1 or 2.

4. A polynucleotide described in claim 3, which comprises a fragment encoding the extracellular domain of human CD16A and has the nucleic acid sequence of SEQ ID NO: 1, and which comprises a fragment encoding the anti-human CD3 single-chain variable fragment and has the nucleic acid sequence of SEQ ID NO:

3.

5. 5. The polynucleotide of claim 3 or 4, further comprising a fragment encoding a secretory signal peptide and having the nucleic acid sequence of SEQ ID NO:

5.

6. The polynucleotide according to any one of claims 3 to 5, comprising the nucleic acid sequence of SEQ ID NO:

7.

7. The polynucleotide according to any one of claims 3 to 6, which is contained in an adeno-associated virus vector.

8. A host cell comprising the polynucleotide of any one of claims 3 to 7.

9. A pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of claim 1 or 2.

10. 10. The pharmaceutical composition of claim 9, further comprising one or more antibodies having an Fc region.

11. 11. The pharmaceutical composition of claim 10 for use in inducing antibody-dependent cellular cytotoxicity in a subject.

12. 12. The pharmaceutical composition of any one of claims 9 to 11 for use in treating cancer, infectious diseases, or post-transplant lymphoproliferative disorders in a subject.

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