Tetrahedral constructs binding folate and CD3

Abstract

The present invention is directed to compositions of novel, tetrahedral antibody constructs comprising one or more FOLRα antigen binding domains and one or more CD3 antigen binding domains, as well as methods of making and using such compositions

Description

Attorney Docket: 53080.4001 / WOTETRAHEDRAL CONSTRUCTS BINDING FOLATE AND CD3 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 720,933, filed November 15, 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 XML format and is hereby incorporated by reference in its entirety. Said ST.26 XML format file was created on October 21, 2025, is named 53080_4001-WO_SL.xml and is 34,212 bytes in size.BACKGROUND OF THE INVENTION

[0003] Antibodies are a diverse family of vertebrate proteins comprising a Y-shaped structure characterized by the presence of two antigen binding (Fab) domains and one effector cell binding (Fc) domain. The arrangement of these three domains around a central hinge region bears a striking resemblance to trigonal molecular geometries in which a central atom is bonded to three peripheral atoms arranged at the comers of a triangle. Such a planar configuration of binding domains is sufficient for antibodies to carry out their normal functions. As such, efforts to date to engineer antibodies and antibody-like molecules have also generally adopted the natural planar configuration of antibody binding domains. However, when the binding domains of an engineered antibody and antibody-like molecules are intended to engage multiple targets, a planar configuration of binding domains is not ideally suited to permit the simultaneous engaging of multiple targets by the binding domains.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, inAttorney Docket: 53080.4001 / WOwhich the principles of the invention are utilized, and the accompanying drawings (also “Fig.”, “FIG.”, “Figure”, “Figures”, “Figs ”, and “FIGs .” herein) of which:

[0005] Fig. 1 shows a schematic overview of the GEM-DIMER™ technology used to create candidate tetraval ent, bi specific antibodies with or without dual enhanced Fc domains using a bivalent, monospecific anti-[target antigen 1] targeting antibody (± S239D / I332E Fc domain mutations) and a bivalent, monospecific anti-[target antigen 2] targeting antibody (± S239D / I332E Fc domain mutations). As will be appreciated by those skilled in the relevant art, the GEM-DIMER™ technology can be used to generate candidate multivalent, multispecific antibodies with varying specificities and / or valences, depending on the parental antibodies used to generate the candidate antibody, such as, for example, antibodies that are trispecific and hexavalent, antibodies that are trispecific and octavalent, and the like.

[0006] Fig. 2 (Figs. 2A-2E) shows a schematic depiction of an exemplary multivalent, multispecific T-cell engager targeting a-FOLRa (folate receptor 1) and a-CD3 (“HB3009”) and the parental 2 + 1 Fab-formatted bispecific antibody (“HB3001”), as well their capacity to induce cytotoxicity and bind target cells expressing FOLRa. Fig. 2A shows a schematic depiction of a 2 + 1 Fab-formatted antibody that is bivalent for binding to a-FOLRa and monovalent for binding to a-CD3 and a schematic depiction of the resultant multispecific T-cell engager, HB3009, generated using the GEM-DEMER™ technology. Fig. 2B shows binding of test articles to FOLRa-expressing IGROV-1 target cells. Fig. 2C shows the results of an apoptosis assay in response to test articles using IGROV-1 ovarian cancer cells (i.e., FOLRa+target cells) and primary human T cells (10:1 effectortarget (E:T) ratio). Apoptotic cells were measured kinetically via caspase 3 / 7 activity over 48 hours in Incucyte. Fig. 2D shows changes in T-cell activation in response to test articles. T-cell activation was analyzed by assessing for changes in CD69 expression of CD3 + cells (left panel) and soluble IFNy (right panel) using flow cytometry and MSD. Fig. 2E shows representative Incucyte images (24 h - first panel; 48 h - second panel; 72 h - third panel; 96 h - fourth panel) of FOLRa (top row) and caspase 3 / 7 expression (bottom row) in IGROV-1 cells in response to test articles.

[0007] Fig. 3 depicts the amino acid sequences for human, mouse, and cynomolgus monkey CD3s, as well as the amino acid sequences for the extracellular domain (ECD) of human / mouse / cynomolgus CD3e.Attorney Docket: 53080.4001 / WO

[0008] Fig. 4 depicts the amino acid sequences for human, mouse, and cynomolgus monkey folate receptor 1.

[0009] Fig. 5 depicts the amino acid sequences for HB3009 and select domains thereof.

[0010] Fig. 6 depicts the amino acid sequences of exemplary peptide linkers.DETAILED DESCRIPTION OF THE INVENTION

[0011] The description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. The section headings used herein are for organization purposes only and are not to be construed as limiting the subject matter described. While various embodiments of the invention(s) of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention(s). It should be understood that various alternatives to the embodiments of the invention(s) described herein may be employed in practicing any one of the inventions(s) set forth herein.

[0012] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.I. Definitions

[0013] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For purposes of the present disclosure, the following terms are defined below. The definitions provided are intended to apply to a given term, as well as other derivative linguistic re-phrasings and grammatical equivalents of the term.

[0014] As used herein, the terms “CD3” or “cluster of differentiation 3,” which can be used interchangeably, is used generally to refer to a protein complex and T-cell co-receptor involved in, inter alia, activation of cytotoxic T-cells, T-helper cells, and others known in the relevant art. The CD3 complex includes a CD3gamma (CD3y) chain, a CD3delta (CD38) chain, and two CD3epsilon (CD3s) chains. Additional information regarding CD3gamma, including amino acid sequences thereof, can be found in, for example, UniProt No. P09693, and GenBank AccessionAttorney Docket: 53080.4001 / WONumbers X04145.1, the contents of which are incorporated by reference in their entirety.Additional information regarding CD3delta, including amino acid sequences thereof, can be found in, for example, UniProt No. P04234, and GenBank Accession Numbers X03934.1, the contents of which are incorporated by reference in their entirety. Additional information regarding CD3 epsilon, including amino acid sequences thereof, can be found in, for example, UniProt No. P07766, and GenBank Accession Numbers X03884.1, the contents of which are incorporated by reference in their entirety. Further, exemplary CD3 sequences are depicted in Fig. 3. Unless otherwise noted, references to CD3 are to the human CD3s sequences.

[0015] As used herein, the terms “Folate Receptor 1,” “Folate Receptor Alpha,” “FOLR1,” “FOLRa,” or “FRa,” which can be used interchangeably, is used generally to refer to a receptor responsible for binding to folic acid and its derivatives. FOLR1 is known to be overexpressed in various cancers, such as, for example, ovarian cancer, breast cancer, renal cancer, lung cancer, colorectal cancer, and various oncological malignancies associated with the brain, and others. As such, FOLR1 can be used as a target of an anti-cancer therapy. Additional information regarding Folate Receptor 1, including amino acid sequences thereof, can be found in, for example, UniProt No. P15328, and GenBank Accession Numbers J05013.1, the contents of which are incorporated by reference in their entirety. Further, exemplary FOLR1 sequences are depicted in Fig. 4. Unless otherwise noted, references to FOLR1 are to human FOLR1.

[0016] As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, and antibody fragments so long as they exhibit the desired biological activity (e.g., Fab and / or single-armed antibodies). The “class” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, and antibody fragments so long as they exhibit the desired biological activity (e.g., Fab and / or single-armed antibodies).

[0017] As used herein, the term “antibody fragment” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constantAttorney Docket: 53080.4001 / WOdomains that correspond to the different classes of immunoglobulins are called a, 8, s, y, and p, respectively.

[0018] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0019] A “blocking” antibody or an “antagonist” antibody is one which significantly inhibits (either partially or completely) a biological activity of the antigen it binds.

[0020] An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.

[0021] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0022] The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), SO-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987).) Exemplary CDRs (CDR-L1 (“vlCDRl”), CDR-L2 (“vlCDR2”), CDR-L3 (“vlCDR3”), CDR-H1 (“vhCDRl”), CDR-H2 (“vhCDR2”), and CDR-H3 (“vhCDR3”)) occur atAttorney Docket: 53080.4001 / WOamino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of Hl, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of Hl, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0023] As used herein, “framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0024] The phrase “N-terminally truncated heavy chain”, as used herein, refers to a polypeptide comprising parts but not all of a full-length immunoglobulin heavy chain, wherein the missing parts are those normally located on the N terminal region of the heavy chain. Missing parts may include, but are not limited to, the variable domain, CHI, and part or all of a hinge sequence. Generally, if the wild type hinge sequence is not present, the remaining constant domain(s) in the N-terminally truncated heavy chain would comprise a component that is capable of linkage to another Fc sequence (i.e., the “first” Fc polypeptide as described herein). For example, said component can be a modified residue or an added cysteine residue capable of forming a disulfide linkage.

[0025] As used herein, “Fc receptor” or “FcR,” which can be used interchangeably, describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIlB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIAAttorney Docket: 53080.4001 / WOcontains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

[0026] The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0027] Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered.WO 2000 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0028] The “hinge region,” “hinge sequence”, and variations thereof, as used herein, includes the meaning known in the art, which is illustrated in, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

[0029] Unless indicated otherwise, the expression “multivalent antibody” is used throughout this specification to denote an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

[0030] An “Fv” fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in thisAttorney Docket: 53080.4001 / WOconfiguration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

[0031] The “Fab” fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0032] The phrase “antigen binding arm”, as used herein, refers to a component part of an antibody fragment that has an ability to specifically bind a target molecule of interest. Generally and preferably, the antigen binding arm is a complex of immunoglobulin polypeptide sequences, e.g., HVR and / or variable domain sequences of an immunoglobulin light and heavy chain.

[0033] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0034] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0035] The expression “linear antibodies” refers to the antibodies described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.Attorney Docket: 53080.4001 / WO

[0036] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0037] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0038] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0039] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.Attorney Docket: 53080.4001 / WO

[0040] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0041] ‘ ‘Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0042] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0043] An “affinity matured” antibody refers to an antibody with one or more alterations in one or more HVRs, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0044] An antibody having a “biological characteristic” of a designated antibody is one which possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies that bind to the same antigen.

[0045] A “functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same. For theAttorney Docket: 53080.4001 / WOmultimeric antibodies herein, the number of functional antigen binding sites can be evaluated using ultracentrifugation analysis as described in Example 2 of U.S. Patent Application Publication No. 2005 / 0186208 Al. According to this method of analysis, different ratios of target antigen to multimeric antibody are combined and the average molecular weight of the complexes is calculated assuming differing numbers of functional binding sites. These theoretical values are compared to the actual experimental values obtained in order to evaluate the number of functional binding sites.

[0046] A “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “binds specifically” to a human antigen (i.e. has a binding affinity (Kd) value of no more than about 1 x 107M, preferably no more than about 1 x ICT8M, and most preferably no more than about 1 x 109 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above. In some embodiments, the species-dependent antibody is a humanized or human antibody.

[0047] An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0048] The term “Fc domain”, as used herein, generally refers to a monomer or dimer complex, comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain. The Fc domain may comprise native or variant Fc sequences. Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc domain is usually defined to stretch from an amino acid residue in the hinge region to the carboxyl terminus of the Fc sequence. The Fc sequence of an immunoglobulin generally comprises two constant regions, a CH2 region and a CH3 region, and optionally comprises a CH4 region. A human Fc domainAttorney Docket: 53080.4001 / WOmay be obtained from any suitable immunoglobulin, such as the TgGl, IgG2, TgG3, or TgG4 subtypes, IgA, IgE, IgD or IgM.

[0049] As used herein, the singular forms “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antigen” includes mixtures of antigens; reference to “a pharmaceutically acceptable carrier” includes mixtures of two or more such carriers, and the like. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0050] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A (alone)”, and “B (alone)”.

[0051] As used herein, the term “about” a value (or parameter) refers to ±10% of a stated value. When referring to a range of values (or parameters), the term “about” refers to +10% of the upper limit and -10% of the lower limit of a stated range of values. When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the present disclosure. Where the stated range includes upper and / or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

[0052] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.Attorney Docket: 53080.4001 / WOTI. Overview

[0053] The present disclosure provides several aspects and embodiments comprising tetrahedral antibody constructs comprising one or more FOLRa ABDs and one or more CD3 ABDs.III. Tetrahedral Antibody Constructs

[0054] The present disclosure provides several aspects and embodiments comprising tetrahedral antibody constructs (sometimes referred to herein as “tetrahedral antibodies” or “tetrahedral T-cell engagers” for the sake of brevity). The tetrahedral antibodies described herein generally comprise: (a) one or more instances of a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus, a first variable heavy (VH1) domain, a first constant heavy (CHIA) domain, a dimerization domain, a first (optional) linker, and a first Fc domain; (b) one or more instances of a second monomer, wherein the second monomer comprises, from N-terminus to C-terminus: a second variable heavy (VH2) domain, a second constant heavy (CHIB) domain, and a second Fc domain; (c) one or more instances of a third monomer, wherein the third monomer comprises, from N-terminus to C-terminus: a first variable light (VL1) domain and a constant light (CL) domain, wherein the first variable heavy (VH1) domain and the first variable light (VL1) domain form a first antigen binding domain (ABD); and (d) one or more instances of a fourth monomer, wherein the fourth monomer comprises, from N-terminus to C-terminus: a second variable light (VL2) domain and a constant light (CL) domain, wherein the second variably heavy (VH2) domain and the second variable light (VL2) domain form a second ABD, and wherein: (1) the first ABD comprises a FOLRa ABD and the second ABD comprises a CD3 ABD, or (2) the first ABD comprises a CD3 ABD and the second ABD comprises a FOLRa ABD. Such tetrahedral antibodies are capable of specifically binding to both: (i) FOLRa-expressing target cells (such as, for example, FOLRa+cancer cells), and (ii) CD3 -expressing target cells (such as, for example, CD3+T-cells), effectuating the recruitment / activation of T-cells and cellular killing of FOLRa+target cells.

[0055] As will be appreciated by those skilled in the art, a subject tetrahedral antibody can take on one of several different “formats” of different valence and / or specificity depending on the parental antibodies used to generate a given tetrahedral antibody. Numerous antibody formats are known in the relevant art, such as, for example, those described in U.S. Patent ApplicationAttorney Docket: 53080.4001 / WOPublication Nos. 2024 / 0158526, 2023 / 0220116, and 2022 / 0289839, the contents of which are incorporated by reference in their entirety, with particularity for descriptions of antibody formats, as well as any figures and amino acid sequences related thereto. As such, in some instances, an antigen binding domain of a given tetrahedral may be configured as a “Fab” (comprising a VH domain, a VL domain, a CHI domain, and a CL domain), a “scFv” (comprising a VH domain, a linker, and a VL domain), a linear antibody, a diabody, or other formats known in the relevant art.

[0056] Due to the modular nature of the tetrahedral antibody’s configuration, a discussion of exemplary components comprising a subject tetrahedral antibody is first provided below (such as, for example, ABDs, Fc domains, dimerization domains, peptide linkers, VH domains, VL domains, etc.), followed by a discussion of exemplary tetrahedral antibody constructs finding utility in some of the embodiments described herein.A. Antigen Binding Domains

[0057] The present disclosure provides anti-FOLRa x anti-CD3 (also referred to herein as “aFOLRa x aCD3,” “anti-CD3 x anti-FOLRa,” “aCD3 x aFOLRa,” “anti-FOLRl x anti-CD3,” “aFOLRl xaCD3,” “anti-CD3 x anti-FOLRl,” and “aCD3 x aFOLRl,” which can all be used interchangeably) tetrahedral antibodies. Such antibodies include at least one FOLRa binding domain (in order to specifically bind to a FOLRa-expressing cell, such as, for example, a FOLRa+cancer cell) and at least one CD3 binding domain (in order to specifically bind to a CD3 -expressing cell, such as, for example, a CD3+T-cell).

[0058] As is more fully outlined herein, these combinations of antigen binding domains (ABDs) can be in a variety of formats (such as, for example, a Fab format, a scFv format, etc.). Further, it is also contemplated that when a subject tetrahedral antibody is multivalent for a given target antigen (such as, for example, FOLR1 or CD3), the individual binding arms that are specific for the given target antigen may be configured in the same format, in different formats, or, for instances where the valence is > 3 for a given target antigen, a combination thereof.

[0059] In some instances, an ABD is characterized using a pair of variable heavy (VH) and variable light (VL) domains (“VH / VL domain pair(s)” or “VH / VL pair(s)”). Alternatively, or in addition to, an ABD can be characterized by a set of 6 CDRs (i.e., vhCDRl, vhCDR2, vhCDR3 (vhCDRl-3), vlCDRl, vlCDR2, and vlCDR3 (vlCDRl-3)). As will be appreciated by thoseAttorney Docket: 53080.4001 / WOskilled in the art, any set of 6 CDRs or VH / VL domain pairs can be in a Fab format or a scFv format, which can then be added to heavy and light constant domains. Such heavy and light constant domains may comprise one or more amino acid substitutions (including, but not limited to, within the CHI domain, as well as the Fc domain(s)).

[0060] For all of the variable heavy and light domains listed herein, further variants can be made. The set of 6 CDRs can have 0, 1, 2, 3, 4, or 5 amino acid modifications (with amino acid substitutions finding particular use in several embodiments), as well as changes in the framework regions of the variable heavy and light domains, as long as the resultant ABD retains specific binding for its target antigen. Further included are ABDs that comprise at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity to any of the ABDs described herein so long as they retain their specific binding for their target antigen (e.g., when the target antigen comprises CD3, an ABD may comprise at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity to a given VH / VL pair described further herein, so long as the ABD comprising the variant VH / VL pair retains specific binding for CD3).

[0061] Binding affinity of a given ABD to its target antigen can be assessed using any number of assays known to those skilled in the relevant art. Non-limiting examples of suitable assays for evaluating binding affinity include: a surface plasmon resonance (SPR) assay, an enzyme-linked immunosorbent assay (ELISA), an ELISpot assay, Biacore assays, KinExA assays, flow cytometry, and the like.1. Folate Receptor 1 :

[0062] Provided herein are tetrahedral antibodies that contain one or more ABDs the bind specifically to FOLRa. In some embodiments, the ABD binds human FOLRa, whereas in other embodiments, the ABD binds mouse FOLRa or cy nomol gus monkey FOLRa. Suitable variable heavy (VH) and variable light (VL) domains that bind human FOLRa are depicted in Fig. 5.

[0063] In some embodiments, the VH domain of the FOLRa ABD is selected from a group including: (i) SEQ ID NO: 13, as shown in Fig. 5. In some embodiments, the VH domain of the FOLRa ABD comprises an amino acid sequence with at least about 90%, about 91%, aboutAttorney Docket: 53080.4001 / WO92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 13.

[0064] In some embodiments, the VH domain of the FOLRa ABD has a set of vhCDRs selected from the vhCDRl, vhCDR2, and vhCDR3 sequences of a VH domain described herein.

[0065] In some embodiments, the VL domain of the FOLRa ABD is selected from a group including: (i) SEQ ID NO: 14, as shown in Fig. 5. In some embodiments, the VL domain of the FOLRa ABD comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.

[0066] In some embodiments, the VL domain of the FOLRa ABD has a set of vlCDRs selected from the vlCDRl, vlCDR2, and vlCDR3 sequences of a VL domain described herein.

[0067] Additional VH / VL domain pairs and sets of 6 CDR sequences capable of specifically binding (human) FOLRa are known in the relevant art. Such VH / VL domain pairs and / or sets of 6 CDR may be optionally and / or independently incorporated into a subject tetrahedral antibody.

[0068] Accordingly, included herein are FOLRa ABDs that have VH / VL pairs selected from a group including: (i) SEQ ID NOs: 13 and 14, respectively, as shown in Fig. 5. In some embodiments, the VH / VL pairs are used in Fabs, whereas in other embodiments, the VH / VL pairs are used in scFvs.

[0069] In some embodiments, a subject tetrahedral antibody includes a FOLRa ABD that includes a variable heavy domain and / or a variable light domain that are variants of a FOLRa ABD VH and / or VL domain disclosed herein. In one embodiment, the variant VH domain and / or the variant VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from a VH domain and / or a VL domain described herein. In an exemplary embodiment, the variant VH domain and / or variant VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from a VH and / or VL domain of one of the following FOLRa binding domain VH / VL pairs: (i) SEQ ID NOs: 13 and 14, respectively. In some embodiments, the changes are in a VH domain described herein. In some embodiments, the changes are in a VL domain described herein. In some embodiments, the changes are in a VH domain and a VL domain described herein. In some embodiments, one or more amino acid changes are in one or more framework regions (FR1, FR2, FR3, and / or FR4) of a VH domain and / or a VL domain described herein. In some embodiments, one or more amino acid changes are in one or more CDRs (vhCDRl, vhCDR2,Attorney Docket: 53080.4001 / WOvhCDR3, vlCDRl , vlCDR3, and / or vlCDR3) of a VH domain and / or a VL domain described herein. In certain embodiments, the (variant) FOLRa ABD of a subject tetrahedral antibody is capable of binding to FOLRa as measured by at least one of: (i) a Biacore assay, (ii) a surface plasmon resonance (SPR) assay, (iii) a biolayer interferometry (BLI) assay (e.g., an Octet assay), (iv) flow cytometry, or any combination thereof. In particular embodiments, the (variant) FOLRa ABD is capable of specifically binding a human FOLRa antigen (see, e.g., Fig. 4; SEQ ID NO: 7).

[0070] In some embodiments, a subject tetrahedral antibody includes a FOLRa ABD comprising a variant VH domain and / or variant VL domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VH domain and / or a VL domain of a FOLRa ABD described herein. In exemplary embodiments, the variant VH domain and / or variant VL domain is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the VH domain and / or VL domain of one of the following FOLRa binding domain VH / VL pairs: (i) SEQ ID NOs: 13 and 14, respectively, as depicted in Fig. 5. In some embodiments, the variant FOLRa ABD includes a variant VH domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VH domain described herein. In some embodiments, the variant FOLRa ABD includes a variant VL domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VL domain described herein. In certain embodiments, the variant FOLRa ABD of a subject tetrahedral antibody is capable of binding to FOLRa, as measured by at least one of: (i) a Biacore assay, (ii) a surface plasmon resonance (SPR) assay, (iii) a biolayer interferometry (BLI) assay (e.g., an Octet assay), (iv) flow cytometry, or any combination thereof. In particular embodiments, the variant FOLRa ABD is capable of binding a human FOLRa antigen (see, e.g., Fig. 4; SEQ ID NO: 7).2. CD3 Epsilon:

[0071] Provided herein are tetrahedral antibodies that contain one or more ABDs the bind specifically to CD3. In some embodiments, the ABD binds human CD3, whereas in otherAttorney Docket: 53080.4001 / WOembodiments, the ABD binds mouse CD3 or cynomolgus monkey CD3. Suitable variable heavy (VH) and variable light (VL) domains that bind human CD3 are depicted in Fig. 5.

[0072] In some embodiments, the VH domain of the CD3 ABD is selected from a group including: (i) SEQ ID NO: 15, as shown in Fig. 5. In some embodiments, the VH domain of the CD3 ABD comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 15.

[0073] In some embodiments, the VH domain of the CD3 ABD has a set of vhCDRs selected from the vhCDRl, vhCDR2, and vhCDR3 sequences of a VH domain described herein.

[0074] In some embodiments, the VL domain of the CD3 ABD is selected from a group including: (i) SEQ ID NO: 14, as shown in Fig. 5. In some embodiments, the VL domain of the CD3 ABD comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.

[0075] In some embodiments, the VL domain of the CD3 ABD has a set of vlCDRs selected from the vlCDRl, vlCDR2, and vlCDR3 sequences of a VL domain described herein.

[0076] Additional VH / VL domain pairs and sets of 6 CDR sequences capable of specifically binding (human) CD3 are known in the relevant art. Such VH / VL domain pairs and / or sets of 6 CDR may be optionally and / or independently incorporated into a subject tetrahedral antibody.

[0077] Accordingly, included herein are CD3 ABDs that have VH / VL pairs selected from a group including: (i) SEQ ID NOs: 15 and 14, respectively, as shown in Fig. 5. In some embodiments, the VH / VL pairs are used in Fabs, whereas in other embodiments, the VH / VL pairs are used in scFvs.

[0078] In some embodiments, a subject tetrahedral antibody includes a CD3 ABD that includes a variable heavy domain and / or a variable light domain that are variants of a CD3 ABD VH and / or VL domain disclosed herein. In one embodiment, the variant VH domain and / or the variant VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from a VH domain and / or a VL domain described herein. In an exemplary embodiment, the variant VH domain and / or variant VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from a VH and / or VL domain of one of the following CD3 binding domain VH / VL pairs: (i) SEQ ID NOs: 15 and 14, respectively. In some embodiments, the changes are in a VH domain described herein. InAttorney Docket: 53080.4001 / WOsome embodiments, the changes are in a VL domain described herein. In some embodiments, the changes are in a VH domain and a VL domain described herein. In some embodiments, one or more amino acid changes are in one or more framework regions (FR1, FR2, FR3, and / or FR4) of a VH domain and / or a VL domain described herein. In some embodiments, one or more amino acid changes are in one or more CDRs (vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR3, and / or vlCDR3) of a VH domain and / or a VL domain described herein. In certain embodiments, the (variant) CD3 ABD of a subject tetrahedral antibody is capable of binding to CD3 as measured by at least one of: (i) a Biacore assay, (ii) a surface plasmon resonance (SPR) assay, (iii) a biolayer interferometry (BLI) assay (e.g., an Octet assay), (iv) flow cytometry, or any combination thereof. In particular embodiments, the (variant) CD3 ABD is capable of specifically binding a human CD3 antigen (see, e g., Fig. 3; SEQ ID NO: 1). In some exemplary embodiments, the (variant) CD3 ABD is capable of specifically binding to the extracellular domain (ECD) of a human CD3 antigen (see, e.g., Fig. 3; SEQ ID NO: 2).

[0079] In some embodiments, a subject tetrahedral antibody includes a CD3 ABD comprising a variant VH domain and / or variant VL domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VH domain and / or a VL domain of a CD3 ABD described herein. In exemplary embodiments, the variant VH domain and / or variant VL domain is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the VH domain and / or VL domain of one of the following CD3 binding domain VH / VL pairs: (i) SEQ ID NOs: 15 and 14, respectively, as depicted in Fig.5. In some embodiments, the variant CD3 ABD includes a variant VH domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VH domain described herein. In some embodiments, the variant CD3 ABD includes a variant VL domain that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a VL domain described herein. In certain embodiments, the variant CD3 ABD of a subject tetrahedral antibody is capable of binding to CD3, as measured by at least one of: (i) a Biacore assay, (ii) a surface plasmon resonance (SPR) assay, (iii) a biolayer interferometry (BLI) assay (e.g., an Octet assay), (iv) flow cytometry, or any combination thereof. In particular embodiments, the variant CD3 ABD is capable of binding aAttorney Docket: 53080.4001 / WOhuman CD3 antigen (see, e.g., Fig. 3; SEQ ID NO: 1). In some exemplary embodiments, the (variant) CD3 ABD is capable of specifically binding to the extracellular domain (ECD) of a human CD3 antigen (see, e.g., Fig. 3; SEQ ID NO: 2).B. Fc Domains

[0080] The present disclosure provides anti-FOLRa x anti-CD3 tetrahedral antibodies. Such antibodies include a first Fc domain and a second Fc domain.

[0081] In some embodiments, the first Fc domain and / or the second Fc domain comprise the CH2-CH3 domains of human immunoglobulin G1 (IgGl), and optionally all or part of the hinge region of human IgGl, as well as fragments thereof. In EU numbering, the CH2-CH3 domain for human IgGl comprises amino acids 231 to 447, and the hinge comprises amino acids 216 to 230. A“Fc fragment” may contain one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids removed from either or both of the N- and C-termini, but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods (such as, for example, non-denaturing chromatography, size exclusion chromatograph, and the like). Nonlimiting examples of Fc domains, as well as variants thereof, are described in U.S. Patent Application Publication Nos. 2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity for their disclosure pertaining to (variant) Fc domains, as well as amino acid sequences thereof described in the figures and the sequence listing.

[0082] In some embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some embodiments, the first Fc domain comprises a first variant Fc domain and the second Fc domain comprises a second variant Fc domain. In some other embodiments, the first Fc domain is a variant Fc domain, and the second Fc domain is not a variant Fc domain, or the reverse where the second Fc domain is a variant Fc domain, and the first Fc domain is not a variant Fc domain.

[0083] In some embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhance FcyR binding activity. In some further embodiments, the one or more amino acid substitutions that enhance FcyR binding activity comprise: 236A, S239D, S239E, I332E, I332D, S239D / I332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243 A, 243L, 264A, 264V or 299T. In an exemplary embodiment, the one orAttorney Docket: 53080.4001 / WOmore amino acid substitutions that enhance FcyR binding activity comprise S239D / I332E.Additional amino acid substitutions that enhance FcyR binding activity finding utility in some of the embodiments described herein are described in, for example, U.S. Patent Application Publication Nos. 2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity for their disclosure pertaining to Fc variants that enhance FcyR binding activity of an antibody.

[0084] In some embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhance FcRn activity and / or half-life of the tetrahedral antibody. In some further embodiments, the one or more amino acid substitutions that enhance FcRn activity and / or half-life of the tetrahedral antibody comprise: (i) M252Y / S254T / T256E (YTE), (ii) L309D / Q311H / N434S (DHS), or (iii) M428L / N434S (LS). Additional amino acid substitutions that enhance FcRn activity and / or half-life finding utility in some of the embodiments described herein are described in, for example, U.S. Patent Application Publication Nos. 2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity for their disclosure pertaining to Fc variants that enhance FcRn activity and / or half-life of an antibody.

[0085] In some embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain. In some further embodiments, the one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain comprise H435R / Y436F (RF). Additional amino acid substitutions that ablate a Protein A binding site of the variant Fc domain finding utility in some of the embodiments described herein are described in, for example, U.S. Patent Application Publication Nos.2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity for their disclosure pertaining to Fc variants that ablate a Protein A binding site of the variant Fc domain of an antibody.

[0086] In some embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that promote heterodimerization of the first (variant) Fc domain with the second (variant) Fc domain. Nonlimiting examples of heterodimerization promoting Fc variants finding utility in some of the embodiments described herein are described in, for example, U.S. Patent Application Publication No. 2022 / 0289839, incorporated by reference in its entirety.Attorney Docket: 53080.4001 / WO

[0087] In some embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate FcyR binding activity. In some further embodiments, the one or more amino acid substitutions that ablate FcyR binding activity comprise: (i) P329G / L234A / L235A (PGLALA), (ii) L234A / L235A (LALA), (iii) P331S / L234A / L235A (PSLALA), (iv) L234F / L235E / P331S (LFLEPS), or (v) L234F / L235E / P329G (LFLEPG). Additional amino acid substitutions that ablate FcyR binding activity finding utility in some of the embodiments described herein are described in, for example, U.S. Patent Application Publication Nos.2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity for their disclosure pertaining to Fc variants that ablate FcyR binding activity of an antibody.C. Dimerization Domains

[0088] The present disclosure provides anti-FOLRa x anti-CD3 tetrahedral antibodies. Such antibodies include one or more dimerization domains. The one or more dimerization domains may be present on one monomer of a subject tetrahedral antibody.

[0089] In some embodiments, the one or more dimerization domains comprise: (i) leucine zipper domain(s), (ii) collectrin-like domain(s), (iii) collectrin domain(s), or (iv) the amino acid sequence of SEQ ID NO: 16, as shown in Fig. 5. Nonlimiting examples of dimerization domains that find utility in some of the embodiments described herein are described in U.S. Patent Application Publication Nos. 2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated by reference, with particularity fortheir disclosure pertaining to dimerization domains, leucine zipper domains, collectrin-like domains, collectrin domains, and any amino acid sequences thereof.D. Peptide Linkers

[0090] The present disclosure provides anti-FOLRa x anti-CD3 tetrahedral antibodies. Such antibodies can include one or more peptide linkers. In some instances, the peptide linker comprises a peptide bond. In other instances, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 17-34. Nonlimiting examples of peptide linkers that find utility in some of the embodiments described herein are described in U.S. Patent Application Publication Nos. 2024 / 0158526 and 2023 / 0220116, the contents of which are incorporated byAttorney Docket: 53080.4001 / WOreference, with particularity fortheir disclosure pertaining to peptide linkers, as well as amino acid sequences thereof.E. Exemplary Embodiments

[0091] [Embodiment A] In one exemplary embodiment, the present disclosure provides a tetrahedral antibody, comprising: (a) one or more of a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: a first variable heavy (VH1) domain, a first constant heavy (CHIA) domain, a dimerization domain, a first (optional) linker, and a first Fc domain; (b) one or more of a second monomer, wherein the second monomer comprises, from N-terminus to C-terminus: a second variable heavy (VH2) domain, a second constant heavy (CHIB) domain, and a second Fc domain; (c) one or more of a third monomer, wherein the third monomer comprises, from N-terminus to C-terminus: a first variable light (VL1) domain and a constant light (CL) domain, wherein the first variable heavy (VH1) domain and the first variable light (VL1) domain form a FOLRa antigen binding domain (ABD); and (d) one or more of a fourth monomer, wherein the fourth monomer comprises, from N-terminus to C-terminus: a second variable light (VL2) domain and a constant light (CL) domain, wherein the second variable heavy (VH2) domain and the second variable light (VL2) domain form a CD3 antigen binding domain (ABD).

[0092] In some further exemplary embodiments, the first variable light (VL1) domain and the second variable light (VL2) domain are identical.

[0093] In some further exemplary embodiments, (i) the second monomer further comprises the first variable heavy (VH1) domain and the first constant heavy (CHIA) domain, or (ii) the first monomer further comprises the second variable heavy (VH2) domain and the second constant heavy (CHIB) domain. In some further embodiments of romanette (i), the second monomer comprises, from N-terminus to C-terminus, the first variable heavy (VH1) domain, the first constant heavy (CHIA) domain, a second (optional) linker, the second variable heavy (VH2) domain, the second constant heavy (CHIB) domain, and the second Fc domain. In some further embodiments of romanette (ii), the first monomer comprises, from N-terminus to C-terminus, the second variable heavy (VH2) domain, the second constant heavy (CHIB) domain, a second (optional) linker, the first variable heavy (VH1) domain, the first constant heavy (CHI A) domain, the dimerization domain, the first (optional) linker, and the first Fc domain. In still some furtherAttorney Docket: 53080.4001 / WOembodiments, the second linker comprises any one of amino acid sequences SEQ ID NOs: 18-34. In some more preferred embodiments, the second linker comprises the amino acid sequence of SEQ ID NO: 19 ((G4S)2).

[0094] In some further exemplary embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 17 (TSTSPTRSMAPGAVHLPQPVSTR).

[0095] In some further exemplary embodiments, (i) the first variable heavy (VH1) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 13; and (ii) the first variable light (VL1) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.

[0096] In some further exemplary embodiments, (i) the second variable heavy (VH2) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 15; and (ii) the second variable light (VL2) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.

[0097] In some further exemplary embodiments, the dimerization domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 16.

[0098] In some further exemplary embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some further exemplary embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhance FcyR binding activity. In still some further exemplary embodiments, the one or more amino acid substitutions that enhance FcyR binding activity comprise: 236A, S239D, S239E, I332E, I332D, S239D / I332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V or 299T. In an even more preferred embodiment, the one or more amino acid substitutions that enhance FcyR binding activity comprise S239D / I332E.

[0099] In some further exemplary embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some further exemplary embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhances FcRn activity and / orAttorney Docket: 53080.4001 / WOhalf-life of the tetrahedral antibody. In still some further exemplary embodiments, the one or more amino acid substitutions that enhances FcRn activity and / or half-life of the tetrahedral antibody comprise: (i) M252Y / S254T / T256E (YTE), (ii) L309D / Q311H / N434S (DHS), or (iii) M428L / N434S (LS).

[0100] In some further exemplary embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some further exemplary embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain. In still some further exemplary embodiments, the one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain comprise H435R / Y436F (RF).

[0101] In some further exemplary embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some further exemplary embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that promote heterodimerization of the first (variant) Fc domain with the second (variant) Fc domain. In still some further exemplary embodiments, the first Fc domain and the second Fc domain each comprise one or more amino acid substitutions that promote heterodimerization of the first (variant) Fc domain with the second (variant) Fc domain.

[0102] In some further exemplary embodiments, the first Fc domain and / or the second Fc domain comprises a variant Fc domain. In some further exemplary embodiments, the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate FcyR binding activity. In still some further exemplary embodiments, the one or more amino acid substitutions that ablate FcyR binding activity comprise: (i) P329G / L234A / L235A (PGLALA), (ii) L234A / L235A (LALA), (iii) P331S / L234A / L235A (PSLALA), (iv) L234F / L235E / P331S (LFLEPS), or(v) L234F / L235E / P329G (LFLEPG).

[0103] In another exemplary embodiment, a nucleic acid composition is provided, wherein the nucleic acid composition comprises one or more nucleic acids encoding the first monomer, the second monomer, the third monomer, and the fourth monomer of the tetrahedral antibody according to any one of the preceding exemplary embodiments.

[0104] In another exemplary embodiment, an expression vector is provided, wherein the expression vector comprises the one or more nucleic acids according to the preceding exemplary embodiment.Attorney Docket: 53080.4001 / WO

[0105] In another exemplary embodiment, a host cell is provided, wherein the host cell is transformed with the expression vector according to the preceding exemplary embodiment.

[0106] In another exemplary embodiment, a method of making a tetrahedral antibody is provided, the method comprising: (a) culturing the host cell according to the preceding exemplary embodiment under conditions wherein the tetrahedral antibody is expressed; and (b) recovering the tetrahedral antibody.

[0107] In another exemplary embodiment, a method of treating a FOLRa+cancer in a human subject is provided, the method comprising administering the tetrahedral antibody according to any of the preceding exemplary embodiments to a human subject in need thereof.

[0108] [Embodiment B] In one exemplary embodiment, the present disclosure provides a tetrahedral antibody, comprising: (a) one or more of a first monomer, wherein the first monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 10; (b) one or more of a second monomer, wherein the second monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 11; (c) two or more of a third monomer, wherein the third monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 12.

[0109] In some further exemplary embodiments, (i) the first monomer comprises SEQ ID NO: 10; (ii) the second monomer comprises SEQ ID NO: 11; and (iii) the third monomer comprises SEQ ID NO: 12.

[0110] In some further exemplary embodiments, (i) the first monomer consists of SEQ ID NO: 10; (ii) the second monomer consists of SEQ ID NO: 11; and (iii) the third monomer consists of SEQ ID NO: 12.[oni] In another exemplary embodiment, a nucleic acid composition is provided, wherein the nucleic acid composition comprises one or more nucleic acids encoding the first monomer, the second monomer, and the third monomer of the tetrahedral antibody according to any one of the preceding exemplary embodiments.Attorney Docket: 53080.4001 / WO

[0112] In another exemplary embodiment, an expression vector is provided, wherein the expression vector comprises the one or more nucleic acids according to the preceding exemplary embodiment.

[0113] In another exemplary embodiment, a host cell is provided, wherein the host cell is transformed with the expression vector according to the preceding exemplary embodiment.

[0114] In another exemplary embodiment, a method of making a tetrahedral antibody is provided, the method comprising: (a) culturing the host cell according to the preceding exemplary embodiment under conditions wherein the tetrahedral antibody is expressed; and (b) recovering the tetrahedral antibody.

[0115] In another exemplary embodiment, a method of treating a FOLRa+cancer in a human subject is provided, the method comprising administering the tetrahedral antibody according to any of the preceding exemplary embodiments to a human subject in need thereof.EXAMPLES

[0116] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.A. Example 1 - Tetravalent anti-FOLRa x anti-CD3 T-cell engagers demonstrate potent cytotoxicity and superior binding to FOLRa target cells.

[0117] A tetravalent anti-FOLRa x anti-CD3 T-cell engager (“HB3009”) was generated as is generally shown in Fig. 1 using a 2 + 1 Fab-formatted anti-FOLRa x anti-CD3 antibody (“HB3001” or “the parental antibody”) and is schematically depicted in Fig. 2A. Subsequently, the cytotoxic capacity and binding affinity of HB3009 compared to HB3001 was assessed.Briefly, IGROV-1 ovarian cancer cells (FOLRa+target cells) were incubated with test articles (HB3009 or HB3001) and primary human T-cells (effector cells) at an effector celktarget cell ratio (E:T) of 10: 1 for 48 h at 37°C. Apoptotic cells were measured kinetically via caspase 3 / 7 activity over 48 h in Incucyte. CD69 upregulation and soluble IFNy was analyzed by flow cytometry and MSD. HB3009 demonstrated potent cytotoxicity and superior binding to FOLRa target cells.

Claims

Attorney Docket: 53080.4001 / WOCLAIMSWHAT IS CLAIMED IS:

1. A tetrahedral antibody, comprising:(a) one or more of a first monomer, wherein:(i) the first monomer comprises, from N-terminus to C-terminus: a first variable heavy (VH1) domain, a first constant heavy (CHIA) domain, a dimerization domain, a first (optional) linker, and a first Fc domain, and(ii) the first variable heavy (VH1) domain and the first variable light (VL1) domain form a FOLRa antigen binding domain (ABD);(b) one or more of a second monomer, wherein:(i) the second monomer comprises, from N-terminus to C-terminus: a second variable heavy (VH2) domain, a second constant heavy (CH 1B) domain, and a second Fc domain, and(ii) the second variable heavy (VH2) domain and the second variable light (VL2) domain form a CD3 antigen binding domain (ABD);(c) one or more of a third monomer, wherein the third monomer comprises, from N- terminus to C-terminus: a first variable light (VL1) domain and a constant light (CL) domain; and(d) one or more of a fourth monomer, wherein the fourth monomer comprises, from N- terminus to C-terminus: a second variable light (VL2) domain and a constant light (CL) domain.

2. The tetrahedral antibody of claim 1, wherein the first variable light (VL1) domain and the second variable light (VL2) domain are identical.

3. The tetrahedral antibody according to claim 1 or 2, wherein:Attorney Docket: 53080.4001 / WO(i) the second monomer further comprises the first variable heavy (VH1) domain and the first constant heavy (CH 1A) domain, or(ii) the first monomer further comprises the second variable heavy (VH2) domain and the second constant heavy (CHIB) domain.

4. The tetrahedral antibody of claim 3, wherein the second monomer comprises, from N-terminus to C-terminus, the first variable heavy (VH1) domain, the first constant heavy (CHIA) domain, a second (optional) linker, the second variable heavy (VH2) domain, the second constant heavy (CHIB) domain, and the second Fc domain.

5. The tetrahedral antibody of claim 3, wherein the first monomer comprises, from N-terminus to C-terminus, the second variable heavy (VH2) domain, the second constant heavy (CHIB) domain, a second (optional) linker, the first variable heavy (VH1) domain, the first constant heavy (CHIA) domain, the dimerization domain, the first (optional) linker, and the first Fc domain.

6. The tetrahedral antibody according to claim 4 or 5, wherein the second linker comprises any one of amino acid sequences SEQ ID NOs: 18-34.

7. The tetrahedral antibody of claim 6, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 19 ((G4S)2).

8. The tetrahedral antibody according to any one of claims 1-7, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 17 (TSTSPTRSMAPGAVHLPQPVSTR).

9. The tetrahedral antibody according to any one of claims 1-8, wherein:(i) the first variable heavy (VH1) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 13; and(ii) the first variable light (VL1) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%>, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.Attorney Docket: 53080.4001 / WO10. The tetrahedral antibody according to any one of claims 1-9, wherein:(i) the second variable heavy (VH2) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 15; and(ii) the second variable light (VL2) domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 14.

11. The tetrahedral antibody according to any one of claims 1-10, wherein the dimerization domain comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 16.

12. The tetrahedral antibody according to any one of claims 1-11, wherein the first Fc domain and / or the second Fc domain comprises a variant Fc domain.

13. The tetrahedral antibody of claim 12, wherein the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhance FcyR binding activity.

14. The tetrahedral antibody of claim 13, wherein the one or more amino acid substitutions that enhance FcyR binding activity comprise: (i) 236A, (ii) S239D, (iii) S239E, (iv) I332E, (v) I332D, (vi) S239D / I332E, (vii) 267D, (viii) 267E, (ix) 328F, (x) 267E / 328F, (xi) 236A / 332E, (xii) 239D / 332E / 330Y, (xiii) 239D, (xiv) 332E / 330L, (xv) 243 A, (xvi) 243L, (xvii) 264A, (xviii) 264V, or (xix) 299T.

15. The tetrahedral antibody of claim 14, wherein the one or more amino acid substitutions that enhance FcyR binding activity comprise S239D / I332E.

16. The tetrahedral antibody according to any one of claims 12-15, wherein the variant Fc domain(s) comprise(s) one or more amino acid substitutions that enhances FcRn activity and / or half-life of the tetrahedral antibody.Attorney Docket: 53080.4001 / WO17. The tetrahedral antibody of claim 16, wherein the one or more amino acid substitutions that enhances FcRn activity and / or half-life of the tetrahedral antibody comprise: (i) M252Y / S254T / T256E (YTE), (ii) L309D / Q311H / N434S (DHS), or (iii) M428L / N434S (LS).

18. The tetrahedral antibody according to any one of claims 12-17, wherein the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain.

19. The tetrahedral antibody of claim 18, wherein the one or more amino acid substitutions that ablate a Protein A binding site of the variant Fc domain comprise H435R / Y436F (RF).

20. The tetrahedral antibody according to any one of claims 12-19, wherein the variant Fc domain(s) comprise(s) one or more amino acid substitutions that promote heterodimerization of the first (variant) Fc domain with the second (variant) Fc domain.

21. The tetrahedral antibody of claim 20, wherein the first Fc domain and the second Fc domain each comprise one or more amino acid substitutions that promote heterodimerization of the first (variant) Fc domain with the second (variant) Fc domain.

22. The tetrahedral antibody according to any one of claims 12-21, wherein the variant Fc domain(s) comprise(s) one or more amino acid substitutions that ablate FcyR binding activity.

23. The tetrahedral antibody of claim 22, wherein the one or more amino acid substitutions that ablate FcyR binding activity comprise: (i) P329G / L234A / L235A (PGLALA), (ii) L234A / L235A (LALA), (iii) P331 S / L234A / L235A (PSLALA), (iv) L234F / L235E / P331 S (LFLEPS), or (v) L234F / L235E / P329G (LFLEPG).

24. A nucleic acid composition, comprising one or more nucleic acids encoding the first monomer, the second monomer, the third monomer, and the fourth monomer of the tetrahedral antibody according to any one of claims 1-23.

25. An expression vector comprising the one or more nucleic acids of claim 24.

26. A host cell transformed with the expression vector of claim 25.Attorney Docket: 53080.4001 / WO27. A method of making a tetrahedral antibody, comprising:(a) culturing the host cell of claim 26 under conditions wherein the tetrahedral antibody is expressed; and(b) recovering the tetrahedral antibody.

28. A method of treating a FOLRa cancer in a human subject comprising administering the tetrahedral antibody according to any one of claims 1-23 to a human subject in need thereof.

29. A tetrahedral antibody, comprising:(a) one or more of a first monomer, wherein the first monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 10;(b) one or more of a second monomer, wherein the second monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 11; and(c) two or more of a third monomer, wherein the third monomer comprises an amino acid sequence with at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 12.

30. The tetrahedral antibody of claim 29, wherein:(i) the first monomer comprises SEQ ID NO: 10;(ii) the second monomer comprises SEQ ID NO: 11; and(iii) the third monomer comprises SEQ ID NO: 12.

31. The tetrahedral antibody of claim 30, wherein:Attorney Docket: 53080.4001 / WO(i) the first monomer consists of SEQ ID NO: 10;(ii) the second monomer consists of SEQ ID NO: 11; and(iii) the third monomer consists of SEQ ID NO: 12.

32. A nucleic acid composition, comprising one or more nucleic acids encoding the first monomer, the second monomer, and the third monomer of the tetrahedral antibody according to any one of claims 29-31.

33. An expression vector comprising the one or more nucleic acids of claim 32.

34. A host cell transformed with the expression vector of claim 33.

35. A method of making a tetrahedral antibody, comprising:(a) culturing the host cell of claim 34 under conditions wherein the tetrahedral antibody is expressed; and(b) recovering the tetrahedral antibody.

36. A method of treating a FOLRa cancer in a human subject comprising administering the tetrahedral antibody according to any one of claims 29-31 to a human subject in need thereof.

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