Engineered VLRB antibodies with immune effector functions

Abstract

Compositions for making and using chimeric variable lymphocyte receptor B (VLRB)-immunoglobulin antibodies are provided. The antibodies are typically composed of two heavy and light chains formed from heavy and light chain fusion proteins. Typically, the heavy chain fusion protein comprises a first variable lymphocyte receptor B (VLRB) antigen binding domain and a CH1 immunoglobulin domain (CH1), and optionally one or more of an immunoglobulin hinge domain (hinge), a CH2 immunoglobulin domain (CH2), a CHS immunoglobulin domain (CHS) and a CH4 immunoglobulin domain (CH4). The heavy chain fusion protein may also comprise a second (VLRB) antigen binding domain, a variable region of an immunoglobulin heavy chain (VH), a monovalent or multivalent single chain variable fragment (ScFv), a polypeptide ligand (L) or a polypeptide receptor (R).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 203,616, filed July 27, 2021, which is hereby specifically incorporated by reference in its entirety.

[0002] Reference to sequence listing The Sequence Listing submitted as an xml file entitled "NOVAB_101_PCT.xml", created on July 27, 2022, and having a size of 68,193 bytes, is hereby incorporated by reference herein in accordance with 37 CFR § 1.834(c)(1).

[0003] FIELD OF THEINVENTION The disclosed invention is generally in the field of variable lymphocyte receptors engineered to have immune effector functions. [Background technology]

[0004] 2. Background of the Invention Unlike Ig antibodies, VLRB antibodies do not have the inherent ability to recruit / activate the biological effector functions required to kill tumor and virus-infected cells or neutralize pathogens, such as complement activation, antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), and do not interact with cellular Fc receptors, including FcRn, which is responsible for Ig recycling and extended serum half-life. The first attempts to "weaponize" VLRB antibodies included expressing them as fusions with human IgG Fc sequences. Nevertheless, improved compositions with the improved biological activities of human IgG1 Fc sequences obtained by FcRn binding, i.e., complement activation, ADCC, ADCP and extended serum half-life, are still desired. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide improved VLRB-based compositions and methods of use thereof. [Means for solving the problem]

[0006] Summary of the Invention Compositions for making and using chimeric variable lymphocyte receptor B (VLRB)-immunoglobulin antibodies are provided.Antibodies are typically composed of two heavy and light chains formed from heavy and light chain fusion proteins.Thus, heavy and light chain fusion proteins are also provided.

[0007] Typically, a heavy chain fusion protein comprises a first variable lymphocyte receptor B (VLRB) antigen-binding domain and a CH1 immunoglobulin domain (CH1) or a CL immunoglobulin domain (CL). Preferably, the heavy chain protein comprises one or more of an immunoglobulin hinge domain (hinge), a CH2 immunoglobulin domain (CH2), a CH3 immunoglobulin domain (CH3) and a CH4 immunoglobulin domain (CH4). The heavy chain fusion protein may also comprise a second (VLRB) antigen-binding domain, a variable region of an immunoglobulin heavy chain (VH), a monovalent or multivalent single-chain variable fragment (ScFv), a polypeptide ligand (L) or a polypeptide receptor (R). Exemplary heavy chain fusion protein domain structures are provided in Table 1. Typically, a light chain fusion protein comprises a variable lymphocyte receptor B (VLRB) antigen binding domain and a CL immunoglobulin domain (CL) or a CH1 immunoglobulin domain (CH1). Exemplary domain structures are provided in Table 2.

[0008] In some embodiments, one or more of the VLRB antigen binding domains and / or one or more of the VL, VH, ScFv, VHH (i.e., nanobody), L or R binds to a cancer or tumor antigen or an antigen expressed by an immune cell type thereon. The immunoglobin domains are typically independently selected from mammalian, optionally human, IgA, IgD, IgE, IgG and IgM, or variants thereof having at least 70% sequence identity thereto. The IgG can be IgG1, IgG2, IgG3 and / or IgG4, and / or the IgA is IgA1 and / or IgA2. In some embodiments, all of the immunoglobin domains are from the same antibody isotype. Any of the fusion proteins may further comprise an active agent cargo conjugated thereto.

[0009] Nucleic acids encoding the fusion proteins and vectors containing same, eg, for recombinant expression thereof, are also provided, as are cells harboring the nucleic acids.

[0010] Chimeric antibodies formed from two heavy chain fusion proteins and two light chain fusion proteins are provided. In some embodiments, the two heavy chains are the same. In other embodiments, the two heavy chains are different. In some embodiments, the two light chains are the same. In other embodiments, the two light chains are different. The desired assembly of the heterotetramer can be facilitated, for example, by incorporating knob-into-hole and / or crossmab strategies (i.e., mutations) into the heavy and / or light chains. Chimeric antibodies can be monospecific, bispecific, or multispecific. Exemplary structures are provided in Tables 3 and 4, and Figures 2, 3A-3D, 4, 5, and 6.

[0011] In some embodiments, one or more of the heavy chain fusion proteins forming the chimeric antibody may not contain a VLRB antigen binding domain, but instead may contain a variable region of an immunoglobulin heavy chain (VH), a monovalent or multivalent single chain variable fragment (ScFv), a polypeptide ligand (L), or a polypeptide receptor (R). Similarly, in some embodiments, one or more of the light chain fusion proteins forming the chimeric antibody may not contain a VLRB antigen binding domain, but instead may contain a variable region of an immunoglobulin heavy chain (VH), a monovalent or multivalent single chain variable fragment (ScFv), a polypeptide ligand (L), or a polypeptide receptor (R). However, when assembled, a chimeric antibody typically contains at least one, and preferably two or more, of the same or different VLRB antigen binding domains.

[0012] In some embodiments, the chimeric antibody can bind to a cancer or tumor antigen. Additionally or alternatively, in some embodiments, the chimeric antibody can bind to an immune cell, such as a T cell, a natural killer (NK) cell, or a macrophage. In some embodiments, the chimeric antibody has antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular phagocytosis (ADCP) activity. The chimeric antibody can have an active drug cargo conjugated thereto.

[0013] Pharmaceutical compositions comprising the antibody are also provided. Typically, the composition has an effective amount of the chimeric antibody to induce a therapeutic or diagnostic result in a subject in need thereof. The formulation is typically suitable for administration to a subject, for example, by parenteral or enteral administration.

[0014] Also provided is a method of treating a subject in need thereof, comprising administering a chimeric antibody composition to the subject. For example, a method comprising administering an effective amount of a chimeric antibody to the subject to increase an immune response in the subject increases (e.g., induces, activates, enhances, etc.) the immune response in the subject in need thereof. A method of treating a subject for cancer may comprise administering an effective amount of a chimeric antibody to the subject to treat one or more symptoms of the cancer. In some such embodiments, the chimeric antibody binds to cells of the cancer, and optionally immune cells, but optionally preferably increases an immune response against the cancer cells. For example, in some embodiments, the composition recruits and / or activates immune cells against the cancer cells. A method of treating a subject for an infectious disease may comprise administering an effective amount of a chimeric antibody to the subject to treat one or more symptoms of the infectious disease. In some such embodiments, the chimeric antibody binds to infected cells, and optionally immune cells, but optionally preferably increases an immune response against the infected cells. In a preferred embodiment, the immune response includes antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) activity, recruiting and / or activating cytotoxic cells. [Brief description of the drawings]

[0015] [Figure 1A-D] Figure 1A is an example of the organization of VLRB antigen binding domains. Figure 1B is a three-dimensional structural representation of the VLRB RBC36-H3 trisaccharide antigen complex. Figure 1C is an example of an IgG structure. Figure 1D is an example of a bivalent VLRB-IgG Fc in which the VLRB and Fc sequences are fused at the hinge domain.

[0016] [Diagram 2] FIG. 2 is an illustration of an engineered VLRB:CL-VLRB:CH1 tetravalent IgG chimera.

[0017] [Figure 3A-B]Figure 3A is an example of an engineered tetravalent VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus of one of the IgG heavy ("H") chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figure 3B is an example of an engineered trivalent VLRB IgG with a monovalent anti-CD3 scFv attached to the N-terminus of one of the IgG H chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figures 3C and 3D show two methods for constructing a VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus (3C) or N-terminus (3D) of one of the IgG light chains, using the inversion of CL and CH1 on one of the H / L chain pairs ("crossmab") and "knob-in-hole" to ensure correct H chain pairing. FIG. 3C is an illustration of an engineered tetravalent VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus of one of the IgG L chains modified by replacement of the CL domain with the H chain CH1 domain, and the complementary H chain ("hole" H chain) modified by replacement of the CH1 domain with a CL domain. The knobs-in-holes method ensures correct H chain pairing. FIG. 3D is an illustration of an engineered trivalent VLRB IgG with a monovalent anti-CD3 scFv attached to the N-terminus of one of the IgG L chains modified by replacement of the CL domain with the H chain CH1 domain, and the complementary H chain ("hole" H chain) modified by replacement of the CH1 domain with a CL domain. The knobs-in-holes method ensures correct H chain pairing. [Figure 3C-D]Figure 3A is an example of an engineered tetravalent VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus of one of the IgG heavy ("H") chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figure 3B is an example of an engineered trivalent VLRB IgG with a monovalent anti-CD3 scFv attached to the N-terminus of one of the IgG H chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figures 3C and 3D show two methods for constructing a VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus (3C) or N-terminus (3D) of one of the IgG light chains, using the inversion of CL and CH1 on one of the H / L chain pairs ("crossmab") and "knob-in-hole" to ensure correct H chain pairing. FIG. 3C is an illustration of an engineered tetravalent VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus of one of the IgG L chains modified by replacement of the CL domain with the H chain CH1 domain, and the complementary H chain ("hole" H chain) modified by replacement of the CH1 domain with a CL domain. The knobs-in-holes method ensures correct H chain pairing. FIG. 3D is an illustration of an engineered trivalent VLRB IgG with a monovalent anti-CD3 scFv attached to the N-terminus of one of the IgG L chains modified by replacement of the CL domain with the H chain CH1 domain, and the complementary H chain ("hole" H chain) modified by replacement of the CH1 domain with a CL domain. The knobs-in-holes method ensures correct H chain pairing.

[0018] [Figure 4] FIG. 4 is an illustration of an engineered bivalent, bispecific VLRB-IgG chimera composed of an anti-CD3 monoclonal antibody with a VLRB fused to the C-terminus of each CH3 domain.

[0019] [Diagram 5] FIG. 5 is an illustration of a CL-VLRB-anti-CD3 bispecific chimeric antibody.

[0020] [Figure 6]FIG. 6 is an illustration of a VLRB:Cl-VLRB:CH tetravalent-CL:ScFv bivalent IgG structure formed from two VLRB-CH1-CH2-CH3 fusion proteins and two VLRB-CL-ScFv fusion proteins.

[0021] [Figure 7] FIG. 7 is an illustration of the domain structure of the VLRB-human IgG1 Fc fusion protein paired with a schematic representation of the bivalent VLRB2-human IgG1 Fc formed therefrom.

[0022] [Figure 8] FIG. 8 is an illustration of the domain structures of VLRB-human IgG1 heavy chain ("H") and light chain ("L") fusion proteins paired with a schematic representation of the tetravalent VLRB4-human IgG1 formed from them.

[0023] [Figure 9] 9 is an illustration of the domain structure of the heavy ("H") and light ("L") chain fusion proteins of monospecific VLRB and bispecific VLRB-scFv arms paired with a schematic representation of the bispecific tetravalent VLRB4-monovalent scFv1 human IgG1 formed therefrom. In this exemplary diagram, the VLRB is MM3 and the scFv is anti-CD3 to form the MM3 VLRB4-anti-CD3 human IgG1 bispecific T cell engager ("BiTe"); the "knob" and "hole" mutations are shown; both heavy ("H") chains have the L234A / L235A / P329G mutations.

[0024] [Figure 10A-B] 10A and 10B are a series of plots showing exemplary flow cytometry data for VLRB fusion proteins and control Ab binding to 10A) Daudi cells, CD38hi, and 10B) KMS-11 CD38-, BJAB CD38lo, Raji CD38int, and Daudi CD38hi. D-MM3 and T-MM3 correspond to MM3 VLRB2 and VLRB4 human IgG1 Fc fusion proteins, respectively.

[0025] [Figure 11] FIG. 11 is a series of plots showing exemplary flow cytometry data for cell line binding of MM3 VLRB4:anti-CD3 scFv BiTe.

[0026] [Figure 12] 12 is a series of plots showing exemplary flow cytometry data for MM3 VLRB4:anti-CD3 scFv BiTe binding to Jurkat and human PBMCs along with an anti-human CD3 antibody comparator. Jurkat TCR / CD3 cells are from Promega's T cell activation kit catalog number 1621.

[0027] [Figure 13] FIG. 13 is a series of plots and corresponding bar graphs showing flow cytometry data for binding of O13 VLRB2 human IgG1 Fc fusion protein (D-O13) to cell lines.

[0028] [Figure 14A-C]Figures 14A-14C show activation of complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) by VLRB fusion proteins. Figure 14A is a flow cytometry plot showing the results of a CDC assay of Daudi cell lysis using 20% ​​human serum and 0.5 μg of N8 and MM3 (D-MM3) VLRB2 human IgG1 Fc and MM3 VLRB4 human IgG1 (T-MM3) fusion proteins and 0.25 μg daratumumab. Dead cells are detected with PI staining. Figure 14B is a plot showing the results of an ADCC assay of Daudi cell activation by Jurkat / FcγRIIIa V158 effector cells measured by NFAT-induced luciferase expression (Promega catalog number G7015). T-MM3 corresponds to MM3 VLRB4 human IgG1 fusion protein and Dara is daratumumab. FIG. 14C is a plot showing the results of an ADCC assay of Daudi cell lysis mediated by NK92 / CD16a effector cells, as measured by the release of LDH into the culture medium. The effector cell to target cell ratio was 5:1. The assay was performed by Genescript (Piscataway, NJ). Anti-human CD20 Ab Rituximab was the positive control (blue line and data points) and MM3 VLRB2 human IgG1 Fc was the test sample (green line and data points).

[0029] [Figure 15A] Figure 15A is a series of flow cytometry plots showing CDC activation by O13 VLRB2 human IgG1 fusion protein, and Figure 15B is a line graph showing ADCC activation by O13 VLRB2 human IgG1 fusion protein. [Figure 15B] Figure 15A is a series of flow cytometry plots showing CDC activation by O13 VLRB2 human IgG1 fusion protein, and Figure 15B is a line graph showing ADCC activation by O13 VLRB2 human IgG1 fusion protein.

[0030] [Figure 16] Figure 16 is a line graph showing activation of Jurkat TCR / CD3 NFAT T cells (Promega catalog number 1621). Target cell lines and activating agents are as indicated.

[0031] [Figure 17A-B] 17A-17D are line graphs showing human PBMC T cell activation by MM3 VLRB2:anti-CD3 scFv BiTe (MM3 / anti-CD3 in the figures). Target cells are as indicated. [Fig. 17C-D] 17A-17D are line graphs showing human PBMC T cell activation by MM3 VLRB2:anti-CD3 scFv BiTe (MM3 / anti-CD3 in the figures). Target cells are as indicated.

[0032] [Figure 18] 18 is a bar graph showing activation of human PBMC T cell cytotoxicity. % Cytotoxicity=100×(Sample−PBMC Spontaneous−Daudi Spontaneous) / (Daudi Max−Daudi Spontaneous), where Sample=MM3 VLRB BiTe+PBMC+Daudi cells, PBMC Spontaneous=MM3 VLRB BiTe+PBMC, Daudi Spontaneous=PBMC+Daudi, and Daudi Max=Lysed Daudi. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Invention I. Definition As used herein, the term "specifically binds" refers to the binding of an antibody to its cognate antigen while not significantly binding to other antigens. Preferably, an antibody binds to its second molecule at least about 10 5 Mol -1 greater than (for example, 10 6 Mol -1 , 10 7 Mol -1 , 10 8 Mol -1, 10 9 Mol -1 , 10 10 Mol -1 , 10 11 Mol -1 and 10 12 Mol -1 A molecule "specifically binds" to an antigen with an affinity constant (Ka) equal to or greater than 0.01.

[0034] As used herein, the term "tumor" or "neoplasm" refers to an abnormal mass of tissue containing neoplastic cells. Neoplasms and tumors can be benign, pre-malignant, or malignant.

[0035] As used herein, the term "cancer" or "malignant neoplasm" refers to cells that exhibit uncontrolled growth, infiltration of adjacent tissues, and often metastasis to other locations in the body.

[0036] As used herein, the term "antineoplastic" refers to a composition, e.g., a drug or biologic, that can inhibit or prevent cancer growth, invasion and / or metastasis.

[0037] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably to refer to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. Thus, the subject may be a human or veterinary patient.

[0038] As used herein, the term "therapeutically effective" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration requires only reduction or alteration, not necessarily elimination. A therapeutically effective amount of a composition for treating cancer is preferably an amount sufficient to cause tumor regression or sensitize tumors to radiation or chemotherapy.

[0039] As used herein, the term "pharmaceutical acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier will necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be known to one of skill in the art.

[0040] As used herein, the term "treatment" refers to the medical management of a patient with the intention of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. This term includes active treatment, i.e., treatment specifically directed toward improving a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed toward removing the cause of an associated disease, pathological condition or disorder. In addition, this term includes symptomatic treatment, i.e., treatment designed to relieve symptoms but not cure a disease, pathological condition or disorder; preventive treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the onset of an associated disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed toward improving an associated disease, pathological condition or disorder.

[0041] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (eg, phosphorylation or glycosylation).

[0042] As used herein, a "variant" polypeptide contains at least one amino acid sequence alteration compared to the amino acid sequence of the corresponding wild-type polypeptide.

[0043] As used herein, an "amino acid sequence alteration" can be, for example, a substitution, deletion or insertion of one or more amino acids.

[0044] As used herein, "fusion protein" refers to a polypeptide formed by the connection of two or more polypeptides via a peptide bond or other linkage formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. Fusion proteins can be formed by chemical coupling of the component polypeptides, or can be expressed as a single polypeptide from a nucleic acid sequence that codes for a single continuous fusion protein. A single-chain fusion protein is a fusion protein that has a single continuous polypeptide backbone. Fusion proteins can be prepared by using conventional techniques in molecular biology to connect two genes in frame into a single nucleic acid sequence, and then expressing the nucleic acid in a suitable host cell under conditions that produce the fusion protein.

[0045] As used herein, a "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors described herein may be expression vectors.

[0046] As used herein, an "expression vector" is a vector that contains one or more expression control sequences.

[0047] As used herein, an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0048] As used herein, "operably linked" means that an expression control sequence is incorporated into a genetic construct so as to effectively control the expression of a coding sequence of interest.

[0049] As used herein, a "fragment" of a polypeptide refers to any subset of a polypeptide that is a shorter polypeptide of the full-length protein. Generally, fragments are 5 amino acids in length or longer.

[0050] As used herein, "valency" refers to the number of available binding sites per molecule.

[0051] As used herein, a "conservative" amino acid substitution is one in which the substituted amino acid has similar structural or chemical properties.

[0052] As used herein, a "non-conservative" amino acid substitution is one that significantly alters the charge, hydrophobicity, or bulk of the substituted amino acid.

[0053] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.

[0054] As used herein, the term "identity" is the relatedness between two or more polypeptide sequences, as determined by comparing the sequences, as known in the art. In the art, "identity" also means the degree of sequence relatedness between polypeptides, as determined by the match between strings of such sequences. "Identity" and "similarity" are used in the context of Computational Molecular Biology, Lesk, AM, Ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, DW, Ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, Eds., Humana Press, New Jersey, 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).

[0055] The preferred method for determining identity is designed to produce the largest match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of Genetics Computer Group, Madison Wis.) incorporating Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST and XBLAST). Default parameters are used to determine identity for the polypeptides of the present disclosure.

[0056] As an example, a polypeptide sequence may be identical to a reference sequence, i.e., 100% identical, or may contain up to a certain integer number of amino acid changes compared to the reference sequence, resulting in a % identity of less than 100%. Such changes are selected from the following: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, which may occur at the amino- or carboxy-terminal position of the reference polypeptide sequence, or anywhere between these terminal positions, either individually between amino acids in the reference sequence, or interspersed in the reference sequence in one or more consecutive groups. The number of amino acid changes for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percentage of each percent identity (divided by 100), and then subtracting the product from the total number of amino acids in the reference polypeptide.

[0057] As used herein, "as needed" or "as needed" means that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes instances when the event, circumstance, or material occurs or exists and instances when it does not occur or exist.

[0058] Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such ranges are expressed, the ranges from one particular value and / or to the other particular value are also specifically contemplated and considered disclosed, unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value constitutes another specifically contemplated embodiment that is to be considered disclosed, unless the context specifically dictates otherwise. It is further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint, unless the context specifically dictates otherwise. It should be understood that all individual values ​​and subranges of values ​​included within an expressly disclosed range are also to be considered specifically contemplated and disclosed, unless the context specifically dictates otherwise. Finally, all ranges should be understood to refer to the recited ranges, both as ranges and as collections of individual numbers, from the first endpoint, inclusive, to the second endpoint, inclusive. In the latter case, it should be understood that any of the individual numbers may be selected as one form of the amount, value, or feature to which the range refers. In this way, a range recites a set of numbers or values ​​from a first endpoint, inclusive, to a second endpoint, inclusive, from which a single member of the set (i.e., a single number) may be selected as one form of the amount, value, or feature to which the range refers. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular instance.

[0059] All compounds disclosed herein are intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.Furthermore, all subgroups that can be identified within this disclosure are intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.As a result, it is specifically contemplated that any compound, or subgroup of compounds, can be specifically included for use or specifically excluded from use, or can be specifically included in or specifically excluded from a list of compounds.

[0060] Disclosed are the components used to prepare the disclosed compositions and the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a particular polypeptide is disclosed and discussed, and several modifications that can be made to some polypeptides are discussed, each and every combination and permutation of the possible polypeptides and modifications is specifically contemplated, unless specifically indicated to the contrary. Thus, when a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of a combination molecule AD is disclosed, each is individually and collectively contemplated, meaning that the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0061] II. VLRB-Ig Chimeras Chimeric molecules are provided that are typically composed of two heavy chains and two light chains, each composed of at least one, preferably two or more VLRB antigen binding domains, linked to an immunoglobulin constant domain(s), and optionally one or more other target moiety binding domains. Referred to herein as chimeric VLRB-Ig, VLRB-Ig, chimeric antibody, or simply antibody, these structures are designed to mimic the natural Y-shaped structure of antibodies, increase the valency of the antigen binding domain, increase the number of targets that can be specifically bound, increase the immune effector response(s), or a combination thereof. The principles of the design, manufacture, and method of use are discussed in more detail below.

[0062] A. Design Strategy In lampreys and hagfish, the only extant jawless vertebrates, variable lymphocyte receptors (VLRs) play a major role in the recognition of foreign antigens. In contrast to the variable, diverse, and joining (VDJ) gene segments of immunoglobulins in jawed vertebrates, jawless vertebrates solve the problem of receptor diversity by somatic DNA rearrangement of diverse leucine-rich repeat (LRR) modules into incomplete vlr genes. The resulting mature vlr gene encodes an N-terminal LRR capping region (LRRNT), the first LRR (LRR1), up to seven 24-residue variable LRRs (LRRVs), a terminal or end LRRV (LRRVe), a connecting peptide (CP), a C-terminal LRR capping region (LRRCT), and a threonine / proline-rich stalk region that links the protein to a glycosylphosphatidylinositol (GPI) anchor and hydrophobic tail to generate the cell surface form of the VRLB (Han, et al., Science, 321(5897):1834-7 (2008)). VLRAs and VLRCs are unique single-pass transmembrane proteins and are not secreted. Secreted VLRBs lack GPI linkages and form a pentamer of dimeric structure, i.e., 10 VLRB-binding domains per molecule (Herrin, et al., Proc Natl Acad Sci US A. 2008;105(6):2040-5. Epub 2008 / 02 / 02. doi: 10.1073 / pnas.0711619105. PubMed PMID: 18238899; PMCID: PMC2542867.).

[0063] The antigen-binding domain of variable lymphocyte receptors (VLRBs) is composed of a variable number of highly diverse tandemly linked leucine-rich repeat (LRR) domains. These consist of an amino-terminal LRR domain (LRRNT), typically 27-39 amino acids long, most typically 32 or 39 amino acids long, a first LRR (LRR1) of 18 amino acid residues, followed by a variable number (from none to eight, but rarely more than three) of LRR domains, typically 24 or 23 amino acid residues each (LRRV1, LRRV2, etc.), and a terminal LRRV (LRRVe), also 24 amino acids long, connected via a short connecting peptide (CP) of typically 13 amino acid residues to a carboxy-terminal LRR domain (LRRCT) of highly variable length, 48-63 amino acid residues, which differs from the LRR1 and LRRV domains, as aligned in FIG. 1A (Herrin and Cooper, J Immunol. 185(3):1367-74 (2010). Epub 2010 / 07 / 28. doi: 10.4049 / jimmunol.0903128. PubMed PMID: 20660361). All VLR LRR domains, except for LRRCT, adopt a canonical beta strand-turn-alpha helix LRR domain tertiary structure such that VLRs form a crescent-shaped "palm-like" protein structure, with the variably present loops in the LRRCT domain forming a "thumb-like" cap at one end of the palm (Figure 1B).X-ray crystal structures of VLR:antigen complexes have localized antigen binding to the concave surface of the VLR, a continuous β-sheet surface formed by the β-strands of each LRR domain (Han, et al., Science, 321(5897):1834-7 (2008). Epub 2008 / 09 / 27. doi: 10.1126 / science.1162484; Collins, et al., Structure, 25(11):1667-78 e4 (2017). Epub 2017 / 10 / 11. doi: 10.1016 / j.str.2017.09.003; Gunn, et al., J Mol Biol., 430(9):1350-67 (2018). Epub 2018 / 03 / 30. doi: 10.1016 / j.jmb.2018.03.016., Kirchdoerfer, et al., Structure, 20:479-486 (2012). [PubMed: 22405006], Collins, et al., Acta Cryst., 682 - 687 (2017), Luo, et al., J Biol Chem. 288(32):23597-606 (2013). Epub 2013 / 06 / 21. doi: 10.1074 / jbc.M113.480467, Velikovsky, Nature Struct. Mol. Biol. 16, 725-730 (2009)). The identified amino acid positions within each LRR domain are solvent exposed on the VLR concave surface and have the potential to form contacts with bound antigen (Figure 1B).

[0064] However, unlike Ig antibodies, VLRB antibodies do not possess the inherent ability to recruit / activate the biological effector functions required for killing tumor and virus-infected cells or neutralizing pathogens, such as complement activation, antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), and do not interact with cellular Fc receptors, including FcRn, which is responsible for Ig recycling and extended serum half-life.

[0065] The basic structure of a naturally occurring antibody molecule is a Y-shaped tetrameric quaternary structure consisting of two identical heavy chains and two identical light chains held together by non-covalent interactions and interchain disulfide bonds, see, e.g., Figure 1C.

[0066] In mammalian species, there are five types of heavy chains: alpha, delta, epsilon, gamma and mu, which determine the immunoglobulin class (isotype): IgA, IgD, IgE, IgG and IgM, respectively. The heavy chain N-terminal variable domain (VH) is followed by a constant region that contains three domains (numbered from N-terminus to C-terminus as CH1, CH2 and CH3) in heavy chains gamma, alpha and delta, whereas the constant region of heavy chains mu and epsilon is composed of four domains (numbered from N-terminus to C-terminus as CH1, CH2, CH3 and CH4). The CH1 and CH2 domains of IgA, IgG and IgD are separated by a flexible hinge that varies in length between the different classes and, in the case of IgA and IgG, between the different subtypes: IgG1, IgG2, IgG3 and IgG4 have hinges of 15, 12, 62 (or 77) and 12 amino acids, respectively, and IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

[0067] There are two types of light chains: lambda and kappa, which can associate with any of the heavy chain isotypes, but in a given antibody molecule, both are of the same type. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a constant region consisting of a single domain called CL.

[0068] The heavy and light chains are paired by protein / protein interactions between the CH1 and CL domains, and the two heavy chains associate by protein / protein interactions between their CH3 domains. The structure of the immunoglobulin molecule is generally stabilized by interchain disulfide bonds between the CH1 and CL domains and between the hinges.

[0069] The clinical effectiveness of therapeutic antibodies depends on both their antigen-binding function and their effector function, which are each associated with different parts of the immunoglobulin molecule. The antigen-binding regions correspond to the arms of a Y-shaped structure, each of which consists of an intact light chain paired with the VH and CH1 domains of a heavy chain, and are called Fab fragments (for antigen-binding fragment). Fab fragments were initially generated from native immunoglobulin molecules by papain digestion, which cleaves the antibody molecule at the hinge region on the amino-terminal side of the interchain disulfide bond to release two identical antigen-binding arms. Other proteases, such as pepsin, also cleave the antibody molecule at the hinge region, but on the carboxy-terminal side of the interchain disulfide bond, to release a fragment consisting of two identical Fab fragments, which remain linked via disulfide bonds; reduction of the disulfide bonds in the F(ab')2 fragment generates the Fab' fragment.

[0070] The part of the antigen-binding region corresponding to the VH and VL domains is called Fv fragment (for variable fragment); it contains CDR (complementarity determining region) that forms the antigen-binding site (also called paratope). In addition to specifically directing the antibody to its goal, upon binding to its target antigen, the antigen-binding region can induce various biological signals that can be positive or negative, depending on both the targeted antigen and the epitope on that antigen that is recognized by the antibody. For use in the field of cancer therapy, antibodies that deliver growth inhibitory or proapoptotic signals that result in cell division arrest or tumor cell death are generally preferred (Verma et al., J Immunol, 186, 3265-76; 2011).

[0071] The effector function of an antibody results from its binding to effector molecules, such as complement proteins, or to the Fc receptors on the surface of immune cells, such as macrophages or natural killer (NK) cells. This results in different effects, such as antibody-dependent phagocytosis (ADP), antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC), which lead to the phagocytosis or lysis of the targeted antigen. The effector region of an antibody, which is responsible for its binding to effector molecules or cells, corresponds to the stem of the Y-shaped structure and contains the paired CH2 and CH3 domains of the heavy chain (or CH2, CH3 and CH4 domains, depending on the class of the antibody), and is called the Fc (for fragment crystallizable) region.

[0072] ADCC, ADP and CDC mediated by the Fc region play a major role in the therapeutic activity of mAbs. The ADCC mechanism appears to be central, since it has been demonstrated that the therapeutic action of two major clinically successful mAbs, anti-HER2 and anti-CD20, against human tumor xenografts was almost completely abolished in nude mice genetically deficient for Fc gamma receptors (Clynes et al., Nat Med, 6, 443-6, 2000). The ADP mechanism has also been shown to be of central importance in several mouse models of human tumors (Uchida et al., J. Exp. Med. 199: 1659-69, 2004), and CDC has also been demonstrated to play a fundamental role in the therapeutic activity of anti-CD20 in vivo (Di Gaetano et al., J Immunol, 171, 1581-7, 2003).

[0073] Strategies are provided for conferring these capabilities to VLRB antibodies.The strategies typically include fusing or linking its antigen-binding fragment, and optionally one or more other cell targeting or binding moieties, to a molecule having a CH1 domain, a CL domain, or a combination thereof to form a VLRB-Ig chimera.In a preferred embodiment, the chimera also includes other Ig constant domains, such as CH2, CH3, hinge region, or a combination thereof, and may further include one or more variable regions of Ig, if necessary.In a preferred embodiment, the molecule adopts a dimeric Y-shaped structure similar to that of an antibody.

[0074] The structures of chimeric VLRB-Igs, fusion proteins that may be used to construct chimeras, and exemplary VLRB and Ig sequences that may be used herein are discussed in more detail below.

[0075] B. VLRB-Ig Chimeric Fusion Protein The disclosed VLRB-Ig chimeras can be assembled using fusion proteins that combine elements of a VLRB antibody and an Ig antibody.

[0076] Heavy chain fusion protein designs include, but are not limited to, the constructs outlined in Table 1, from N-terminus to C-terminus: [Table 1-1] [Table 1-2] [Table 1-3]

[0077] Light chain fusion protein designs include, but are not limited to, the constructs outlined in Table 2, from N-terminus to C-terminus: [Table 2]

[0078] In the disclosed fusion proteins, "VLRB" refers to a domain that comprises a segment, fragment or variant of a VLRB antibody that can bind to an antigen. Thus, typically, the VLRB domain comprises at least an antigen-binding domain of VLRB (see, e.g., FIG. 1A and the description herein and elsewhere in the art), or an antigen-binding variant thereof that has, for example, at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the parent VLRB antigen-binding domain. In some embodiments, the VLRB domain of the fusion protein is the entire VLRB antibody, or an antigen-binding variant thereof that has, for example, at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the parent VLRB antibody.

[0079] In the disclosed fusion proteins, "CH1", "CH2", "CH3" and "CH4" refer to the CH1, CH2, CH3 and CH4 domains of a mammalian Ig, and variants thereof. In some embodiments, the domain(s) and / or variants thereof maintain one or more immunological functions of the constant region of a mammalian Ig. Preferred retained functions include, but are not limited to, ADCC, ADP and / or CDC. The Ig can be any IgA, IgD, IgE, IgG and IgM, respectively, including all subclasses thereof.

[0080] In the disclosed fusion proteins, "hinge" refers to an Ig hinge region or domain, or another flexible linker, such as those discussed below. The sequence / length of the hinge varies between different classes and subclasses of Ig. For example, IgG1, IgG2, IgG3 and IgG4 have 15, 12, 62 (or 77) and 12 amino acid hinges, respectively, and IgA1 and IgA2 have 20 and 7 amino acid hinges, respectively. Each of the constant domains can be independently selected from all of the available Ig constant domains of appropriate type (e.g., CH1, CH2, CH3, CH4, hinge, etc.). Thus, isotype mixing of constant domains is contemplated. However, in a preferred embodiment, all constant domains are from the same isotype.

[0081] In the disclosed fusion proteins, "VH" and "VL" refer to the variable regions of the heavy and light chains, respectively, of an antibody or antigen-binding fragment thereof, which typically have at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the parent VH or VL domain. Collectively referred to as Fv fragments (for variable fragment), these domains contain three CDRs (complementarity determining regions), each of which forms the antigen-binding site of an Ig. Preferably, the "VH" and / or "VL" domains of the fusion protein contain all three of their parent VH and / or VL CDR sequences, or variant sequence(s) thereof, which have at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity thereto.

[0082] In the disclosed fusion proteins, "ScFv" refers to a fusion protein, or an antibody-binding fragment thereof, comprising a variable region of a heavy chain ("VH") and a variable region of a light chain ("VL"), as defined in the preceding paragraph, connected together by a flexible peptide linker. The scFv may be in a VH-linker-VL or VL-linker-VH orientation, but may be assembled in any manner appropriate for the scFv to form the desired structure for antigen binding (i.e., the V of a single polypeptide chain). H and VL V associates with each other to form Fv. L and V H The regions may be derived from a parent antibody or may be chemically or recombinantly synthesized. "ScFv" as used herein refers not only to monovalent single chain variable fragments (i.e., mono-scFv), but also to multivalent single chain variable fragments, e.g., di-scFv, tri-scFv, etc., each of which is specifically disclosed alone and in combination. Di-scFvs and tri-scFvs may be monospecific, bispecific, trispecific, etc., each of which is specifically disclosed alone and in combination.

[0083] In the disclosed fusion proteins, "VHH" refers to nanobodies, which are small recombinantly produced antigen-binding VHH fragments derived from alpaca heavy chain IgG antibodies (HCAbs).

[0084] In the disclosed fusion proteins, "R" and "L" refer to non-antibody targeting moieties or domains, such as receptors ("R") or ligands ("L"). Thus, in some embodiments, the fusion protein comprises a polypeptide receptor or polypeptide ligand in place of the antigen-binding domain of an antibody. Similar to the antigen-binding domain, the R / L domain can target and / or link the chimeric antibody to a cell surface ligand or receptor that is specifically expressed, for example, on immune cells, e.g., on CD8+ cytotoxic T cells, on tumor cells or tumor-associated neovasculature, or that is overexpressed on tumor cells or tumor-associated neovasculature compared to normal tissue.

[0085] In the disclosed fusion proteins, "-" refers to the link between the domains of the chimeric fusion protein, and may also indicate an optional linker domain. Typically, the domains are linked by peptide bond(s), either directly between adjacent domains or through an intervening flexible linker, although chemical linkages for some or all of the links are also contemplated.

[0086] In the disclosed fusion proteins, " / " indicates an option, as also discussed in more detail below with respect to Table 3. For example, ScFv / VHH / R / L indicates that the domain can be an "ScFv" domain or an "R" domain or an "L" domain as defined herein.

[0087] Each of the above domains, and examples thereof, are discussed in more detail below.

[0088] C. Chimeric VLRB-Ig structure The disclosed fusion proteins are typically used to assemble dimers or tetramers that resemble the overall structure of a partial or complete antibody. The disclosed chimeric antibodies contain one or more VLRB antigen binding domains, and therefore many include one or more antibody variable domains, ScFv, VHH, R, L, etc.

[0089] In some embodiments, a chimeric antibody comprises one, preferably two, heavy chains in the absence of a light chain. Such antibodies typically comprise a hinge-CH2-CH3 / CH4 structure, preferably as a monomer or as a heterodimer or more preferably as a homodimer. Such a structure is also referred to herein as IgG Fc (see, for example, FIG. 1D). Preferably, a chimeric antibody comprises at least one, preferably two, heavy chains and one, preferably two, light chains, although higher order structures may also be formed.

[0090] In a preferred embodiment, the chimeric antibody forms a traditional antibody structure consisting of two heavy chains and two light chains. As in naturally occurring antibodies, disulfide bonds formed between the two heavy chains and between pairs of heavy and light chains can link the chains into tetrameters.

[0091] Typically, the N-terminus of the heavy and light chain(s) exhibits one or more antigen binding domains (e.g., VLRB antigen binding domain, antibody variable domain, ScFv, VHH, R, L, etc.). In some embodiments, the C-terminus of the heavy and / or light chain(s) alternatively or additionally exhibits an antigen binding domain (e.g., VLRB antigen binding domain, ScFv, VHH, R, L, etc.).

[0092] A variety of homodimeric and heterodimeric constructs are envisioned. For example, in some embodiments, the resulting tetramer has two identical halves that together form a Y-like shape. Each end of the fork contains an identical antigen-binding site.

[0093] In other embodiments, the halves are not identical. Thus, various combinations of identical and non-identical heavy and light chains are provided. VLRB antigen binding domains can be combined with variable domains and / or ScFvs and / or VHHs and / or Rs and / or Ls. Furthermore, when two or more classes of antigen binding domains or other targeting moieties (e.g., VLRBs, antibody variable domains, ScFvs, VHHs, Rs and / or Ls) are present, each domain can be the same or different. In this way, chimeric antibodies can be multivalent for the same antigen and / or multivalent for different antigens or other targets. Thus, chimeric antibodies can be monospecific, bispecific or multispecific with adjustable valency.

[0094] Heterodimerization technology can be incorporated to drive the assembly of desired components and desired locations. For example, in some embodiments, the heavy chain and optionally the light chain(s) feature "knobs-into-holes" technology, whereby complementary mutations are made in one or more of the domains of the heavy chain (e.g., the CH3 chain domain) and / or the domains of the light / heavy chain pair (e.g., the CL and CH1 domains) (see, e.g., Merchant, et al. "An efficient route to human bispecific IgG." Nat Biotechnol. 1998;16:677-81. doi: 10.1038 / nbt0798-677). These non-covalent interactions, together with disulfide bridges in the hinge region, drive the assembly towards heterodimerization of antibodies with the same or different light chains (Spiess, et al., "Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies." Nat Biotechnol. 2013;31:753-8. doi: 10.1038 / nbt.2621.).See, Shatz, "Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity," MAbs. 2013 Nov 1; 5(6): 872-881. doi: 10.4161 / mabs.26307; Rouet, et al., "Stability engineering of the human antibody repertoire", FEBS Lett. 2014 Jan 21;588(2):269-77. doi: 10.1016 / j.febslet.2013.11.029. Epub 2013 Nov 28; and Rouet and Christ, "Bispecific antibodies with native chain structure." Nat Biotechnol 32, 136-137 (2014), each of which is hereby specifically incorporated by reference in its entirety. See also doi.org / 10.1038 / nbt.2812.

[0095] In some embodiments, the antibody is a "crossmab". Based on the knob-into-hole technology that facilitates the heterodimerization of heavy chains, the correct association of light chains with their cognate heavy chains is achieved by the exchange of heavy and light chain domains within the antigen-binding fragment (Fab) of one half of a bispecific (or multispecific) antibody. This "crossover" preserves the antigen-binding domain but makes the two arms different such that light chain mispairing can no longer occur. See, for example, Schaefer, et al., "Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies", PNAS, 108(27):11187 - 11192 (2011), pnas.org / cgi / doi / 10.1073 / pnas.1019002108, specifically CH1-CL crossmab, which is hereby specifically incorporated by reference in its entirety.

[0096] For example, in some embodiments, an antibody is formed from two heavy chains from Table 1 and two light chains from Table 2, and the antibody comprises at least one VRLB domain. Table 3 below provides non-limiting exemplary antibody genus structures with eight locations for the targeting moiety domain.

[0097] [Table 3]

[0098] More specifically, structures 3a, 3b and 3c show the N- and C-termini of the CL domain, the N-terminus of the CH1 domain, and the C-terminus of the CH3 or CH4 domain as fusion points for binding / targeting moieties, indicated by Roman numerals i, ii, iii, iv, v, vi, vii and vii. The binding / targeting moieties (i.e., each Roman numeral) can be VLRB, VH, VL, ScFv, VHH, R or L, as discussed above. Each of these domains can be unoccupied or occupied by any of the targeting moiety domains described above, provided that at least one VLRB is present in all possible combinations.

[0099] All subgenera and each such specific structure encompassed by the structures in Table 3 are specifically disclosed. When two or more of the same domains are present, they can target the same or different antigens or binding partners. By way of illustration, in the structure in which all of the Roman numerals i, ii, iii, iv, v, vi, vii and vii are occupied by VLRB, each VLRB can be the same or different from the others, such as only one VLRB is present 8 times, or as many as eight VLRBs are present once each, or any subcombination thereof, in any desired location. Arabic numerals can be used to indicate different VLRBs. For example, when eight different VLRBs are present, they can be indicated as VLRB1, VLRB2, VLRB3, VLRB4, VLRB5, VLRB6, VLRB7 or VLRB8. Similarly, when the same VLRB is used 8 times, all eight VLRBs can be indicated as VLRB1.

[0100] Higher order combinations with VLRB, VH, VL, ScFv, VHH, R and / or L, each directed against the same or different targets, can also be treated in the same way and labeled in the same way in subgenera and species structures derived from the genus structure with the same or different VH, VL, ScFv, VHH, R and / or L at any desired position, again using Arabic numerals to indicate different binding targets, provided that at least one VLRB is present. Positions may be left unoccupied. Typically, if a VL is present, it is fused to the N-terminus of the CL, adjacent to the paired VH fused to the N-terminus of the CH1.

[0101] At another annotated position, "CH3 / CH3-CH4" means that either CH3 or CH3-CH4 can be independently selected at the referenced position.

[0102] Other terms, including but not limited to VLRB, VL, VH, ScFv, VHH, R, L, CHL, CH1, hinge, CH2, CH3 and CH4, are defined as discussed above for fusion proteins.

[0103] Structures 3b and 3c provide exemplary "crossmab" designs to drive specific, desired dimerization of light and heavy chain pairs.

[0104] Table 4 provides non-limiting exemplary species of the genera in Table 3, which are also shown in the figures as indicated.

[0105] [Table 4-1] [Table 4-2] [Table 4-3]

[0106] D. Exemplary Domains 1. VLRB sequence The VLRB domain comprises at least the antigen-binding component of a VLRB antibody. Exemplary VLRB constructs and sequences are provided, for example, in U.S. Patent Application Publication Nos. 2011 / 0165584, 2012 / 0189640, 2017 / 0081385, and 2017 / 0008947, each of which is specifically incorporated by reference in its entirety.

[0107] The structure of VLRB is discussed above, and such information there, here, and elsewhere herein may be used in the disclosed design strategies. The VLRB domain may include an N-terminal leucine-rich repeat (LRRNT), one or more leucine-rich repeats (LRR) (herein referred to as internal LRRs), a C-terminal leucine-rich repeat (LRRCT), and a connecting peptide, which constitutes an alpha helix. The length of the polypeptide may have as little as about 130 or 137 amino acids or as much as about 225 or 285 amino acids.

[0108] Optionally, a linking peptide is located at the N-terminus of the LRRCT, more specifically, between the internal LRR and the LRRCT. The linking peptide can be linked to the internal LRR and the LRRCT. Thus, in some embodiments, the VLRB domain comprises an LRRNT, one or more internal LRRs, a linking peptide, and an LRRCT in that order. In some embodiments, the internal LRR region between the LRRNT and the LRRCT comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 leucine-rich repeats, and the LRR1 is located adjacent to or near the LRRNT. As used herein, the LRR1, 2, 3, 4, 5, 6, 7, 8 or 9 are considered to be arranged consecutively from the LRRNT to the LRRCT. Thus, the disclosed domains can include, in that order, an LRRNT, 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, or 1-9 LRRs, a connecting peptide, and an LRRCT.

[0109] Leucine-rich repeats (LRRs) are short sequence motifs that typically participate in protein-protein interactions, and LRRs contain multiple leucine residues. LRRs contain leucine or other aliphatic residues, for example, at positions 2, 5, 7, 12, 16, 21, and 24. However, it is understood and contemplated herein that leucine or other aliphatic residues can be present at other positions in addition to or instead of residues at positions 2, 5, 7, 12, 16, 21, and 24. For example, leucine can be present at position 3 instead of position 2. Structurally, it is also understood that the motif forms a beta-sheet structure. Thus, for example, the disclosed VLRB domain can include an LRRNT, five LRRs, an LRRCT, and a connecting peptide, and can include seven beta-sheet structures, and an alpha helix of the connecting peptide.

[0110] It is understood that the length and sequence of each LRR may vary from the other LRRs in the domain, as well as from the LRRNT and LRRCT. For example, in some embodiments, a VLRB domain comprises an LRRNT, 1-9 LRRs, a connecting peptide, and an LRRCT, where the first internal LRR is LRR1, where LRR1 comprises less than about 20 amino acids, e.g., about 18 amino acids. Optionally, the domain further comprises LRR2-9, where LRR2-9 are less than about 25 amino acids each. In some embodiments, LRR2-9 each comprise about 24 amino acids. LRR1-9 may be the same or different from each other in a given domain, both in length and in specific amino acid sequence.

[0111] The terminal LRRs, denoted LRRNT and LRRCT, are typically longer than each internal LRR. The LRRNT and LRRCT contain invariant regions (regions that have little variation relative to the remainder of the polypeptide compared to similar variable lymphocyte receptors). The variable regions provide specificity to the receptor, while the invariant regions and the overall structural similarity across the receptor help maintain protective immune function. The domain may include an LRRNT, where the LRRNT comprises less than about 40 amino acids. Thus, the LRRNT optionally comprises the amino acid sequence CPSQCSC (SEQ ID NO: 1), CPSRCSC (SEQ ID NO: 2), CPAQCSC (SEQ ID NO: 3), CPSQCLC (SEQ ID NO: 4), CPSQCPC (SEQ ID NO: 5), NGATCKK (SEQ ID NO: 6), or NEALCKK (SEQ ID NO: 7), with or without one or more conservative amino acid substitutions. The domain may include an LRRCT, where the LRRCT is less than about 60 amino acids, optionally 40-60 amino acids in length. In some embodiments, the LRRCT comprises the sequence KNWIVQHASIVN-(P / L)-X-(S / Y / N / H)-GGVDNVK (SEQ ID NO: 8) or KNWIVQHASIVN-(P / L)-XX-(S / Y / N / H)-GGVDNVK (SEQ ID NO: 9), where (P / L) means either P or L at that position, X means any amino acid, and (S / Y / N / H) means either S, Y, N or H at that position. In particular, specifically disclosed are polypeptides where the LRRCT comprises the amino acid sequence TNTPVRAVTEASTSPSKCP (SEQ ID NO: 10), SGKPVRSIICP (SEQ ID NO: 11), SSKAVLDVTEEEAAEDCV (SEQ ID NO: 12), or QSKAVLEITEKDAASDCV (SEQ ID NO: 13), with or without conservative amino acid substitutions.

[0112] It is understood that, as with all peptides, polypeptides and proteins, substitutions may be made in the amino acid sequences of LRRCT and LRRNT that do not alter the properties or function of the peptide, polypeptide or protein, including conservative amino acid substitutions.

[0113] The disclosed compositions can also include a linking peptide. Typically, such peptides are short peptides less than 15 amino acids long and can form alpha helices. Thus, for example, 10, 11, 12, 13, 14 and 15 amino acids long linking peptides that form alpha helices are specifically disclosed. It is understood that linking peptides function to link structural components of a polypeptide. It is further understood that linking peptides of a polypeptide can be linked to LRRCT.

[0114] The polypeptide may comprise a stalk region, which typically comprises a threonine-proline rich region, optionally present in a membrane-bound form of the polypeptide, together with a GPI anchor and a hydrophobic tail.

[0115] Endogenous VLRB antibodies typically contain a glycosyl-phosphatidyl-inositol (GPI) anchor, which maintains the polypeptide on the membrane surface, and a hydrophobic tail.

[0116] Since the disclosed fusion proteins are typically and preferably soluble, the VLRB domain of the fusion protein and chimeric antibody construct may lack one, two or all three of the stalk, GPI anchor and hydrophobic tail domains. In some embodiments, the VLRB domain lacks the GPI anchor and hydrophobic tail and includes part or all of the stalk domain (e.g., 7 amino acids). Such a partial stalk domain may serve as a restriction enzyme site to facilitate VLRB domain replacement in an expression construct.

[0117] The VLRB domain has a desired function. The polypeptides of the VLRB domain described herein selectively bind to an antigen or drug in the same way that an antibody selectively binds to an antigen or drug. The polypeptide is optionally a variable lymphocyte receptor (naturally occurring or non-naturally occurring) or a fragment or variant thereof. The term "variable lymphocyte receptor" is used broadly herein and includes various versions with various specificities, as well as the term "antibody". The polypeptides can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their therapeutic, diagnostic or other purified activity can be tested according to known testing methods.

[0118] The polypeptides can bind to extracellular agents (e.g., pathogens) or antigens. The agents or antigens can include, but are not limited to, peptides, polypeptides, lipids, glycolipids, and proteins. The agents or antigens can originate from a variety of sources, including, but not limited to, pathogenic organisms. Binding to an agent or antigen is understood to be selective. "Selectively binds" or "specifically binds" means binding to one agent or antigen to the partial or complete exclusion of other antigens or agents. "Binds" means detectable binding at least about 1.5 times the background of the assay method. For selective or specific binding, such detectable binding can be detected for a given antigen or agent, but not for a control antigen or agent. Thus, disclosed are VLRB polypeptides that selectively bind to, for example, viral, bacterial, fungal, or protozoan antigens or agents.

[0119] Examples of polypeptides that can be used or modified for use as the VLRB antigen binding domain of the disclosed fusion proteins and VLRB-Ig antibodies include the antigen binding domains or complete sequences of sequence identifiers 1-43, 45-52, 54, 56, 60-65, 68-72, 75, 77-78, 81-119, 122-125, 129-132, 134-144 and 146-155 of U.S. Patent Application Publication No. 2011 / 0165584, which is hereby specifically incorporated by reference in its entirety, with or preferably without the stalk, GPI anchor and hydrophobic tail domains, and GenBank Accession Nos. AY577941-AY578059 and CK988414-CK988652, each of which is hereby specifically incorporated by reference in its entirety. The sequence comprising the amino acid sequence of sequence identifiers 1-20 of US Patent Application Publication No. 2011 / 0165584 provides an example of a full-length VLR. The sequence comprising the amino acid sequence of sequence identifier 43 of US Patent Application Publication No. 2011 / 0165584 is an example of a full-length VLR with a signal peptide. Each of these published applications is also incorporated by reference in its entirety, including its sequence listing.

[0120] Other specific VLR antigen-binding domains may be derived from: Sequence identifier 20 of U.S. Patent Application Publication No. 2012 / 0189640, which is hereby specifically incorporated by reference in its entirety, including its sequence listing and all sequences disclosed therein, provides an antigen-specific polypeptide that specifically binds to blood group determinant H; Sequence identifiers 5, 22, 47, 49, 51, 53, 55, 57, 59, or 61 of U.S. Patent Application Publication No. 2012 / 0189640, which is hereby specifically incorporated by reference in its entirety, including its sequence listing and all sequences disclosed therein, provide antigen-specific polypeptides, wherein the binding polypeptide specifically binds to a pathogen, e.g., a Bacillus anthracis cell surface polypeptide, e.g., BclA;

[0121] In some embodiments, the VLRB domain comprises or consists of the antigen binding domain of VLRB MM3.

[0122] Exemplary MM3 antigen binding domains include the following: ACPSQCSCPGTDVNCHERRLASVPAEIPTTTKILRLYINQITKLEPGVFD RLTQLTQLGLWDNQLQALPEGVFDRLVNLQKLYLNQNQLLALPVGVFDKLTQLTYLDLNNNQLKSIPRGAFDNLKSLTHIWLYGNPWDCECSDILYLKNWIVQHASIVNPHPYGGVDNVKCSGTNTPVRAVTEASTSPSKCPG (SEQ ID NO: 14), which is the polypeptide sequence of sequence identifier 56 of U.S. Patent Application Publication No. 2019 / 0202887, which is hereby specifically incorporated by reference in its entirety, including its sequence listing and all sequences disclosed therein; or ACPSQCSCPGTDVNCHERRLASVPAEIPTTTKILRLYINQITKLEPGVFDRLTQLTQLGLWDNQLQALPEGVFDRLVNLQKLYLNQNQLLALPVGVFDKLTQLTYLDLNNNQLKSIPRGAFDNLKSLTHIWLYGNPWDCECSDILYLKNWIVQHASIVNPHPYGGVDNVKCSGTNTPVRAVTEASTSPSKCPGYVATTT (SEQ ID NO: 15, which contains the MM3 antigen binding domain and part of the stalk); or a humanized antigen-binding domain thereof, e.g. CPAACTCSNNIVDCRGKGLTEIPTNLPETITEIRLEQNTIKVIPPGAFSP YKKLRILRLYINQISELAPDAFQGLRSLNQLVLWDNKITELPKSLFEGLFSLQLLYLNANKINCLRVDAFQDLHNLNYLDLNNNKLQTIAKGTFSPLRAIQHIWLYGNPFICDCHLKWLADYLHASIVNPHPYGGVDNARCTSPRRLAN (SEQ ID NO: 16), which is the polypeptide sequence of sequence identifier 64 in U.S. Patent Application Publication No. 2019 / 0202887. CPAACTCSNNIVDCRGKGLTEIPTNLPETITEIRLEQNTIKVIPPGAFSP YKKLRILRLYINQISELAPDAFQGLRSLNQLVLWDNKITELPKSLFEGLFSLQLLYLNANKINCLRVDAFQDLHNLNYLDLNNNKLQTIAKGTFSPLRAIQHIWLYGNPFICDCHLKWLADYLHHASIVNPHPYGGVDNARCTSPRRLAN (SEQ ID NO: 17), which is the polypeptide sequence of sequence identifier 74 in U.S. Patent Application Publication No. 2019 / 0202887; CPAACTCSNNIVDCRGKGLTEIPTNLPETITEIRLEQNTIKVIPPGAFSP YKKLRILRLYINQISELAPDAFQGLRSLNQLVLWDNKITELPKSLFEGLFSLQLLYLNANKINCLRVDAFQDLHNLNYLDLNNNKLQTIAKGTFSPLRAIQHIWLYGNPFICDCHLKWLADYLHTHASIVNPHPYGGVDNRCTSPRRLAN (SEQ ID NO: 18), which is the polypeptide sequence of sequence identifier 75 in U.S. Patent Application Publication No. 2019 / 0202887; CPAACTCSNNIVDCRGKGLTEIPTNLPETITEIRLEQNTIKVIPPGAFSP YKKLRILRLYINQISELAPDAFQGLRSLNQLVLWDNKITELPKSLFEGLFSLQLLYLNANKINCLRVDAFQDLHNLNYLDLNNNKLQTIAKGTFSPLRAIQHIWLYGNPFICDCHLKWLADYLHTNHASIVNPHPYGGVDNARCTSPRRLAN (SEQ ID NO: 19), which is the polypeptide sequence of sequence identifier 76 in U.S. Patent Application Publication No. 2019 / 0202887; CPAACTCSNNIVDCRGKGLTEIPTNLPETITEIRLEQNTIKVIPPGAFSP YKKLRILRLYINQISELAPDAFQGLRSLNQLVLWDNKITELPKSLFEGLFSLQLLYLNANKINCLRVDAFQDLHNLNYLDLNNNKLQTIAKGTFSPLRAIQHIWLYGNPFICDCHLKWLADYLHTNPHASIVNPHPYGGVDNRCTSPRRLAN (SEQ ID NO:20), which is the polypeptide sequence of sequence identifier 77 in U.S. Patent Application Publication No. 2019 / 0202887; or a variant thereof having, for example, 80, 85, 90, 95, 97, 98% percent identity or similarity thereto.

[0123] It is understood that these sequences are examples of a genus of polypeptides, based on structure and function as taught herein and otherwise known in the art. It is understood that disclosed are full-length VLRs and fragments thereof that can be used as the VLRB domain(s) of the disclosed fusion proteins and VLRB-Ig antibodies.

[0124] Other examples of VLRB antibodies and antigen binding domains are disclosed in U.S. Patent Application Publication Nos. 2020 / 0308247, 2019 / 0256574, 2019 / 0202897, 2019 / 0202887, 2017 / 0081385, 2017 / 0008947, 2016 / 0376348, 2012 / 0189640, 2012 / 0107929, and 2011 / 0165584, each of which is hereby specifically incorporated by reference in its entirety. Any of the VLRB domains may be humanized, e.g., as discussed in U.S. Patent Application Publication No. 2019 / 0202887. Thus, in some embodiments, the VLRB domain is a humanized VLRB antigen binding domain.

[0125] Methods for generating antigen-specific proteins with selected antigen specificity are also known in the art and can be used to prepare the VLR antigen-binding domains of the disclosed fusion proteins and chimeric antibodies. These methods are described, for example, in US Patent Application Publication No. 2012 / 0189640 and may include administering to the lamprey or hagfish one or more target antigens (such as, for example, a target carbohydrate, a target protein, a target pathogen, a target glycoprotein, a target lipid, a target glycolipid, a target tumor antigen, a target ligand, a target receptor, a target cell, or any combination thereof including, for example, two carbohydrates, one carbohydrate and one protein); isolating antigen-specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; amplifying antigen-specific protein-encoding cDNA from the isolated RNA; cloning the cDNA into an expression vector; expressing the expression vector in bacteria transformed with the expression vector; isolating the cDNA clones; transfecting cultured cells with the cDNA clones; screening the culture supernatants for the ability to bind to the target antigens, and isolating antigen-specific proteins that bind to the target antigens from the supernatants.

[0126] In other embodiments, the method of production may involve preparation of total RNA, preparation of cDNA using an olig-dT primer, and then amplification of the cDNA using VLRB gene-specific primers that also contain a region complementary to the vector sequence to facilitate cloning.

[0127] A transfection / screening process called transfectoma can be used, but this is low throughput, with hundreds of transfectants screened, and has been largely supplanted by yeast display and phage / phagemid display, with millions (yeast) or hundreds of millions (phage) screened. For VLRB phage display, see, for example, Hassan, et al., "Generation of lamprey monoclonal antibodies ("Lambribodies") using a phage display system," Biomolecules, 9, 868 (2019); doi:10.3390 / biom9120868; for yeast display, see Tasumi, et al., "High-affinity lamprey VLRA and VLRB monoclonal antibodies," Proc. Natl. Acad. Sci. USA, 106, 12891-12896 (2009). Each of which is hereby specifically incorporated by reference in its entirety.

[0128] Another exemplary VLRB antigen binding domain is ACPSQCSCSGTTVNCKSKSLASVPAGIPTTTRVLYLNDNQITKLEPGVFDRLVNLQTLWLNNNQLTSLPAGLFDSLTQLTILALDSNQLQALPVGVFGRLVDLQQLYLGSNQLSALPSAVFDRLVHLKELLMCCNKLTELPRGIERLTHLTHLALDQNQLKSIPHGAFDRLSSLTHAYLFGNPWDCECRDIMYLRNWVADHTSIVMRWDGKAVNDPDSAKCAGTNTPVRAVTEASTSPSKCPGYVATTT (SEQ ID NO: 21, O13 VLRB antigen binding domain).

[0129] 2. Antibody variable and receptor / ligand domains Exemplary antibodies are provided from which the VH, VL, ScFv and VHH antigen binding domains may be utilized in the disclosed fusion proteins and VLRB antibodies. Such antibodies include, but are not limited to, daratumumab (DARZALEX®) and Reichert, Mabs,3(1): 76-99 (2011), e.g., AIN-457, bapineuzumab, brentuximab vedotin, briakinumab, dalotuzumab, epratuzumab, farletuzumab, girentuximab (WX-G250), naptumomab estafenatox, necitumumab, obinutuzumab, otelixizumab, pagibaximab, pertuzumab, ramucirumab, REGN88, reslizumab, solanezumab, T1h, teplizumab, trastuzumab emtansine, tremelimumab, vedotin, Lizumab, Zalutumumab and Zanolimumab, as well as others including, but not limited to: Rituximab (Rituxan®, IDEC / Genentech / Roche), a chimeric anti-CD20 antibody approved to treat non-Hodgkin's lymphoma (see, e.g., U.S. Pat. No. 5,736,137); HuMax-CD20, an anti-CD20 currently under development by Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT / US2003 / 040426, entitled "Immunoglobulin Variants and UsesThereof), trastuzumab (Herceptin®, Genentech), a humanized anti-Her2 / neu antibody approved for treating breast cancer (see, e.g., U.S. Pat. No. 5,677,171); pertuzumab (rhuMab-2C4, Omnitarge), currently under development by Genentech; anti-Her2 antibodies described in U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone), a chimeric anti-EGFR antibody in clinical trials for various cancers (U.S. Pat. No. 4,943,533; PCT Publication No. WO 2013 / 023362); 96 / 40210); ABX-EGF currently being developed by Abgenix-Immunex-Amgen (U.S. Patent Application No. 6,235,883); HuMax-EGFr currently being developed by Genmab (U.S. Patent Application No. 10 / 172,317); 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); 1CR62 (Institute of Cancer Research) (PCT WO 95 / 20045;Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46;Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53;Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US Pat. No. 5,891,996; US Pat. No. 6,506,883; Mateo et al,1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MRI-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCT WO 01 / 88138); alemtuzumab (Campath®, Millenium), a humanized mAb currently approved for the treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson, IDEC / Schering ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by AG; gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; and anti-LFA-3 (LFA-3) antibody developed by Biogen. Alefacept (Amcvive®), an Fc fusion; abciximab (ReoPro®), developed by Centocor / Lilly; basiliximab (Simulect®), developed by Novartis; palivizumab (Synagis®), developed by Mediimmune; infliximab (Remicade®), an anti-TNF alpha antibody developed by Centocor; adalimumab (Humira®), an anti-TNF alpha antibody developed by Abbott; Humicade®, an anti-TNF alpha antibody developed by Celltech; golimumab (CNTO-148), a fully human TNF antibody developed by Centocor; p75 (Synagis®), developed by Immunex / Amgen;Etanercept (Enbrel®), a TNF receptor Fc fusion; Lenercept, a p55 TNF receptor Fc fusion previously developed by Roche; ABX-CBL, an anti-CD147 antibody being developed by Abgenix; ABX-IL8, an anti-IL8 antibody being developed by Abgenix; ABX-MAI, an anti-MUC18 antibody being developed by Abgenix; Pemtumomab (R1549,90Y-muHMFG1), an anti-MUC1 being developed by Antisoma; Therex (R1550), an anti-MUC1 antibody being developed by Soma; AngioMab (AS1405), being developed by Antisoma; HuBC-1, being developed by Antisoma; Thioplatin (AS1407), being developed by Antisoma; Antegrene (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen; VLA-1, an anti-VLA-1 integrin antibody being developed by Biogen; mAb, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-beta 2 antibody being developed by Cambridge Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an anti-TGF beta 1 antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-eotaxin-1 (Eotaxinl) antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., LyntphoStat-B® anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc.TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by Genentech; Avastin® (bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech; anti-HER receptor family antibodies being developed by Genentech; anti-tissue factor (ATF), an anti-tissue factor antibody being developed by Genentech; Xolair® (Omalizurnab), an anti-IgE antibody being developed by Genentech; Raptiva® (Efalizurnab), an anti-CD11a antibody being developed by Genentech and Xoma; MLN-02 antibody (formerly LDP-02), being developed by Genentech and Millenium Pharmaceuticals; HuMax, an anti-CD4 antibody being developed by Genmab HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen; HuMax-Inflam, an anti-heparanase I antibody being developed by Genmab and Medarex and Oxford GcoSciences; HuMax-Cancer, an anti-heparanase I antibody being developed by Genmab and Amgen; HuMax-Lymphoma, an anti-CD40L antibody being developed by Genmab and Amgen; HuMax-TAC, an anti-CD40 antibody being developed by Genmab; IDEC-131, an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-151 (clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody being developed by IDFC Pharmaceuticals; IDEC-152, an anti-CD23 antibody being developed by IDEC Pharmaceuticals; IDECImclone is developing a new anti-VE-cadherin antibody, and Imclone is developing a new anti-inflammatory drug, Imclone. ... (epratuzumab), AFP-Cide being developed by Immunomedics, MyelomaCide being developed by Immunomedics, LkoCide being developed by Immunomedics, ProstaCide being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Medarex and Immuno-Designed Osidem® (IDM-I), an anti-Her2 antibody being developed by Molecules; HuMaxe-CD4, an anti-CD4 antibody being developed by Medarex and Genmab; HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab; CNTO 148, an anti-TNFα antibody being developed by Medarex and Centocor / J&J; CNTO 1275, an anti-cytokine antibody being developed by Centocor / J&J; MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys; MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys; and Protein Design Labs. Thus, Nuvion® (vidilizumab), an anti-CD3 antibody under development, HuZAFO, an anti-gamma interferon antibody under development by Protein Design Labs, anti-alpha5beta1 integrin under development by Protein Design Labs, anti-IL-12 under development by Protein Design Labs, ING-1, an anti-Ep-CAM antibody under development by Xoma, Xolair® (omalizumab), a humanized anti-IgE antibody under development by Genentech and Novartis, and MLN01, an anti-beta2 integrin antibody under development by Xoma. In another embodiment, the therapeutic agent includes: KRN330 (Kirin); huA 33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrin, Centocor); MEDI-522 (alpha V133 integrin, Medinum); Volociximab (αVβ1 integrin, Biogen / PDL); human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD19×CD3, Medinum); 4G7×H22 (bispecific B cell×Fc gamma R1, Meclarex / Merck KGa); rM28 (bispecific CD28×MAPG, U.S. Patent EP 1444268); MDX447 (EMD 82633) (bispecific CD64 x EGFR, Medarex); catumaxomab (removah) (bispecific EpCAM x anti-CD3, Trion / Fres); ertumaxomab (bispecific HER2 / CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); siplizumab (MEDI-507) (CD2, Medimmune);ofatumumab (Humax-CD20) (CD20, Genmab); rituximab (Rituxan) (CD20, Genentech); THIOMAB (Genentech); veltuzumab (hA20) (CD20, Immunomedics); epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1 (CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genetics); SGN-33 (lintuzumab) (CD33, Seattle Genetics;zanolimumab (HuMax-CD4) (CD4, Genmab);HCD 122 (CD40, Novartis);SGN-40 (CD40, Seattle Genetics);Campathlh (alemtuzumab) (CD52, Genzyme);MDX-1411 (CD70, Medarex);hLL1 (EPB-I) (CD74.38, Immunomedics);galiximab (IDEC-144) (CD80, Biogen);MT293 (TRC093 / D93) (truncated collagen, Tracon);HuLuc63 (CS1, PDL Pharma);ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb; Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); 1-IGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (Lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8 (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio);panitumumab (Vectabix) (EGFR, Amgen); zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam Glaxo / Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); trastuzumab (Herceptin) (HER2, Celldex); pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); Apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); Anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871(IGF1-R, Pfizer);IMC-A12(IGF1-R, Imclone);B1111022(Biogen);Mik-beta-1(IL-2Rb(CD122), Hoffman LaRoche);CNTO 328 (IL6, Centocor); anti-KIR (1-7F9) (killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research);hCBE-11 (LTβR, Biogen);HuHMFG1 (MUC1, Antisoma / NCI);RAV 12 (N-linked carbohydrate epitope, Raven);CAL (parathyroid hormone-related protein (PTH-rP), University of California);CT-011 (PD1, CtireTech);MDX-1106 (ono-4538) (PDL Nileclarox / Ono);MAb CT-011 (PD1, Curetech);IMC-3G3 (PDGFRa, Imclone);bavituximab (phosphatidylserine, Peregrine);huJ591 (PSMA, Cornell Research Foundation);muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); infliximab (Remicade) (TNFα, Centocor); A27.15 (Transferrin receptor, Salk Institute, INSERN WO 2005 / 111082); E2.3 (Transferrin receptor, Salk Institute); bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab-WO / 2000 / 034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); and IMC-1121 (VEGFR2, Imclone).

[0130] Other antibodies include bispecific T cell engagers in which one or both of the antigen binding domains are incorporated into a VLRB-Ig as discussed herein. Examples of suitable bispecific and other antibodies are discussed in Tian, ​​et al., "Bispecific T cell engagers: an emerging therapy for management of hematologic malignancies", Journal of Hematology & Oncology volume 14, Article number: 75 (2021), but are not limited to those in Table 5. [Table 5-1] [Table 5-2]

[0131] An exemplary non-VLRB antigen binding domain is the low affinity TR66 anti-CD3 scFv (KD=100 nM) and is used in the following experiments: DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKS (SEQ ID NO:22) (see also WO 2007 / 073499A2, which is hereby specifically incorporated by reference in its entirety) and anti-CD3 high affinity VH-L-VL scFv [ka] (See also WO 2017 / 091656, which is hereby specifically incorporated by reference in its entirety), where the CDRs are identified in bold and the linker is shown in lower case.

[0132] In some embodiments, the fusion protein comprises an R / L domain that binds to a cell surface receptor or ligand that is specifically expressed on immune cells, tumor cells, or tumor-associated neovasculature, or that is overexpressed on tumor cells or tumor-associated neovasculature compared to normal tissue. Tumors also secrete a number of ligands into the tumor microenvironment that affect tumor growth and development. Thus, the R / L can be a receptor(s) that binds to a ligand that is typically expressed on the surface of cells, e.g., immune cells, such as members of the histocompatibility antigen family, or a ligand expressed on the surface of a tumor, including, but not limited to, growth factors, cytokines, and chemokines. In other embodiments, the R / L is a ligand that binds to a receptor expressed on the surface of an immune cell or tumor.

[0133] Thus, in some embodiments, the fusion protein contains a domain that specifically binds to a chemokine or chemokine receptor. Chemokines are soluble, small molecular weight (8-14 kDa) proteins that bind to their cognate G protein-coupled receptors (GPCRs) to elicit a cellular response, usually directional migration or chemotaxis. Tumor cells secrete and respond to chemokines, which facilitate growth achieved by increased endothelial cell recruitment and angiogenesis, subversion of immunological surveillance, and steering the tumor leukocyte profile to skew it such that chemokine release allows tumor growth and metastasis to distant sites. Thus, chemokines are crucial for tumor progression.

[0134] Based on the positioning of the two conserved N-terminal cysteine ​​residues of chemokines, they are classified into four groups: CXC, CC, CX3C and C chemokines. CXC chemokines can be further classified into ELR+ and ELR- chemokines based on the presence or absence of the motif "glu-leu-arg (ELR motif)" preceding the CXC sequence. CXC chemokines bind to and activate their cognate chemokine receptors on neutrophils, lymphocytes, endothelial and epithelial cells. CC chemokines act on some subsets of dendritic cells, lymphocytes, macrophages, eosinophils and natural killer cells, but do not stimulate neutrophils, since neutrophils other than mouse neutrophils lack the CC chemokine receptor. There are approximately 50 chemokines and only 20 chemokine receptors, and thus there is considerable redundancy in this system of ligand / receptor interactions.

[0135] Chemokines produced from tumor and stromal cells bind to chemokine receptors present on tumor and stromal cells. Autocrine loops of tumor cells and paracrine stimulatory loops between tumor and stromal cells facilitate tumor progression. In particular, CXCR2, CXCR4, CCR2 and CCR7 play major roles in tumorigenesis and metastasis. CXCR2 plays a pivotal role in angiogenesis, and CCR2 plays a role in recruiting macrophages into the tumor microenvironment. CCR7 is involved in the metastasis of tumor cells into sentinel lymph nodes, since lymph nodes have CCL21, a ligand for CCR7. CXCR4 is primarily involved in the metastatic spread of a wide variety of tumors.

[0136] 3. Constant Domains In certain embodiments, the CHL, CH1, hinge, CH2, CH3 and / or CH4 are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat and human sequences. These may be endogenous sequences to the subject to which the chimeric antibody is administered. In preferred embodiments, the sequences are human sequences. In certain embodiments, the CHL, CH1, CH2 and / or CH3 sequences are from IgA1, IgA2, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 isotypes, and / or the CH4 sequences are from IgE or IgM isotypes. Any of the CHL, CH1, hinge, CH2, CH3 and / or CH4 sequences may be naturally occurring sequences or variants thereof having at least 70, 75, 80, 85, 90, 95 or more sequence identities thereto. Preferred variants are orthogonal mutations that improve an activity or performance of one or more of the constant domains or of the chimeric antibody as a whole, or alternatively, eliminate an activity, such as binding to one or more FcRs or activation of complement.

[0137] The CL amino acid sequence can be a lambda light chain constant domain sequence. In certain embodiments, the CL amino acid sequence is a human lambda light chain constant domain sequence, such as the lambda light chain sequence of UniProt Accession No. P0CG04, the entire contents of which are hereby specifically incorporated by reference herein. GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:23) is the polypeptide sequence of P0CG04·IGLC1_HUMAN, immunoglobulin lambda constant 1. Thus, in some embodiments, the CL domain is SEQ ID NO:23, or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0138] The CL amino acid sequence may be a kappa light chain constant domain sequence. In a preferred embodiment, the CL amino acid sequence is a human kappa light chain constant domain sequence, for example, the kappa light chain sequence is UniProt Accession No. P01834, the entire contents of which are hereby specifically incorporated by reference herein. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:24) is the polypeptide sequence of P01834·IGKC_HUMAN immunoglobulin kappa constant. Thus, in some embodiments, the CL domain is SEQ ID NO:24, or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0139] In some embodiments, one or more of the heavy chain domain sequences are derived from the human IgG1 sequence of UniProt Accession Nos. P0DOX5 or P01857, each of which is hereby specifically incorporated by reference in its entirety herein, the sequences of which are provided below: [ka] is the polypeptide sequence of P0DOX5·IGG1_HUMAN, an immunoglobulin gamma-1 heavy chain, which also provides the following domain / region annotation: [Table 6] [ka] is the polypeptide sequence of P01857·IGHG1_HUMAN, immunoglobulin heavy chain constant gamma 1, which also provides the following domain / region annotation: [Table 7]

[0140] SEQ ID NO:25, P0DOX5·IGG1_HUMAN, immunoglobulin gamma-1 heavy chain, and SEQ ID NO:26, P01857·IGHG1_HUMAN, immunoglobulin heavy chain constant gamma 1, are related to SEQ ID NO:26, which lacks the variable (V) domain involved in antigen recognition of SEQ ID NO:25. This variable domain of SEQ ID NO:25 is typically not utilized (e.g., absent or substituted) in the disclosed constructs. Exemplary mutation sites, some of which are discussed in more detail below, are shown in bold in SEQ ID NOs:25 and 26.

[0141] Thus, in some embodiments, the fusion protein comprises one or more of the CH1, hinge, CH2 and / or CH3 region(s) of SEQ ID NO:26, or the corresponding sequence of SEQ ID NO:25, or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0142] In some embodiments, the fusion protein preferably comprises the variable domain of SEQ ID NO:25 (e.g., amino acids 120-444) or SEQ ID NO:26 without the corresponding sequence, or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the variant comprises a mutation in one or more of the bolded residues, optionally one or more of the mutations discussed in more detail below.

[0143] In an exemplary embodiment, the CH1 sequence is from an IgG1 isotype. In a specific example, the CH1 sequence is UniProt Accession No. P01857 (SEQ ID NO: 26), amino acids 1-98: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:57), or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0144] In an exemplary embodiment, the hinge sequence is from an IgG1 isotype. In a specific example, the CH1 sequence is UniProt Accession No. P01857 (SEQ ID NO: 26), amino acids 99-110, EPKSCDKTHTCP (SEQ ID NO: 58), or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0145] In certain embodiments, the CH1 sequence and the CL sequence separately contain orthogonal modifications in the endogenous CH1 and CL sequences, respectively, such as modifications that introduce engineered disulfide bridges, charge pair mutations, or combinations thereof.See, for example, U.S. Patent Nos. 8,053,562, 9,527,927, 8,592,562, 9,248,182, and 9,358,286, each of which is hereby incorporated by reference in its entirety.

[0146] An exemplary CH2 sequence is UniProt Accession No. P01857 (SEQ ID NO:26), amino acids 111-223: [ka] or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the variant comprises a mutation in one or more of the bolded residues, optionally one or more of the mutations being discussed in more detail below. Orthologous CH2 amino acid sequences useful for the disclosed antibodies are described in more detail in International PCT Applications WO2017 / 011342 and WO2017 / 106462, each of which is hereby incorporated by reference in its entirety.

[0147] The CH2 sequence may include or be otherwise linked via an N-terminal hinge region peptide that links the N-terminal variable domain-constant domain (e.g., CH1) to the CH2 domain. In addition, the hinge region typically provides both flexibility between the N-terminal variable domain-constant domain segment and the CH2 domain, as well as amino acid sequence motifs that form disulfide bridges between the heavy chains (e.g., between the first and third polypeptide chains).

[0148] In some embodiments, the CH3 sequence is from an IgG isotype, e.g., an IgG1 isotype. In some embodiments, the CH3 sequence is from an IgA isotype. In certain embodiments, the CH3 sequence is from UniProt Accession No. P01857 (SEQ ID NO: 26), amino acids 224 to 330: [ka] or a fragment or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the variant comprises a mutation in one or more of the bolded residues, optionally one or more of the mutations discussed in more detail below.

[0149] In some embodiments, the CH3 sequence is a segment of an endogenous CH3 sequence, or an engineered or modified sequence.

[0150] One or more heavy chain mutations (e.g., insertions, deletions and / or substitutions) can be incorporated into the sequences or fragments utilized in the disclosed constructs. Exemplary mutations include those provided below with reference to UniProt Accession Number P0DOX5 (SEQ ID NO:25), P01857 (SEQ ID NO:26) and corresponding residues in the exemplary domains in parentheses (SEQ ID NOs:62-64), as well as those identified in or applied to corresponding residues in other IgG reference sequences. For example, in certain embodiments, the CH3 sequence has an endogenous CH3 sequence lacking the N-terminal amino acids G343 (224) and Q344 (225). In certain embodiments, the CH3 sequence has an endogenous CH3 sequence lacking the C-terminal amino acids P447 (328), G448 (329) and K449 (330). In certain embodiments, the CH3 sequence has an endogenous CH3 sequence that lacks both the N-terminal amino acids G343(224) and Q344(225) and the C-terminal amino acids P447(328), G448(329) and K449(330).

[0151] In certain embodiments, the CH3 sequence is a naturally occurring sequence with one or more substitutions. In certain embodiments, the mutation is one or more orthogonal mutations introduced into the endogenous CH3 sequence to guide the specific pairing of a particular CH3 sequence, to reduce immunogenicity, or a combination thereof. In some embodiments, the CH3 sequence comprises a knob-hole orthogonal mutation; an isoallotype mutation of either S356(237)C or Y351(232)C mutation that forms a disulfide bridge engineered together with the CH3 domain containing the orthogonal mutation, or a combination thereof. In some preferred embodiments, the knob-hole orthogonal mutation combined with the isoallotype mutation is the following mutational change: D358(239)E, L360(241)M, T368(249)S, L370(251)A and Y409(290)V. CH3 sequences engineered to reduce the immunogenicity of chimeric antibodies by replacing specific amino acids of one allotype with specific amino acids of another allotype are referred to herein as isoallotypic mutations, as described in detail by Stickler et al. (Genes Immun. 2011 April; 12(3): 213-221), which is hereby incorporated by reference for all it teaches. In certain embodiments, specific amino acids of the G1m1 allotype are replaced. In preferred embodiments, isoallotypic mutations D358(239)E and L360(241)M are made in the CH3 sequence. In some specific examples, the human IgG1 CH3 amino acid sequence may have the following mutational changes: P345(226)V; Y351(232)C; and tripeptide insertions 447(328)P, 448(329)G, 449(330)K. In another embodiment, the human IgG1 CH3 sequence has the following mutational changes: T368(249)K; and the tripeptide insertions 447(328)K, 448(329)S, 449(330)C.In other embodiments, the human IgG1 CH3 has a sequence with the following mutational changes: a human IgG1 CH3 sequence with a Y349(230)C and a tripeptide insertion, 447(328)P, 448(329)G, 449(330)K, or 449(330)C mutation incorporated into an otherwise endogenous CH3 sequence.

[0152] In some embodiments, the Fc region (e.g., the CH2-CH3(-CH4) domain(s)) is modified to increase Fc-mediated effector function. For example, in some embodiments, the Fc domain may contain one or more amino acid insertions, deletions, or substitutions that enhance binding to a specific Fc receptor that is specifically expressed on tumors or tumor-associated neovasculature, or that is overexpressed on tumors or tumor-associated neovasculature compared to normal tissue.

[0153] Therapeutic outcomes in patients treated with rituximab (a chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin's lymphoma or Waldenstrom's macroglobulinemia correlated with the individual's expression of allelic variants of Fcγ receptors that have distinct unique affinities for the Fc domain of human IgG1. In particular, patients with high affinity alleles of the low affinity activating Fc receptor CD16A (FcγRIIIA) showed higher response rates and, in the case of non-Hodgkin's lymphoma, improved progression-free survival. In another embodiment, the Fc domain may contain one or more amino acid insertions, deletions, or substitutions that reduce binding to the low affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain wild-type levels of or enhance binding to the low affinity activating Fc receptor CD16A (FcγRIIIA). In a preferred embodiment, the Fc domain contains an amino acid insertion, deletion, or substitution that enhances binding to CD16A. A number of substitutions in the Fc domain of human IgG1 that increase binding to CD16A and reduce binding to CD32B are known in the art and are described in Stavenhagen, et al., Cancer Res., 57(18):8882-90 (2007). Exemplary variants of human IgG1 Fc domains with reduced binding to CD32B and / or increased binding to CD16A contain F245(130)L, R294(175)P, Y302(183)L, V307(188)I or P398(279)L substitutions. These amino acid substitutions may be present in any combination in the human IgG1 Fc domain. In one embodiment, the human IgG1 Fc domain variant contains F245(130)L, R294(175)P and Y302(183)L substitutions. In another embodiment, the human IgG1 Fc domain variant contains F245L, R294P, Y302L, V307(188)I and P398(279)L substitutions.

[0154] Another embodiment is an IgG antibody that reduces binding to FcRs, increasing their half-life. 2-4 Hybrid and IgG 2-4Representative IGs including mutants 2-4 Hybrids and IgG4 mutants are described in Angal, S. et al. (1993) "A Single Amino Acid Substitution Abolishes The Heterogeneity Of Chimeric Mouse / Human (Igg4) Antibody," Molec. Immunol. 30(1):105-108; Mueller, JP et al. (1997) "Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric Igg2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells," Mol. Immun. 34(6):441-452; and U.S. Pat. No. 6,982,323. In some embodiments, IgG 1 and / or IgG 2 Domains have been deleted; for example, Angal, S. et al. have described an IgG in which serine 241(122) was replaced by proline. 1 and IgG 2 The following is stated.

[0155] The substitutions, additions or deletions in the chimeric antibody may be in the Fc region of the antibody, thereby functioning to modify the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies with modified binding to one or more FcγRs are known in the art, see, for example, PCT Publication Nos. WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO 99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089, and U.S. Patent Nos. 5,843,597 and 5,642,821. In one particular embodiment, modification of the Fc region results in an antibody with altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement dependent cytotoxicity (CDC), phagocytic activity, or any combination thereof.

[0156] In some embodiments, the molecules include antibodies whose Fc regions have been modified to exhibit altered Fc receptor (FcR) binding activity, e.g., decreased activity towards activating receptors, e.g., FcγRIIA or FcγRIIIA, or increased activity towards inhibitory receptors, e.g., FcγRIIB. Preferably, such antibodies exhibit decreased antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity (compared to wild-type Fc receptors).

[0157] Modifications that affect Fc-mediated effector functions are well known in the art (see U.S. Pat. No. 6,194,551 and WO 00 / 42072; Stavenhagen, JB et al. (2007) "Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors," Cancer Res. 57(18):8882-8890; Shields, RL et al. (2001) "High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR," J. Biol. Chem. 276(9):6591-6604). Exemplary variants of human IgG1 Fc domains that have reduced binding to FcγRIIA or FcγRIIIA but unchanged or enhanced binding to FcγRIIB include S241(122)A, H270(151)A, S269(150)G, E271(152)A, E295(176)A, E295(176)D, Y298(179)F, R303(184)A, V305(186)A, A329(210)G, K324(205)A, E335(216)A, K336(217)A, K340(221)A, A341(222)A, D378(259)A.

[0158] See also U.S. Patent Application Publication Nos. 2021 / 0179734, 20110150867, 2005 / 0037000, and 2005 / 0064514, each of which is hereby specifically incorporated by reference in its entirety.

[0159] Further mutations include, but are not limited to, those in Table 8: [Table 8]

[0160] See, Lo, et al., "Effector attenuating substitutions that maintain antibody stability and reduce toxicity in mice." J Biol Chem. (2017) 292:3900-8. doi: 10.1074 / jbc.M116.767749; Oganesyan, et al., "Structural characterization of a human Fc fragment engineered for lack of effector functions." Acta Crystallogr D Biol Crystallogr. (2008) 64:700-4. doi: 10.1107 / S0907444908007877; and Chu, et al., "Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies." Mol Immunol 45:3926-3933, all of which are hereby specifically incorporated by reference in their entireties. (2008) are also included.

[0161] Exemplary sequences include, but are not limited to, the following: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ((SEQ ID NO: 61) human IgG1 hinge-CH2-CH3 Fc sequence), and fragments and variants thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ((SEQ ID NO: 24) Human Ig kappa CL sequence), and fragments and variants thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ((SEQ ID NO: 62) Human IgG1 C H 1-hinge-C H 2-C H3 sequences), as well as fragments and variants thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; [ka] Human IgG1 CH1-hinge-CH2-CH3 sequence (with L117A / L118A / P212G "LALA PG" FcR1, RII, RIII silencing; S237C / T249W "knob" mutations shown in italics / bold / single underline), as well as fragments and variants thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and [ka] [ka] Human IgG1 C H 1-hinge-C H 2-C H 3 sequences (Italics / Bold / Single Underlined L117A / L118A / P212G "LALA PG" FcR1, RII, RIII silencing; Italics with Y232C / T249S / L251A / Y290V "Hole" mutations), as well as fragments and variants thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0162] 4. Linker and signal sequences Signal sequence Signal peptides (sometimes called signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences or leader peptides) are short peptides (usually 16-30 amino acids long) present at the N-terminus (or occasionally, non-classically, at the C-terminus or internally) of most newly synthesized proteins destined for the secretory pathway. These proteins include those that are either present inside certain organelles (endoplasmic reticulum, Golgi or endosomes), those that are secreted from the cell, or those that are inserted into most cell membranes. At the end of a signal peptide there is typically a stretch of amino acids that is recognized and cleaved by a signal peptidase and is therefore called the cleavage site.

[0163] All disclosed fusion proteins are expressly disclosed with and without a signal sequence. An exemplary, non-limiting signal sequence used in the constructs of the following examples is MEWSWVFLFFLSVTTGVHS (SEQ ID NO:27).

[0164] b. Linker The term "linker" as used herein includes, without limitation, peptide linkers. Peptide linkers can be of any size, provided that they do not interfere with the binding of the epitope by the variable region or VLRB antigen binding domain. In some embodiments, the linker comprises one or more glycine and / or serine amino acid residues. The linker is selected based on its intended purpose, which may include linking the VLRB and / or ScFv antigen binding domain to the CL and / or C1 and / or CH3 and / or CH4 domains of the light and heavy chains, and / or binding the heavy and light chain(s) of the ScFv together in their appropriate conformational orientation. Bivalent, trivalent and other multivalent scFvs typically include three or more linkers. The linkers can be the same or different in length and / or amino acid composition. Thus, the number of linkers, the composition of the linker(s) and the length of the linker(s) can be determined based on the desired valency of the scFv, as known in the art. The linker(s) may enable or drive the formation of bivalent, trivalent and other multivalent scFvs. Exemplary flexible linkers include those having the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:28), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO:29), (Gly 4 -Ser) 2 (SEQ ID NO: 30) and (Gly 4 -Ser) 4 (SEQ ID NO: 31), (Gly-Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 32), and Gly 4 -Ala-Gly 4 (SEQ ID NO: 33). Other linkers include those in Table 9 (V. Mallajosyula et al., Sci. Immunol. 10.1126 / sciimmunol.abg5669 (2021)).

[0165] [Table 9]

[0166] 5. Cargo Any of the disclosed fusion proteins or chimeric antibodies can be further modified to include one or more cargoes.Typically, the cargo is an active agent.The delivered agent includes therapeutic, nutritional, diagnostic and preventive compounds.Proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules and organic molecules, as well as diagnostic agents can be delivered.

[0167] Active agents include drugs and imaging agents. Therapeutic agents include antibiotics, antivirals, antiparasitic (helminths, protozoa) drugs, anticancer drugs (referred to herein as "chemotherapeutic drugs" and including cytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5FU, methotrexate, adriamycin, camptothecin, epothilones A-F, and taxol), antibodies and biologically active fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations, peptide drugs, anti-inflammatory drugs, dietary supplements such as vitamins, and oligonucleotide drugs (including DNA, mRNA, antisense, siRNA, miRNA, anti-miRNA, piRNA, aptamers, ribozymes, RNA including external guide sequences for ribonuclease P, and triplex forming agents such as tcPNAs). In some embodiments, the active agent is a vector, plasmid, or other polynucleotide encoding an oligonucleotide, such as those discussed above.

[0168] Exemplary drugs that can be delivered include antiangiogenic agents, antiproliferative agents, and chemotherapeutic agents. Such compositions can be called antibody-drug conjugates (ADCs). By combining antibody targeting with therapeutic drugs (e.g., the cancer-killing ability of cytotoxic drugs), ADCs allow for highly sensitive differentiation between healthy and diseased tissues.

[0169] Non-limiting examples of anti-neoplastic drugs that damage DNA or inhibit DNA repair include carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, idarubicin, ifosfamide, lomustine, mechlorethamine, mitoxantrone, oxaliplatin, procarbazine, temozolomide, and valrubicin. In some embodiments, the anti-neoplastic drug is temozolomide, a DNA damaging alkylating agent commonly used for glioblastoma. In some embodiments, the anti-neoplastic drug is a PARP inhibitor that inhibits a step during base excision repair of DNA damage. For example, the PARP inhibitor is olaparib (C 24 H 23 FN 4 O 3 ).

[0170] In some embodiments, the anti-neoplastic drug is a histone deacetylase inhibitor, which suppresses DNA repair at the transcription level and disrupts chromatin structure.In some embodiments, the anti-neoplastic drug is a proteasome inhibitor, which suppresses DNA repair by disrupting ubiquitin metabolism in cells.Ubiquitin is a signaling molecule that regulates DNA repair.In some embodiments, the anti-neoplastic drug is a kinase inhibitor, which suppresses DNA repair by changing DNA damage response signaling pathway.

[0171] Additional antineoplastic drugs include alkylating agents (e.g., temozolomide, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil, gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), some mitotic inhibitors, and vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines, and cyclosporines. These include, but are not limited to, cyclosporines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycins, such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), and topoisomerase inhibitors (including camptothecins, such as irinotecan and topotecan, and derivatives of epipodophyllotoxins, such as amsacrine, etoposide, etoposide phosphate, and teniposide), and cytoskeleton-targeting drugs, such as paclitaxel.

[0172] Prophylactic agents may include compounds that reduce swelling, compounds that reduce radiation damage, and anti-inflammatory agents.

[0173] Representative classes of diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Exemplary materials include, but are not limited to, metal oxides, such as iron oxide, metal particles, such as gold particles, and the like. Biomarkers can also be conjugated to surfaces for diagnostic applications.

[0174] For example, for imaging, radioactive materials, such as technetium 99 ( 99m Tc) or magnetic materials, e.g. Fe 2 O 3can be used. Examples of other materials include gases or gas-generating compounds that are radiopaque. The most common imaging agents for brain tumors include iron oxide and gadolinium. Diagnostic agents can be radioactive, magnetic, or x-ray or ultrasound detectable. Other detectable labels include, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), elemental particles (e.g., gold particles) or contrast agents. These can be encapsulated in, dispersed in, or conjugated to the polymer.

[0175] For example, fluorescent labels can be chemically conjugated to the polymer of the nanocarrier to obtain fluorescently labeled polymers. In other embodiments, the label is a contrast agent. A contrast agent refers to a substance used in medical imaging to enhance the contrast of structures or fluids in the body. Contrast agents are known in the art and include, but are not limited to, agents that function based on X-ray attenuation and magnetic resonance signal enhancement. Suitable contrast agents include iodine and barium.

[0176] The active agent can be selected based on the type of treatment being used. Exemplary active agents for treating cancer, infections and injuries.

[0177] Chimeric antibodies can be chemically linked to the polypeptide by peptide bonds or by chemical or peptide linker molecules of a type well known in the art. Methods for attaching drugs or other small molecule pharmaceuticals to antibody fragments are well known and include bifunctional chemical linkers, such as N-succinimidyl (4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-A-inverted-(2-pyridyldithio)toluene; sulfosuccinimidyl-6-[alpha-methyl-A-inverted-(pyridyldithiol)-toluamido]hetero; xanoate;N-Succinimidyl-3-(-2-pyridyldithio)-proprionate;Succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido]hexanoate;Sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyldithio)-propionylhydrazide, Ellman's reagent, dichlorotriazinic acid acid), S-(2-thiopyridyl)-L-cysteine, etc. Additional bifunctional linking molecules are discussed, for example, in U.S. Patent Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

[0178] The linker can be cleavable or non-cleavable. A highly stable linker can reduce the amount of payload that falls off in circulation, thus improving the safety profile and ensuring that more payload reaches the target cells. The linker can be based on chemical motifs, including disulfides, hydrazones or peptides (cleavable), or thioethers (non-cleavable), to control the distribution and delivery of active agents to target cells. Cleavable and non-cleavable linkers have been proven safe in preclinical and clinical studies (see, for example, brentuximab vedotin, which contains an enzyme-sensitive linker that can be cleaved by cathepsin; and trastuzumab emtansine, which contains a stable non-cleavable linker). In certain embodiments, the linker is a peptide linker that can be cleaved by Edman degradation (Bachor, et al., Molecular diversity, 17 (3): 605-11 (2013)).

[0179] E. Antigen target The disclosed chimeric antibodies can be monospecific to one target, or bispecific or multispecific to two or more targets. In some embodiments, the chimeric antibodies bind to antigens specific to tumor cells or tumor-associated neovasculature, or antigens that are upregulated in tumor cells or tumor-associated neovasculature compared to normal tissue. In some embodiments, the chimeric antibodies bind to antigens specific to immune tissues, such as those involved in regulating B and / or T cell activation in response to infectious disease causative agents, cancer, etc. In some embodiments, the chimeric antibodies are bispecific or multispecific, and bind to both tumor and immune cell targets.

[0180] Some embodiments may facilitate the recruitment and / or activation of T cells. For example, bispecific chimeric antibodies may interact with T cell receptors (anti-CD3 antibodies or scFv) and with cancer cells that are recognized by VLRB antibodies, preferably recruiting and activating T cells to lyse the cancer cells.

[0181] 1. Immune cell targets The antigen can be a checkpoint ligand or receptor expressed by an immune cell or tumor cell, such as CTLA4, PD-1, PD-L1, PD-L2, B7-H3 (e.g., MGA271), B7-H4, BTLA, HVEM, TIM3, GALS, LAGS, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands.

[0182] The antigen can be a costimulatory ligand or receptor expressed by an immune cell or tumor cell, for example, costimulatory molecules include MHC class I molecules, BTLA, Toll ligand receptor, OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LF A-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD40 and CD19a.

[0183] Other immune cell antigens include, but are not limited to, CD3, CD4, CD8, NKG2A, TLR and IDO.

[0184] 2. Tumor targeting Antigens expressed by tumors may be tumor-specific or may be expressed at higher levels on tumor cells compared to non-tumor cells. Antigenic markers, such as serologically defined markers known as tumor-associated antigens, that are either uniquely expressed by cancer cells or are present at significantly higher levels (e.g., statistically significantly elevated) in subjects with malignant conditions compared to appropriate controls, are contemplated for use in certain embodiments.

[0185] Tumor-associated antigens can include, for example, cellular oncogene-encoded products or aberrantly expressed proto-oncogene-encoded products (e.g., products encoded by the neu, ras, trk and kit genes), or mutated forms of growth factor receptors or receptor-like cell surface molecules (e.g., the surface receptor encoded by the c-erb B gene). Other tumor-associated antigens include molecules that may be directly involved in transformation events, or that may not be directly involved in oncogenic transformation events but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melanoma-associated antigens, etc.) (see, e.g., U.S. Pat. No. 6,699,475; Jager, et al., Int. J. Cancer, 106:817-20 (2003); Kennedy, et al., Int. Rev. Immunol., 22:141-72 (2003); Scanlan, et al. Cancer Immun., 4:1 (2004)).

[0186] Genes encoding cellular tumor-associated antigens include cellular oncogenes and aberrantly expressed proto-oncogenes. In general, cellular oncogenes encode products that are directly related to cell transformation, and due to this, these antigens are particularly preferred targets for immunotherapy. An example is the oncogenic neu gene, which encodes a cell surface molecule involved in oncogenic transformation. Other examples include the ras, kit and trk genes. The products of proto-oncogenes (normal genes that are mutated to form oncogenes) can be aberrantly expressed (e.g., overexpressed), and this aberrant expression can be associated with cell transformation. Thus, the products encoded by proto-oncogenes can be targeted. Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules that are expressed on the tumor cell surface. An example is the cell surface receptor encoded by the c-erbB gene. Other tumor-associated antigens may or may not be directly involved in malignant transformation. However, these antigens are expressed by certain tumor cells, and therefore may provide effective targets. Some examples are carcinoembryonic antigen (CEA), CA125 (associated with ovarian cancer) and melanoma-specific antigen.

[0187] In ovarian cancer and other carcinomas, for example, tumor-associated antigens are detectable in easily obtained biological fluids, such as serum or mucosal secretion samples.One such marker is CA125, a carcinoma-associated antigen that also falls into the bloodstream and is detectable in serum (e.g., Bast, et al., N. Eng. J. Med., 309:883 (1983); Lloyd, et al., Int. J. Canc., 71:842 (1997)). CA125 levels in serum and other biological fluids have been measured along with other markers, such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN mucin (STN), and placental alkaline phosphatase (PLAP), in an attempt to provide a diagnostic and / or prognostic profile of ovarian cancer and other carcinomas (e.g., Sarandakou, et al., Acta Oncol., 36:755 (1997); Sarandakou, et al., Eur. J. Gynaecol. Oncol., 19:73 (1998); Meier, et al., Anticancer Res., 17(4B):2945 (1997); Kudoh, et al., Gynecol. Obstet. Invest., 47:52 (1999)). Elevated serum CA125 may also occur in association with neuroblastoma (e.g., Hirokawa, et al., Surg. Today, 28:349 (1998)), while elevated CEA and SCC may occur in association with colorectal cancer, among others (Gebauer, et al., Anticancer Res., 17(4B):2939 (1997)).

[0188] The tumor-associated antigen mesothelin, defined by its reactivity with monoclonal antibody K-1, is present on the majority of squamous cell carcinomas, including epithelial ovarian, cervical and esophageal tumors, as well as on mesotheliomas (Chang, et al., Cancer Res., 52:181 (1992); Chang, et al., Int. J. Cancer, 50:373 (1992); Chang, et al., Int. J. Cancer, 51:548 (1992); Chang, et al., Proc. Natl. Acad. Sci. USA, 93:136 (1996); Chowdhury, et al., Proc. Natl. Acad. Sci. USA, 95:669 (1998)). Using MAb K-1, mesothelin is detectable only as a cell-associated tumor marker and is not found in a soluble form in serum from ovarian cancer patients or in medium conditioned by OVCAR-3 cells (Chang, et al., Int. J. Cancer, 50:373 (1992)). However, structurally related human mesothelin polypeptides also include tumor-associated antigen polypeptides, such as distinct mesothelin-related antigen (MRA) polypeptides that are detectable as naturally occurring soluble antigens in biological fluids from patients with malignant disease (see WO 00 / 50900).

[0189] Tumor antigens can include cell surface molecules. Tumor antigens of known structure and known or described function include the following cell surface receptors: HER1 (GenBank Accession No. U48722), HER2 (Yoshino, et al., J. Immunol., 152:2393 (1994); Disis, et al., Canc. Res., 54:16 (1994); GenBank Accession Nos. X03363 and M17730), HER3 (GenBank Accession Nos. U29339 and M34309), HER4 (Plowman, et al., Nature, 366:473 (1993);GenBank accession numbers L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank accession numbers U48722 and KO3193), vascular endothelial growth factor (GenBank accession number M32977), vascular endothelial growth factor receptor (GenBank accession numbers AF022375, 1680143, U48801, and X62568), insulin-like growth factor-I (GenBank accession numbers X00173, X56774, X56773, X06043, European Patent No. GB2241703), insulin-like growth factor-II (GenBank accession numbers X03562, X00910, M17863, and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981);GenBank accession numbers X01060 and M11507), estrogen receptor (GenBank accession numbers M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank accession numbers X51730, X69068 and M15716), follicle-stimulating hormone receptor (FSH-R) (GenBank accession numbers Z34260 and M65085), retinoic acid receptor (GenBank accession numbers L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes, et al., Proc. Nat. Acad. Sci.USA, 86:7159 (1989);GenBank accession nos. M65132 and M64928), NY-ESO-1 (GenBank accession nos. AJ003149 and U87459), NA 17-A (PCT publication no. WO 96 / 40039), Melan-A / MART-1 (Kawakami, et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994);GenBank accession nos. U06654 and U06452), tyrosinase (Topalian, et al., Proc. Nat. Acad. Sci. USA, 91:9461 (1994);GenBank accession no. M26729;Weber, et al., J. Clin. Invest, 102:1258 (1998)), Gp-100 (Kawakami, et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994); GenBank accession number S73003, Adema, et al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen, et al., Science, 254:1643 (1991); GenBank accession numbers U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735, and M77481), BAGE (GenBank accession number U19180; U.S. Patent No. 5,683,886 and no. 5,571,711), GAGE ​​(GenBank accession nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, and U19142), any of the receptors of the CTA class, which notably includes the HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank accession nos. X86175, U90842, U90841, and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985); GenBank Accession Nos. M59710, M59255 and M29540), and PyLT (GenBank Accession Nos. J02289 and J02038); p97 (melanotransferrin) (Brown, et al., J. Immunol., 127:539-46 (1981); Rose, et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986)).

[0190] Additional tumor-associated antigens include thymic leukemia antigen (TL), prostate surface antigen (PSA) (U.S. Pat. Nos. 6,677,157 and 6,673,545); β-human chorionic gonadotropin β-HCG) (McManus, et al., Cancer Res., 36:3476-81 (1976); Yoshimura, et al., Cancer, 73:2745-52 (1994); Yamaguchi, et al., Br. J. Cancer, 60:382-84 (1989): Alfthan, et al., Cancer Res., 52:4628-33 (1992)); glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase (GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989);Ando, ​​et al., Int. J. Cancer, 40:12-17 (1987);Tsuchida, et al., J. Natl. Cancer, 78:45-54 (1987);Tsuchida, et al., J. Natl. Cancer, 78:55-60 (1987));NUC18(Lehmann, et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989); Lehmann, et al., Cancer Res., 47:841-45 (1987)); melanoma antigen gp75 (Vijayasardahi, et al., J. Exp. Med., 171:1375-80 (1990); GenBank accession number X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali, et al., Cancer, 59:55-63 (1987); keratin 19 (Datta, et al., J. Clin. Oncol., 12:475-82 (1994)).

[0191] Tumor antigens of interest include those antigens that are considered in the art to be immunogenic "cancer / testis" (CT) antigens in subjects with a malignant condition (Scanlan, et al., Cancer Immun., 4:1 (2004)). CT antigens include at least 19 different families of antigens that contain one or more members and are capable of inducing an immune response, including, but not limited to, the following: MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE ​​(CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).

[0192] Additional tumor antigens that may be targeted, including tumor-associated or tumor-specific antigens, include, but are not limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomerase, Bage-1, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, Mage-A1, 2, 3, 4, 6, 10, 12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, as well as TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29 / BCAA), CA 195, CA 242, CA-50, CAM43, CD68 / KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, CD19 (e.g., expressed in B cell acute leukemia), EpCAM (e.g., expressed in CTCs), CD20 (e.g., expressed in B cell acute leukemia), and CD45 (e.g., expressed in CTCs). Other tumor-associated and tumor-specific antigens are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.

[0193] The antigen may be specific to tumor neovasculature or may be expressed at a higher level in tumor neovasculature as compared to normal vasculature. Exemplary antigens that are overexpressed by tumor-associated neovasculature compared to normal vasculature include VEGF / KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin, and α 5 β 3 Other antigens that are overexpressed by tumor-associated neovasculature compared to normal vasculature are known to those of skill in the art and are suitable for targeting with the disclosed fusion proteins, including but not limited to integrin / vitronectin.

[0194] In another embodiment, the chimeric antibody specifically binds to a chemokine or chemokine receptor. Chemokines are soluble, small molecular weight (8-14 kDa) proteins that bind to their cognate G protein-coupled receptors (GPCRs) and elicit a cellular response, usually directional migration or chemotaxis. Tumor cells secrete and respond to chemokines, which facilitate growth achieved by increased endothelial cell recruitment and angiogenesis, subversion of immunological surveillance, and steering the tumor leukocyte profile to skew it such that chemokine release allows tumor growth and metastasis to distant sites. Thus, chemokines are crucial for tumor progression.

[0195] Based on the positioning of the two conserved N-terminal cysteine ​​residues of chemokines, they are classified into four groups: CXC, CC, CX3C and C chemokines. CXC chemokines can be further classified into ELR+ and ELR- chemokines based on the presence or absence of the motif "glu-leu-arg (ELR motif)" preceding the CXC sequence. CXC chemokines bind to and activate their cognate chemokine receptors on neutrophils, lymphocytes, endothelial and epithelial cells. CC chemokines act on some subsets of dendritic cells, lymphocytes, macrophages, eosinophils and natural killer cells, but do not stimulate neutrophils, since neutrophils other than mouse neutrophils lack the CC chemokine receptor. There are approximately 50 chemokines and only 20 chemokine receptors, and thus there is considerable redundancy in this system of ligand / receptor interactions.

[0196] Chemokines produced from tumor and stromal cells bind to chemokine receptors present on tumor and stromal cells. Autocrine loops of tumor cells and paracrine stimulatory loops between tumor and stromal cells facilitate tumor progression. In particular, CXCR2, CXCR4, CCR2 and CCR7 play major roles in tumorigenesis and metastasis. CXCR2 plays a pivotal role in angiogenesis, and CCR2 plays a role in recruiting macrophages into the tumor microenvironment. CCR7 is involved in the metastasis of tumor cells into sentinel lymph nodes, since lymph nodes have CCL21, a ligand for CCR7. CXCR4 is primarily involved in the metastatic spread of a wide variety of tumors.

[0197] III. Nucleic acids A. Isolated Nucleic Acid Molecules Encoding Fusion Proteins Nucleic acids are disclosed, including isolated nucleic acids encoding fusion proteins and variants thereof. As used herein, "isolated nucleic acid" refers to a nucleic acid that is separated from other nucleic acid molecules present in a mammalian genome, including the nucleic acids that are normally adjacent to the nucleic acid on one or both sides in the mammalian genome.

[0198] An isolated nucleic acid can be, for example, a DNA molecule, provided that one of the nucleic acid sequences normally found immediately adjacent to the DNA molecule in a naturally occurring genome has been removed or is absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as a recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. Additionally, an isolated nucleic acid can include a recombinant DNA molecule that is part of an engineered nucleic acid, e.g., a hybrid or fusion nucleic acid. For example, a nucleic acid that exists among hundreds to millions of other nucleic acids in a cDNA or genomic library, or a gel slice containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.

[0199] The nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence that encodes the disclosed fusion proteins.

[0200] The nucleic acid may be DNA, RNA (e.g., mRNA) or a nucleic acid analog. The nucleic acid analog may be modified at the base moiety, sugar moiety or phosphate backbone. Such modifications may, for example, improve the stability, hybridization or solubility of the nucleic acid. Modifications at the base moiety may include deoxyuridine instead of deoxythymidine, and 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine instead of deoxycytidine. Modifications at the sugar moiety may include modification of the 2' hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone may be modified to produce morpholino nucleic acids in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, e.g., Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidite, or an alkyl phosphotriester backbone.

[0201] B. Vectors and host cells expressing fusion proteins Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to cause the replication of the inserted segment. A vector can be an expression vector. An "expression vector" is a vector that contains one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0202] The nucleic acid in the vector may be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct such that the expression control sequence effectively controls the expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that is typically comprised of a region of a DNA molecule within 100 nucleotides upstream of the point where transcription begins (generally near the initiation site for RNA polymerase II). To place a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translation reading frame of the polypeptide between 1 nucleotide and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. Enhancers can also be located downstream of the transcription initiation site. A coding sequence is "operably linked" in a cell and "under the control" of an expression control sequence if RNA polymerase is capable of transcribing the coding sequence into mRNA that can then be translated into the protein encoded by the coding sequence.

[0203] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus and adeno-associated virus. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA) and Invitrogen Life Technologies (Carlsbad, CA).

[0204] The expression vector may contain a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags may be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A.

[0205] The vector containing the nucleic acid to be expressed can be transferred into a host cell. The term "host cell" is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, "transformed" and "transfected" encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of several techniques. Without being limited to a particular technique, some of these techniques are well established in the art. Prokaryotic cells can be transformed with nucleic acid, for example, by electroporation or calcium chloride-mediated transformation. Nucleic acid can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection. Host cells (e.g., prokaryotic or eukaryotic cells, e.g., CHO cells) can be used, for example, to produce the fusion proteins described herein.

[0206] IV. Method of Manufacturing A. Methods for Producing Isolated Nucleic Acid Molecules Encoding Fusion Proteins An isolated nucleic acid molecule encoding a fusion protein can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant costimulatory polypeptide. PCR is a technique in which a target nucleic acid is enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be used to design oligonucleotide primers that are identical in sequence to opposing strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14-40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. Common PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification can also be used to obtain isolated nucleic acid.See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

[0207] The isolated nucleic acid can be chemically synthesized (e.g., using phosphoramidite technology for automated DNA synthesis in the 3' to 5' direction) either as a single nucleic acid molecule or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) containing the desired sequence can be synthesized, each pair containing a short segment of complementarity (e.g., about 15 nucleotides) that forms a duplex when the oligonucleotide pair anneals. A DNA polymerase can be used to extend the oligonucleotides to produce a single double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated into a vector. The isolated nucleic acid can also be obtained by mutagenesis. Fusion protein-encoding nucleic acids can be mutated using standard techniques, including PCR-mediated oligonucleotide-directed mutagenesis and / or site-directed mutagenesis. See Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992. Examples of amino acid positions that can be modified include those described herein.

[0208] B. Methods for Making Fusion Proteins Fusion proteins can be obtained, for example, by chemical synthesis or by recombinant production in a host cell. To recombinantly produce a fusion protein, a nucleic acid containing a nucleotide sequence encoding a polypeptide can be used to transform, transduce or transfect a bacterial or eukaryotic host cell (e.g., insect, yeast or mammalian cell). In general, the nucleic acid construct comprises a regulatory sequence operably linked to the nucleotide sequence encoding the fusion protein. Regulatory sequences (also referred to herein as expression control sequences) typically do not code for a gene product, but instead affect the expression of the nucleic acid sequence to which it is operably linked.

[0209] Prokaryotic and eukaryotic systems useful for expressing and producing polypeptides are well known in the art and include, for example, Escherichia coli strains, such as BL-21, and cultured mammalian cells, such as CHO cells.

[0210] In eukaryotic host cells, several viral expression systems can be used to express fusion proteins. Viral expression systems are well known in the art and include, but are not limited to, baculovirus, SV40, retrovirus or vaccinia-based viral vectors.

[0211] The mammalian cell line that stably expresses variant costimulatory polypeptide can be produced using an expression vector with suitable control elements and selectable marker.For example, the eukaryotic expression vector pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expressing variant costimulatory polypeptide in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells and human vascular endothelial cells (HUVEC).After introducing the expression vector by electroporation, lipofection, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran, or other suitable transfection method, stable cell line can be selected (for example, by antibiotic resistance against G418, kanamycin or hygromycin). The transfected cells can be cultured so that the polypeptide of interest is expressed, and the polypeptide can be recovered, for example, from the cell culture supernatant or from lysed cells. Alternatively, the fusion protein can be produced by (a) ligating the amplified sequence into a mammalian expression vector, for example, pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0212] Fusion proteins can be isolated, for example, using chromatographic methods, such as DEAE ion exchange, gel filtration and hydroxylapatite chromatography. For example, costimulatory polypeptides in cell culture supernatants or cytoplasmic extracts can be isolated using a protein G column. In some embodiments, variant costimulatory polypeptides can be "engineered" to contain amino acid sequences that allow the polypeptide to be captured on an affinity matrix. For example, tags, such as c-myc, hemagglutinin, polyhistidine or Flag™ (Kodak), can be used to aid in polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus. Other fusions that can be useful include enzymes, such as alkaline phosphatase, that aid in the detection of the polypeptide. Immunoaffinity chromatography can also be used to purify costimulatory polypeptides.

[0213] Methods for introducing random mutations to produce variant polypeptides are known in the art.Random peptide display libraries can be used to screen for desired fusion protein variants.Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Patent No. 5,223,409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al., U.S. Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5,571,698), and random peptide display libraries and kits for screening such libraries are commercially available.

[0214] C. Methods for Producing VLRB-Ig Antibodies The disclosed chimeric antibodies can be prepared using any suitable method known in the art. For example, recombinant chimeric antibodies can be produced by transfecting a desired combination(s) of one or more vectors encoding the disclosed fusion proteins into a host cell. Exemplary descriptions of recombinant means of generating and producing antibodies include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition); U.S. Pat. No. 4,816,397 (Boss et al.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.), UK Patent GB 2,188,638 (Winter et al.) and UK Patent GB 2,209,757, Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering, W. H. Freeman (1992). Further information regarding the generation, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0215] Antibodies can also be produced by suppliers that specialize in custom recombinant production.

[0216] V. Formulations Pharmaceutical compositions comprising VLRB-Ig are provided. Pharmaceutical compositions containing peptides or polypeptides may be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation) or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration. The compositions may also be administered using biodegradable inserts and delivered directly to appropriate lymphatic tissues (e.g., spleen, lymph nodes, or mucosa-associated lymphatic tissues) or directly to organs or tumors. The compositions may be formulated in dosage forms appropriate for each route of administration. In some embodiments, the compositions are formulated for enteral administration.

[0217] The term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or to otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary according to a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0218] In some embodiments, VLRB-Ig is administered in the range of 0.1-20 mg / kg, based on tumor modeling and extrapolation from bioavailability. The most preferred range is 5-20 mg / kg VLRB-Ig. Generally, for intravenous injection or infusion, dosages may be lower than when administered by alternative routes.

[0219] A. Formulations for Parenteral Administration In a preferred embodiment, the disclosed compositions, including those containing peptides and polypeptides, are administered in aqueous solution by parenteral injection or infusion. Formulations can also be in the form of suspensions or emulsions. In general, a pharmaceutical composition is provided that contains an effective amount of a peptide or polypeptide, and optionally includes a pharma- ceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally additives, such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oil, such as olive oil and corn oil, gelatin, and injectable organic ester, such as ethyl oleate.The preparation can be lyophilized and redissolved / resuspended immediately before use.The preparation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0220] B. Formulations for Enteral Administration VLRB-Ig can also be formulated for oral delivery. Oral solid dosage forms are known to those skilled in the art. Solid dosage forms include tablets, capsules, pills, troches or lozenges, sachets, pellets, powders or granules, or incorporating materials into particulate preparations such as polymeric compounds, e.g., polylactic acid, polyglycolic acid, or into liposomes. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the proteins and derivatives of the present invention. See, for example, Remington's Pharmaceutical Sciences, 21st Ed. (2005, Lippincott, Williams & Wilins, Baltimore, Md. 21201) pages 889-964. The compositions can be prepared in liquid form or can be in dry powder (e.g., lyophilized) form. Liposomal or polymer encapsulation can be used to formulate the compositions. See also Marshall, K. In: Modern Pharmaceutics Edited by GS Banker and CT Rhodes Chapter 10, 1979. In general, the formulations include an active drug and inactive ingredients that protect the VLRB-Ig in the stomach environment and protect the release of biologically active materials in the intestine.

[0221] Liquid dosage forms for oral administration, including pharma- ceutically acceptable emulsions, solutions, suspensions, and syrups, may contain inert diluents; adjuvants, such as wetting agents, emulsifying and suspending agents; and other components including sweetening, flavoring, and perfuming agents.

[0222] C. Controlled Delivery Polymeric Matrices Compositions containing one or more VLRB-Igs may be administered in controlled release formulations. Controlled release polymeric devices may be made for systemic long-term release after implantation of the polymeric device (rods, cylinders, films, disks) or injection (microparticles). The matrix may be in the form of microparticles, e.g., microspheres, in which the peptide is dispersed within a solid polymeric matrix or microcapsules, the core being of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin plate or film ranging from a few nanometers to four centimeters, a powder produced by milling or other standard techniques, or even a gel, e.g., a hydrogel. Matrices may also be incorporated into or onto medical devices to modulate immune responses, prevent infections in immunocompromised patients (e.g., catheterized elderly, or premature infants), or aid healing in the case of matrices used to facilitate healing of pressure sores, decubitis ulcers, and the like. Either non-biodegradable or biodegradable matrices may be used for delivery of VLRB-Ig, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight) and may be crosslinked with multivalent ions or polymers, if desired.

[0223] Matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.Biodegradable microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, as described, for example, by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987);Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987);and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0224] Controlled release oral formulations may be desirable. VLRB-Ig may be incorporated into an inert matrix, such as a film or gum, that allows release by either diffusion or leaching mechanisms. Slowly disintegrating matrices may also be incorporated into the formulation. Another form of controlled release is where the drug is encapsulated in a semipermeable membrane that allows water to enter and push the drug out through a single small opening due to osmotic effects. For oral formulations, the location of release may be the stomach, small intestine (duodenum, jejunem or ileum) or large intestine. Preferably, release avoids the deleterious effects of the gastric environment, either by protection of the active agent (or derivative) or by release of the active agent beyond the gastric environment, such as in the intestine. To ensure complete gastric resistance, an enteric coating (i.e., impermeable to at least a pH of 5.0) is essential. These coatings may be used as mixed films or as capsules, such as those available from Banner Pharmacaps.

[0225] The devices can be formulated for local release to treat the area of ​​implantation or injection, typically delivering a much smaller dosage than that for systemic treatment. The devices can also be formulated for systemic delivery. They can be implanted or injected subcutaneously.

[0226] VI. Method of Use A. Therapeutic and Diagnostic Methods Typically, VLRB-Ig is designed to bind to one or more target cells, for example, by containing one or more domains (e.g., VLRB, ScFv, VHH, VH / VL, R and / or L domains) that bind to an antigen, receptor, ligand or other moiety on the target cell, as discussed above. This may function to increase, induce or enhance ADCC and / or CDC and / or ADCP, to increase delivery of cargo to more and more target cells, to increase or enhance the proximity or communication between two different target cells, to recruit effector cells, e.g., cytotoxic T cells, to form complexes with target cells, e.g., tumor cells, and / or to induce aggregation of one or more different types of targets. For embodiments in which two or more cells are targeted (e.g., to increase or enhance the proximity or communication between two different target cells, to recruit effector cells to target cells, to induce aggregation of one or more different types of targets, etc.), typically bispecific or multispecific antibodies are utilized that include one or more domains (e.g., VLRB, ScFv, VH / VL, R and / or L domains) that target two or more different antigens, receptors, ligands or other moieties on two or more different target cells.

[0227] For embodiments in which one or more different cell types may be targeted, e.g., for increased delivery of cargo to one or more target cells, as well as induced or enhanced ADCC and / or CDC, the chimeric antibody may be monospecific, bispecific or multispecific. Thus, the disclosed methods typically include administering to a subject in need thereof an effective amount of a VLRB-Ig antibody that binds to one or more target cell types. Such methods are preferably effective to achieve a diagnostic result (e.g., detect the location or amount of target cells in a subject) or a therapeutic result (e.g., reduce or prevent one or more symptoms of a disease or disorder).

[0228] For example, the VLRB-Ig antibodies provided herein may be useful in vivo and ex vivo as immune response stimulating therapeutics, e.g., to treat cancer or infectious diseases.

[0229] In some embodiments, VLRB-Ig is designed to facilitate VLRB-Ig-induced antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0230] Also called antibody-dependent cell-mediated cytotoxicity, ADCC is a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells that have specific antibodies bound to their membrane surface antigens. This is one mechanism by which antibodies, as part of the humoral immune response, can be utilized to limit and contain infections or cancer.

[0231] ADCC is independent of the immune complement system, which also lyses targets, but does not require any other cells. ADCC requires effector cells classically known to be natural killer (NK) cells, which typically interact with immunoglobulin G (IgG) antibodies. However, macrophages, neutrophils, and eosinophils can also mediate ADCC, for example, eosinophils kill certain parasites known as helminths via IgE antibodies.

[0232] Thus, in some embodiments, the disclosed chimeric antibodies may exhibit ADCC activity or improved ADCC activity compared to a control. ADCC activity refers to the ability of an antibody to initiate an antibody-dependent cellular cytotoxicity (ADCC) response. ADCC is a cell-mediated reaction in which antigen-nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils and macrophages) recognize antibodies bound to the surface of target cells and subsequently cause lysis (i.e., "killing") of the target cells. The primary mediator cell in ADCC is the natural killer (NK) cell. NK cells express FcγRIII, FcγRIIA is an activating receptor, and FcγRIIIB is an inhibitory receptor. Monocytes express FcγRI, FcγRII and FcγRIII.

[0233] CDC is an effector function of IgG and IgM antibodies. When they are bound to surface antigens on target cells (e.g., bacteria- or virus-infected cells), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, resulting in the formation of the membrane attack complex (MAC) and target cell lysis. The complement system is efficiently activated by human IgG1, IgG3 and IgM antibodies, weakly activated by IgG2 antibodies, and not activated by IgG4 antibodies. ADCC and CDC are two mechanisms of action by which therapeutic antibodies can achieve antitumor effects.

[0234] Thus, additionally or alternatively, the disclosed chimeric antibodies may exhibit CDC activity or may exhibit improved CDC activity compared to a control. CDC activity refers to the reaction of one or more components of the complement system to recognize bound antibodies on a target cell and subsequently cause lysis of the target cell.

[0235] ADCP is a potent mechanism for the elimination of antibody-coated foreign particles, e.g., microorganisms or tumor cells. Engagement of FcγRIIa and FcγRI expressed on macrophages triggers a signaling cascade that leads to phagocytosis of IgG-opsonized particles. See, e.g., Tay, et al., Front Immunol., 10: 332, doi: 10.3389 / fimmu.2019.00332 (2019).

[0236] Thus, additionally or alternatively, the disclosed chimeric antibodies may exhibit ADCP activity or may exhibit improved ADCP activity compared to a control. ADCP activity refers to the ability of an antibody to elicit an antibody-dependent cellular phagocytosis (ADCP) response.

[0237] Additionally or alternatively, the disclosed bispecific or multispecific chimeric antibodies can be used to bring two or more target cell types into close proximity. Examples of paired target cells include, but are not limited to, immune cells and cancer cells, such as cytotoxic T cells and tumor cell targets, two different immune cells, and the like. In this way, the antibody can facilitate signaling between the two cells. Examples of cell-cell interactions and / or signaling include, but are not limited to: increased immune response activation induced by antigen-presenting cell (APC) targets interacting with T cell targets; Increased immune response effector function induced by NK cells or macrophages targeting tumor or infected cell targets, e.g., ADCC and / or CDC and / or ADCP; and Anti-CD8: Recruitment of CD8 cytotoxic T cells to tumors by tumor-specific VLRB.

[0238] Additionally or alternatively, the disclosed chimeric antibodies can deliver a conjugated cargo to a target cell, as introduced above, such as a cytotoxic or diagnostic agent to a tumor cell, an immune response inducer, such as a proinflammatory cytokine to an immune cell, etc.

[0239] B. Target Cells and Diseases for Treatment Thus, the antigen binding domain, ligand or receptor presented by the VLRB-Ig antibody can be selected based on the intended use and designed to target the desired cell type(s). In some embodiments, one or more of the target cell types are cancer cells. Cancers whose cells can be targeted include carcinomas, gliomas, sarcomas, blood and lymphatic system (including leukemias, lymphomas, e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, solitary plasmacytoma, multiple myeloma), genitourinary system cancers (including prostate cancer, bladder cancer, kidney cancer, urethral cancer, penile cancer, testicular cancer), nervous system cancers (including meningiomas, gliomas, glioblastomas, ependymomas), head and neck cancers (including oral, nasal, nasopharyngeal, oropharyngeal ... These include, but are not limited to, cancers of the following organs: uterine cavity, larynx, and paranasal sinuses (including squamous cell carcinoma), lung cancer (including small cell and non-small cell lung cancer), gynecological cancers (including cervical, endometrial, vaginal, vulvar, ovarian, and fallopian tube cancer), gastrointestinal cancers (including gastric, small intestine, colorectal, liver, hepatobiliary, and pancreatic cancer), skin cancers (including melanoma, squamous cell carcinoma, and basal cell carcinoma), breast cancer (including ductal and lobular carcinoma), and childhood cancers (including neuroblastoma, Ewing's sarcoma, Wilms' tumor, and medulloblastoma).

[0240] In some embodiments, one or more of the target cell types are immune cells, including, but not limited to, T lymphocytes, natural killer cells, dendritic cells, antigen-presenting cells, B cells, and macrophages.

[0241] In some preferred embodiments, the chimeric antibody is bispecific or multispecific for a tumor cell type and an immune cell type.

[0242] In some embodiments, the chimeric antibody is a bispecific T cell engager (BiTE). BiTEs are reviewed in Tian, ​​et al. "Bispecific T cell engagers: an emerging therapy for management of hematologic malignancies." J Hematol Oncol 14, 75 (2021), pages 1-18, doi.org / 10.1186 / s13045-021-01084-4, the entirety of which is hereby specifically incorporated by reference herein. BiTEs typically target immune cell targets, e.g., CD3 and tumor antigens simultaneously. BiTEs can be used to induce immune responses against cancers (e.g., tumors) bearing tumor antigens. In some embodiments, the VLRB domain targets the tumor antigen and another binding / targeting domain (e.g., ScFv, VHH, VH / VL, R and / or L domains) targets the immune cell. In other embodiments, the VLRB domain targets immune cells and another binding / targeting domain (e.g., ScFv, VHH, VH / VL, R and / or L domains) targets a tumor antigen. Exemplary BiTE constructs are discussed herein and experimentally demonstrated in the Examples below.

[0243] C. Combination therapy The disclosed chimeric antibody compositions can be administered in conjunction with prophylactic or therapeutic vaccines that can be used to initiate or enhance a subject's immune response to an existing antigen, e.g., a tumor antigen in a subject with cancer. The combination can be administered in the same or separate mixtures.

[0244] The desired outcome of preventive, therapeutic or desensitized immune response may vary according to the disease, according to the principles well known in the art. Similarly, immune response against cancer, allergens or infectious agents may treat the disease completely, alleviate symptoms, or be one aspect of the overall therapeutic intervention against the disease. For example, the stimulation of immune response against cancer may be coupled with surgical, chemotherapeutic, radiological, hormonal and other immunological approaches to affect treatment. Treatment administered in addition to the first therapeutic agent to eradicate tumors may be called adjuvant therapy. Adjuvant treatment is given to reinforce the primary treatment, e.g., surgery or radiation, to reduce the possibility of cancer recurrence. This additional treatment may result in the amplification of the primary response, as evidenced by a stronger and / or prolonged response.

[0245] There are five main types of adjuvant therapy (note that some of these may also be used as primary / solo therapy): 1.) chemotherapy, which uses drugs to kill cancer cells, either by preventing their proliferation or by causing the cells to self-destruct; 2.) hormonal therapy, which reduces hormone production and prevents cancer growth; 3.) radiation therapy, which uses high-powered rays to kill cancer cells; and 4.) immunotherapy, which attempts to influence the body's own immune system to attack and eradicate any remaining cancer cells. Immunotherapies can either stimulate the body's own defenses (cancer vaccines) or complement them (passive administration of antibodies or immune cells), while targeted therapies target specific molecules present in cancer cells, leaving normal healthy cells alone. For example, many cases of breast cancer are caused by tumors that produce excessive amounts of a protein called HER2. Trastuzumab (Herceptin) is used as an adjuvant therapy to target HER2-positive tumors.

[0246] Typically, adjuvant treatments are co-administered or given in conjunction with primary treatments to induce multiple mechanisms and increase the likelihood of eradicating the tumor. Immunotherapy, and vaccines in particular, offer the unique advantage of inducing long-lasting antitumor effects with excellent specificity and the ability to circumvent existing immune tolerance.

[0247] In some embodiments, the disclosed chimeric antibody is administered as a secondary therapeutic agent after administration of a first therapeutic agent, e.g., a cancer therapeutic agent. In other embodiments, the disclosed chimeric antibody is a primary therapeutic agent and is administered before a secondary therapeutic agent. The timing of administration of the secondary therapeutic agent can range from day 0 to day 14 after the primary treatment and can include single or multiple treatments. In certain embodiments, the VLRB-Ig antibody is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after administration of the primary treatment or before the secondary agent.

[0248] In some embodiments, the second agent is one of the cargos discussed above. For example, in some embodiments, representative therapeutic agents include chemotherapeutic agents and pro-apoptotic agents, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin ... These include, but are not limited to, covorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof, and / or immune checkpoint inhibitors, such as PD-1, CTLA4 and B7-H1 antagonists, such as anti-PD-1, anti-B7-H1 and anti-CTLA4 antibodies.

[0249] D. Further Methods Additional therapeutic, diagnostic and research-based methods are also provided. For example, chimeric antibodies can be used to detect selected agents, to block the activity of selected agents, to purify agents, as imaging tools, and as therapeutic agents.

[0250] Provided herein is a method for detecting a drug in a sample, comprising contacting the sample with a chimeric antibody under conditions that allow the composition to bind to the drug in the sample, and detecting the composition bound to the drug in the sample. The bound composition indicates the drug in the sample. Detection methods are well known in the art. For example, the chimeric antibody can be labeled with a detectable tag. Detection methods can be used to indicate the presence or absence of a drug in a sample. However, detection methods can be further combined with quantification methods. In vitro assay methods include colorimetric assays, such as ELISA, that allow quantification of the drug based on comparison with a control sample, or a sample with a known amount of drug, which can be used to establish the amount compared to a standard. These methods can also include radiometric assays that allow quantification based on emitted radiation, and fluorescent assays, or any of the above visualization and quantification means.

[0251] The sample can be any sample to be tested, including any biological sample. Samples can include fluid samples (e.g., water, blood, urine, etc.), tissue samples, culture samples, cellular samples, etc.

[0252] Chimeric antibodies can also be used to block the activity of any drug to which they bind, in comparison to blocking antibodies. Thus, also disclosed is a method of blocking the activity of a drug, comprising contacting the drug with a chimeric antibody under conditions in which the composition binds to the drug. The binding of the composition to the drug blocks the activity of the drug. The contacting step can be in vivo or in vitro. Thus, for example, to reduce sample contamination, a chimeric antibody that binds to a toxin can be added to the sample and block the toxin activity.

[0253] Chimeric antibodies can also be used to enhance the activity of the agent to which they bind, comparable to agonistic antibodies. Thus, also disclosed is a method of enhancing the activity of an agent, comprising contacting the agent with a composition under conditions in which the composition binds to the agent. Binding of the composition to the agent enhances the activity of the agent.

[0254] The chimeric antibodies disclosed herein can be used to determine the function of genes with unknown function. Thus, methods of using the disclosed chimeric antibodies in protein knockdown assays are disclosed herein. For example, the disclosed compositions can be expressed in the cytoplasm of cells that contain genes of unknown function. When RNA transcripts are translated in the cytoplasm of cells, the disclosed compositions can bind to the protein product of the gene in question. By monitoring the effect that loss of protein expression has on cells, the function of the protein can be determined. Thus, chimeric antibodies specific to gene products of unknown function are specifically disclosed. Also provided is a method of determining the function of a gene, comprising introducing a chimeric antibody specific to the protein product of the gene into the cytoplasm of cells expressing the gene, and monitoring the effect caused by the loss of the protein product of the gene with unknown function.

[0255] The chimeric antibody can also be used in imaging methods. For example, the imaging method can include administering an effective amount of the disclosed composition to a subject and detecting the localization of the bound composition in the subject. Examples of imaging methods are described above.

[0256] Methods of purification are provided. Disclosed herein are methods of purifying drugs from samples, which may include contacting the sample with a chimeric antibody under conditions in which the composition binds to the drug and forms a composition / drug complex; and isolating the drug from the composition / drug complex. For example, the composition can be bound to a column, and the sample can be passed through the column under conditions that allow the drug in the sample to bind to the bound composition. The drug can subsequently be eluted from the column in a desired eluent. Purification methods are useful as research methods and commercial methods. For example, such methods are useful in removing contaminants from pharmacological compounds.

[0257] VII. Exemplary Embodiments Non-limiting exemplary embodiments are shown in Figures 2-6. Figure 2 is an illustration of an engineered VLRB:CL-VLRB:CH1 tetravalent monospecific IgG chimera. Tetravalent monospecific MM3 VLRB human IgG1, kappa constructs have been produced by recombinant expression in high yield CHO cell lines by replacing the VL and VH sequences of the human IgG1 VHCH genes and the human VLC kappa genes with the MM3 VLRB sequences.

[0258] Figure 3A is an example of an engineered tetravalent VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus of one of the IgG H chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figure 3B is an example of an engineered trivalent VLRB IgG with a monovalent anti-CD3 scFv attached to the N-terminus of one of the IgG H chains (the "knob" H chain), using the knob-in-hole method to ensure correct H chain pairing. Figures 3C and 3D show two methods for constructing a VLRB IgG with a monovalent anti-CD3 scFv attached to the C-terminus (3C) or N-terminus (3D) of one of the IgG light chains, using the inversion of CL and CH1 on one of the H / L chain pairs ("crossmab") and "knob-in-hole" to ensure correct H chain pairing.

[0259] FIG. 4 is an illustration of an engineered bivalent, bispecific VLRB-IgG chimera composed of an anti-CD3 monoclonal antibody with a VLRB fused to the C-terminus of each CH3 domain.

[0260] FIG. 5 is an illustration of a bivalent anti-CD3, bivalent VLRB antibody having 2× anti-CD3 mAb VL and VH, and 2× VLRB fused to the C-terminus of the CL domain, where the heavy chain further comprises a CH1, CH2 and CH3 domain.

[0261] FIG. 6 is an illustration of a VLRB-scFv chimera, where the VLRB domains can be 4× monospecific, 2× bispecific, 1× monospecific and 1× trispecific, or 1× tetraspecific, and the scFvs can be the same (i.e., bivalent monospecific) or different (i.e., monovalent bispecific).

[0262] In an alternative exemplary embodiment, the anti-CD3 or anti-CD3 scFv domains of the above figures are replaced with an anti-CD8 antibody (or scFv) or CD8 ligand domain. An exemplary CD8 ligand is a thymic leukemia antigen. These antibodies are designed to recruit cytotoxic T cells, all of which express CD8 and CD3, but not regulatory or other inhibitory T cells, which also express CD3 but not CD8, and preferably avoid recruitment of CD4 T cells. See, e.g., Clement, et al., J Immunol., 187(2): 654-663. doi:10.4049 / jimmunol.1003941 (2011); Tsujimura, et al., International Immunology, Vol. 15, No. 11, pp. 1319-1326 doi: 10.1093 / intimm / dxg131 (2003); and WO 2014 / 164553, each of which is hereby specifically incorporated by reference in its entirety.

[0263] In these embodiments, MM3 VLRB can bind to plasmacytoma tumor cells and recruit and activate T cells (via binding to CD3 or CD8) to lyse the tumor cells.

[0264] Exemplary, non-limiting fusion proteins include those utilized in the examples below: (1) VLRB human IgG1 Fc fusion protein (a) Bivalent MM3 VLRB 2 Human IgG1 Fc Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 Hinge-C H 2-C H 3 Fc sequence (italics) [ka] [ka] * The C-terminal lysine was removed according to Cai, et al. (2011) Biotechnol Bioeng 108: 404 - 412 doi: 10.1002 / bit.22933.

[0265] Other VLRB human IgG1 Fc fusion proteins have been reported to inhibit O13 VLRB 2 A human IgG1 Fc fusion protein was generated by replacing the MM3 VLRB sequence with that of a new VLRB, as shown below, while maintaining all other sequences of the above construct.

[0266] (b) Bivalent O13 VLRB 2 Human IgG1 Fc Signal sequence (dashed underline) O13 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 Hinge-C H 2-C H 3 Fc sequence (italics) [ka] * The C-terminal lysine was removed according to Cai, et al. (2011) Biotechnol Bioeng 108: 404 - 412 doi: 10.1002 / bit.22933.

[0267] (2) Tetravalent MM3 VLRB 4 Human IgG1 (a) MM3 VLRB human IgG1 heavy chain construct Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 Array (Italics) [ka]

[0268] (b) MM3 VLRB human Ig kappa light chain construct Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human Ig kappa C L Array (Italics) [ka]

[0269] (3) MM3 VLRB 4 :TR66 anti-CD3 scFv human IgG1 BiTe Utilizing "knobs-in-holes" technology to facilitate precise H-chain pairing and LALA PG mutations to silence FcR1, II and III binding, while retaining the desired blood T of IgG1 antibodies. 1 / 2 FcRn binding is maintained to confer All constructs are designed to increase / enhance tumor binding by incorporating multivalent MM3 VLRB presentation, as well as to avoid T cell activation in the absence of tumor engagement, i.e., cross-linking of CD3 leading to non-specific T cell activation and killing, by incorporating monovalent anti-CD3 scFv presentation.

[0270] (a) MM3 VLRB human IgG1 H chain S354C / T366W "knob" with C-terminal TR66 anti-CD3 scFv construct with L234A / L235A / P329G FcR1, II, III silencing changes Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 sequences (Italics / bold / single underlined L344A / L345A / P439G (also referred to elsewhere as "L234A / L235A / P329G") "LALA PG" FcR1, RII, RIII silencing; Italics with S464C / T476W (also referred to elsewhere as "S354C / T366W") "knob" mutations) TR66 anti-CD3 scFv sequence (double underlined) [ka]

[0271] (b) MM3 VLRB human IgG1 H chain Y349C / T366S / L368A / Y407V "hole" construct with L234A / L235A / P329G FcR1, II, III silencing changes Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 sequences (italics / bold / single underlined with L344A / L345A / P439G (also referred to elsewhere as "L234A / L235A / P329G") "LALA PG" FcR1, RII, RIII silencing; italics with Y459C / T476S / L478A / Y517V (also referred to elsewhere as "Y349C / T366S / L368A / Y407V") "hole" mutations) [ka]

[0272] (4) MM3 VLRB 4 :High affinity anti-CD3 scFv human IgG1 BiTe (a) MM3 VLRB knob heavy chain with a high affinity anti-CD3 scFv at the C-terminus Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 Sequence (italic / bold / italic with "knob" mutations shown in single underline) High affinity anti-CD3 scFv sequence (double underlined) [ka]

[0273] (b) Knob H chain with a high affinity anti-CD3 scFv attached to its N-terminus Signal sequence (dashed underline) High affinity anti-CD3 scFv sequence (double underlined) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 Sequence (italic / bold / italic with "knob" mutations shown in single underline) [ka] [ka]

[0274] In some embodiments, SEQ ID NO:53 forms a tetrameric antibody with itself and / or one or more MM3 VLRB L chains (eg, SEQ ID NO:54).

[0275] In some embodiments, SEQ ID NO:55 forms a tetrameric antibody with a MM3 VLRB hole heavy chain (eg, SEQ ID NO:56) and / or one or more MM3 VLRB light chains (eg, SEQ ID NO:54).

[0276] In some embodiments, SEQ ID NO:65 forms a tetrameric antibody with a MM3 VLRB hole heavy chain (eg, SEQ ID NO:56) and / or one or more MM3 VLRB light chains (eg, SEQ ID NO:54).

[0277] In some embodiments, SEQ ID NO:66 forms a tetrameric antibody with a MM3 VLRB hole heavy chain (eg, SEQ ID NO:56) and / or one or more MM3 VLRB light chains (eg, SEQ ID NO:54).

[0278] The disclosed invention can be further understood by the following numbered paragraphs: Paragraph 1. A heavy chain fusion protein comprising one or more variable lymphocyte receptor B (VLRB) antigen binding domains and a CH1 immunoglobulin domain (CH1) or a CL immunoglobulin domain (CL), optionally wherein the VLRB is at the N-terminus of the fusion protein, the C-terminus of the fusion protein, or a combination thereof. Paragraph 2. The heavy chain fusion protein of Paragraph 1, further comprising an immunoglobulin hinge domain (hinge). Paragraph 3. The heavy chain fusion protein of paragraphs 1 or 2, further comprising a CH2 immunoglobulin domain (CH2). Paragraph 4. The heavy chain fusion protein of any one of paragraphs 1 to 3, further comprising a CH3 immunoglobulin domain (CH3). Paragraph 5. The heavy chain fusion of any one of Paragraphs 1 to 4, further comprising a CH4 immunoglobulin domain (CH4). Paragraph 6. The heavy chain fusion protein of any one of Paragraphs 1 to 5, further comprising a second (VLRB) antigen binding domain. Paragraph 7. A heavy chain fusion protein according to any one of Paragraphs 1 to 6, further comprising a variable region of an immunoglobulin heavy chain (VH), optionally wherein the VH is at the N-terminus of the fusion protein, and wherein the VLRB antigen binding domain and the VH domain are fused to different termini of the fusion protein. Paragraph 8. A heavy chain fusion protein according to any one of paragraphs 1 to 7, optionally further comprising one or more of a mono- or multivalent single chain variable fragment (ScFv), a VHH, a polypeptide ligand (L) or a polypeptide receptor (R) at the N-terminus or C-terminus of the fusion protein, wherein the VLRB antigen binding domain and the mono- or multivalent single chain variable fragment (ScFv), VHH, polypeptide ligand (L) or polypeptide receptor (R) are at different ends of the fusion protein. Paragraph 9. A heavy chain fusion protein according to any one of paragraphs 1 to 9, comprising a domain structure of Table 1. Paragraph 10. A heavy chain fusion protein comprising a VLRB antigen binding domain and a structure of Table 1. Paragraph 11. The heavy chain fusion protein of any one of Paragraphs 1 to 10, wherein one or both of the VLRB antigen binding domains bind to a cancer or tumor antigen or an antigen expressed by an immune cell. Paragraph 12. A heavy chain fusion protein according to any one of paragraphs 8 to 11, wherein one or more of the VH, ScFv, VHH, L or R binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon. Paragraph 13. The heavy chain fusion protein of any one of Paragraphs 1 to 12, wherein each of said immunoglobin domains is independently selected from mammalian, optionally human, IgA, IgD, IgE, IgG and IgM, or a variant thereof having 70% sequence identity thereto. Paragraph 14. The heavy chain fusion protein of Paragraph 13, wherein said IgG is IgG1, IgG2, IgG3 and / or IgG4, and / or said IgA is IgA1 and / or IgA2. Paragraph 15. A heavy chain fusion protein according to any one of Paragraphs 1 to 14, comprising the structure CH1-hinge-CH2-CH3 or CL-hinge-CH2-CH3, together with an N-terminal and / or C-terminal VLRB domain. Paragraph 16. The heavy chain fusion protein of any one of Paragraphs 1 to 15, wherein the CL domain is SEQ ID NO: 23 or 24, or a variant thereof having at least 70% sequence identity thereto. Paragraph 17. A heavy chain fusion protein according to any one of Paragraphs 1 to 16, wherein the CH1, CH2 and / or CH3 comprises a CH1, CH2 and / or CH3 sequence of SEQ ID NO: 25, 26, 62, 63 or 64, or a variant thereof having at least 70% sequence identity thereto. Paragraph 18. A heavy chain fusion protein according to any one of Paragraphs 1 to 17, comprising an amino acid sequence of SEQ ID NO: 25, 26, 62, 63, 64, 65 or 66, or a variant thereof having at least 70% sequence identity thereto. Paragraph 19. A heavy chain fusion protein according to any one of paragraphs 1 to 18, comprising the amino acid sequence of SEQ ID NO: 53, 55 or 56, with or without a signal sequence, or a variant thereof having at least 70% sequence identity thereto, optionally wherein the VLRB antigen binding domain is not mutated compared to SEQ ID NO: 53, 56 or 56. Paragraph 20. A heavy chain fusion protein comprising the amino acid sequence of one of SEQ ID NOs: 53, 55 or 65, with or without a signal sequence. Paragraph 21. A light chain fusion protein comprising one or two variable lymphocyte receptor B (VLRB) antigen binding domains and a CL or CH1 domain, wherein said VLRB antigen binding domains are the same or different, and optionally said VLRB antigen binding domain is N-terminal to the CL or CH1 domain, C-terminal to the CL or CH1 domain, or a combination thereof. Paragraph 22. The light chain fusion protein of Paragraph 21, optionally further comprising a variable region of an immunoglobulin light chain (VL), an ScFv, a VHH, an L or an R at the N-terminus or C-terminus of the fusion protein, wherein the VLRB antigen binding domain and the monovalent or multivalent single chain variable fragment (ScFv), VHH, polypeptide ligand (L) or polypeptide receptor (R) are at different ends of the fusion protein. Paragraph 23. A light chain fusion protein according to Paragraph 22, comprising a domain structure in Table 2. Paragraph 24. A light chain fusion protein comprising one or more VLRB antigen binding domains and a domain structure of Table 2. Paragraph 25. The light chain fusion protein of any one of Paragraphs 21 to 24, wherein the VLRB antigen binding domain binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon. Paragraph 26. The light chain fusion protein of any one of Paragraphs 22 to 25, wherein the variable region (VL) of an immunoglobulin light chain, ScFv, VHH, L or R binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon. Paragraph 27. The light chain fusion protein of any one of Paragraphs 1 to 26, wherein each of said immunoglobin domains is independently selected from mammalian, optionally human, IgA, IgD, IgE, IgG and IgM, or a variant thereof having 70% sequence identity thereto. Paragraph 28. The light chain fusion protein of Paragraph 27, wherein said IgG is IgG1, IgG2, IgG3 and / or IgG4, and / or said IgA is IgA1 and / or IgA2. Paragraph 29. A light chain fusion protein according to any one of Paragraphs 21 to 28, consisting of one or two of said VLRB antigen binding domains or one VLRB antigen binding domain and, optionally, an immunoglobulin light chain (VL), ScFv, VHH, L or R at the N-terminus or C-terminus of said fusion protein, wherein said VLRB antigen binding domain and a monovalent or multivalent single chain variable fragment (ScFv), VHH, polypeptide ligand (L) or polypeptide receptor (R) are fused to the N-terminus and C-terminus of the CL or CH domain. Paragraph 30. The light chain fusion protein of any one of Paragraphs 21 to 29, wherein the CL domain is SEQ ID NO: 23 or 24, or a variant thereof having at least 70% sequence identity thereto. Paragraph 31. The light chain fusion protein of any one of Paragraphs 21 to 29, wherein the CH1 comprises the sequence of CH1 of SEQ ID NO: 25, 26, 62, 63, 64, 65 or 66, or a variant thereof having at least 70% sequence identity thereto. Paragraph 32. A light chain fusion protein according to any one of Paragraphs 21 to 31, comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least 70% sequence identity thereto. Paragraph 33. A light chain fusion protein according to any one of paragraphs 1 to 18, comprising the amino acid sequence of SEQ ID NO: 54, with or without a signal sequence, or a variant thereof having at least 70% sequence identity thereto, optionally wherein the VLRB antigen binding domain is not mutated compared to SEQ ID NO: 54. Paragraph 34. A light chain fusion protein comprising the amino acid sequence of SEQ ID NO:54, with or without a signal sequence. Paragraph 35. A heavy chain according to any one of Paragraphs 1 to 20 and / or a heavy chain according to any one of Paragraphs 21 to 33, further comprising an active drug cargo conjugated thereto. Paragraph 36. A nucleic acid encoding a fusion protein according to any one of paragraphs 1 to 34. Paragraph 37. The nucleic acid of Paragraph 36, further comprising an expression control sequence. Paragraph 38. A cell comprising a nucleic acid according to Paragraph 36 or 37. Paragraph 39. A chimeric antibody comprising two heavy chain fusion proteins according to any one of paragraphs 1 to 20 and two light chain fusion proteins according to any one of paragraphs 21 to 34. Paragraph 40. The antibody of Paragraph 39, wherein the two heavy chain fusion proteins are the same. Paragraph 41. The antibody of Paragraph 39, wherein the two heavy chain fusion proteins are different. Paragraph 42. The antibody of any one of Paragraphs 39 to 41, wherein the two light chain fusion proteins are the same. Paragraph 43. The antibody of any one of Paragraphs 39 to 42, wherein the two light chain fusion proteins are different. Paragraph 44. The antibody of any one of Paragraphs 39 to 43, which is monospecific. Paragraph 45. An antibody according to any one of Paragraphs 39 to 43 which is bispecific. Paragraph 46. An antibody according to any one of Paragraphs 39 to 43, which is multispecific. Paragraph 47. An antibody according to any one of Paragraphs 39 to 47, comprising a structure according to any one of Table 3, Table 4, or Figures 2, 3A-3D, 4, 5 or 6. Paragraph 48. A chimeric antibody comprising SEQ ID NO:53 by itself and / or one or more MM3 VLRB light chains, optionally with SEQ ID NO:54, which forms a tetrameric antibody structure. Paragraph 49. A chimeric antibody comprising at least one VLRB antigen binding domain, optionally comprising an MM3 VLRB whole heavy chain, optionally SEQ ID NO: 56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO: 55, which form a tetrameric antibody with SEQ ID NO: 54. Paragraph 50. A chimeric antibody comprising at least one VLRB antigen binding domain, optionally comprising a MM3 VLRB whole heavy chain, optionally SEQ ID NO:56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO:65, which form a tetrameric antibody structure with SEQ ID NO:54. Paragraph 51. A chimeric antibody comprising at least one VLRB antigen binding domain, optionally comprising a MM3 VLRB whole heavy chain, optionally SEQ ID NO:56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO:66, which form a tetrameric antibody structure with SEQ ID NO:54. Paragraph 52. A chimeric antibody comprising two heavy chains independently selected from Table 1 and two light chains independently selected from Table 2, wherein the chimeric antibody comprises at least one VLRB antigen binding domain. Paragraph 53. A chimeric antibody comprising a structure as set forth in any of Table 3, Table 4, or Figures 2, 3A-3D, 4, 5 or 6, wherein the chimeric antibody comprises at least one VLRB antigen binding domain. Paragraph 54. A chimeric antibody comprising a dimer of a VLRB antigen-binding domain fused to a hinge-CH2-CH3. Paragraph 55. The antibody of Paragraph 54, comprising a dimer of the amino acid sequence of any one of SEQ ID NOs: 51 or 52. Paragraph 56. An antibody according to any one of Paragraphs 39 to 55, which is capable of binding to a cancer or tumor antigen. Paragraph 57. An antibody according to any one of Paragraphs 39 to 56, which is capable of binding to an immune cell. Paragraph 58. The antibody of any one of Paragraphs 39 to 57, which is capable of binding to both a cancer or tumor antigen and an immune cell. Paragraph 59. The antibody of Paragraph 57 or 58, wherein the immune cell is a natural killer (NK) cell or a macrophage. Paragraph 60. The antibody of any one of Paragraphs 39 to 59, having antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP) activity. Paragraph 61. The antibody of any one of Paragraphs 39 to 62, which is capable of inducing T cell activation, T cell proliferation, T cell killing of target cells, or a combination thereof. Paragraph 62. The antibody of any one of Paragraphs 39 to 61, comprising an active drug cargo conjugated thereto. Paragraph 63. A composition comprising an antibody according to any one of Paragraphs 39 to 62. Paragraph 64. A composition according to Paragraph 63, in an effective amount for inducing a therapeutic or diagnostic result in a subject in need thereof. Paragraph 65. A composition according to Paragraph 63 or 64 in a formulation suitable for parenteral or enteral administration. Paragraph 66. A method of treating a subject in need thereof, comprising administering to the subject a composition described in any one of Paragraphs 63 to 65. Paragraph 67. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition according to any one of Paragraphs 63 to 65. Paragraph 68. A method of treating a subject for cancer, comprising administering to the subject a composition described in any one of Paragraphs 63 to 65. Paragraph 69. The method of Paragraph 68, wherein the antibody binds to cells of the cancer. Paragraph 70. A method of treating a subject for an infectious disease, comprising administering to the subject a composition described in any one of Paragraphs 63 to 65. Paragraph 71. The method of Paragraph 70, wherein the antibody binds to an infected cell. Paragraph 72. The method of any one of Paragraphs 66 to 71, wherein the antibody binds to one or more immune cell types. Paragraph 73. The method of any one of Paragraphs 66 to 72, comprising an immune response selected from antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP) activity. Paragraph 74. Any of the preceding paragraphs, wherein the antibody comprises an anti-CD3 antigen binding domain / antibody specific for T cells, or comprises an anti-CD8 antigen binding domain or a CD8 ligand T-cell antigen and is specific for cytotoxic T cells. Paragraph 75. The fusion protein and / or antibody of any of the preceding paragraphs, wherein one or more of said VLRB domains is an ScFv domain comprising an anti-CD3 or anti-CD8 antigen binding domain or an MM3 VLRB antigen binding domain optionally combined with a CD8 ligand, optionally wherein said ligand is a thymic leukemia antigen. Paragraph 76. Any of the preceding paragraphs comprising an anti-CD3 binding domain of SEQ ID NO:22 or SEQ ID NO:34. Paragraph 77. An antibody described in any one of Paragraphs 68 to 70, having the structure of any one of Figures 3A, 3B, 3C, 3D, 4, 5 or 6. Paragraph 78. A fusion protein comprising a VLRB antigen-binding domain fused to a hinge-CH2-CH3. Paragraph 79. A fusion protein according to Paragraph 78, comprising the amino acid sequence of any one of SEQ ID NOs: 51 or 52. EXAMPLES

[0279] Example 1 Construction of chimeric VLRB-IgG antibodies, their binding, and their immune response modulation material and method Cells and reagents. All human hematopoietic cell lines were provided by M. Cooper (Emory University, Atlanta, Georgia, USA) and cultured in RPMI 1640 (complete medium) supplemented with glutamine, 100 U / ml penicillin-streptomycin, 50 μM 2-mercaptoethanol and 10% FBS. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2. De-identified anticoagulant-treated human blood samples and serum were provided by Yerkes Primate Research Center (Emory University). Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation in Lymphoprep™ gradient medium (Stem Cell Technologies, Cat. No. 07801). Mouse monoclonal Abs against cell surface antigens CD19, CD38, CD3, BCMA, CD4, CD8, CD25 and CD69; the CD38-specific therapeutic antibody Darzalex (daratumumumab) and fluorophore-labeled goat anti-mouse IgG secondary Abs were commercially sourced.

[0280] Monoclonal VLRB 2 Human IgG1 Fc, MM3 VLRB 4 Human IgG1 and MM3 VLRB 4 :Anti-CD3 scFv human IgG1 fusion protein. Monoclonal VLRB Ab MM3(Yu, et al., "Identification of human plasma cells with a lamprey monoclonal antibody." JCI Insight. 2016;1(3). Epub 2016 / 05 / 07. doi: 10.1172 / jci.insight.84738. PubMed PMID: 27152361; PMCID: PMC4854299.), N8(Chan, et al., "A tyrosine sulfation-dependent HLA-I modification identifies memory B cells and plasma cells." Sci Adv. 2018;4(11):eaar7653. Epub 2018 / 11 / 13. doi: 10.1126 / sciadv.aar7653. PubMed PMID: 30417091; PMCID: PMC6221509.) and O13 (Collins, et al., "Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates." Structure. 2017;25(11):1667-78 e4. Epub 2017 / 10 / 11. doi: 10.1016 / j.str.2017.09.003. PubMed PMID: 28988747; PMCID: PMC5677568.) have been previously described. 2 Human IgG1 Fc and tetravalent MM3 VLRB 4Human IgG1 fusion proteins (Figures 7 and 8), as well as the MM3 VLRB:anti-CD3 scFv human IgG1 bispecific T cell engager (BiTe) (Figure 9) were produced by Curia-LakePharma using their TunaCHO™ recombinant protein expression technology and purified from culture medium by Protein A affinity chromatography. The BiTe design incorporates "knobs-in-holes" mutations to facilitate precise H-chain pairing (Carter, et al., "Bispecific IgG by Design." J. Immunol. Meth. 248: 7 - 15, (2001)) and "LALA PG" mutations to silence FcRI, FcRII and FcRIII binding (Lo, et al., "Effector-Attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice." J. Biol. Chem. 292: 3900 - 3908, (2017)).

[0281] The BiTe structure is tetravalent for the MM3 VLRB, designed to improve tumor cell binding, and monovalent for the anti-CD3 scFv, designed to avoid CD3 cross-linking and T cell activation in the absence of MM3 VLRB tumor cell engagement. The anti-CD3 scFv sequence is from sequence identifier 65 of WO 2007 / 073499A2. The MM3 VLRB sequence is from sequence identifier 57 of US Pat. No. 10,167,330 B2.

[0282] O13 VLRB antibodies recognize the human O blood group type 2 H trisaccharide antigen (H3), fucose β1,2 galactose β1,4 N-acetylglucosamine.

[0283] Unless otherwise indicated, genes for all constructs were synthesized by LakePharma and the encoded proteins were produced by transient expression using their proprietary vector / CHO cell recombinant protein expression technology and purified from the culture medium by Protein A chromatography.

[0284] (1) VLRB human IgG1 Fc fusion protein (a) Bivalent MM3 VLRB 2 Human IgG1 Fc Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 Hinge-C H 2-C H 3 Fc sequence (italics) [ka] * The C-terminal lysine was removed according to Cai, et al. (2011) Biotechnol Bioeng 108: 404 - 412 doi: 10.1002 / bit.22933.

[0285] Other VLRB human IgG1 Fc fusion proteins have been reported to inhibit O13 VLRB 2 A human IgG1 Fc fusion protein was generated by replacing the MM3 VLRB sequence with that of a new VLRB, while maintaining all other sequences of the above construct, as shown below for the human IgG1 Fc fusion protein.

[0286] (b) Bivalent O13 VLRB 2 Human IgG1 Fc Signal sequence (dashed underline) O13 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 Hinge-C H 2-C H 3 Fc sequence (italics) [ka] [ka] * The C-terminal lysine was removed according to Cai, et al. (2011) Biotechnol Bioeng 108: 404 - 412 doi: 10.1002 / bit.22933.

[0287] (2) Tetravalent MM3 VLRB 4 Human IgG1 (a) MM3 VLRB human IgG1 heavy chain construct Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 Array (Italics) [ka]

[0288] (b) MM3 VLRB human Ig kappa light chain construct Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human Ig kappa C L Array (Italics) [ka]

[0289] (3) MM3 VLRB 4 :TR66 anti-CD3 scFv human IgG1 BiTe Utilizing "knobs-in-holes" technology to facilitate precise H-chain pairing and LALA PG mutations to silence FcR1, II and III binding, while retaining the desired blood T of IgG1 antibodies. 1 / 2 FcRn binding is maintained to confer

[0290] All constructs are designed to increase / enhance tumor binding by incorporating multivalent MM3 VLRB presentation, as well as to avoid T cell activation in the absence of tumor engagement, i.e., cross-linking of CD3 leading to non-specific T cell activation and killing, by incorporating monovalent anti-CD3 scFv presentation.

[0291] (a) MM3 VLRB human IgG1 H chain S354C / T366W "knob" with C-terminal TR66 anti-CD3 scFv construct with L234A / L235A / P329G FcR1, II, III silencing changes Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 sequences (Italics / bold / single underlined L344A / L345A / P439G (also referred to elsewhere as "L234A / L235A / P329G") "LALA PG" FcR1, RII, RIII silencing; Italics with S464C / T476W (also referred to elsewhere as "S354C / T366W") "knob" mutations) TR66 anti-CD3 scFv sequence (double underlined) [ka] [ka]

[0292] (b) MM3 VLRB human IgG1 H chain Y349C / T366S / L368A / Y407V "hole" construct with L234A / L235A / P329G FcR1, II, III silencing changes Signal sequence (dashed underline) MM3 VLRB sequence (bold) Linker sequence (lowercase) Human IgG1 C H 1-hinge-C H 2-C H 3 sequences (italics / bold / single underlined with L344A / L345A / P439G (also referred to elsewhere as "L234A / L235A / P329G") "LALA PG" FcR1, RII, RIII silencing; italics with Y459C / T476S / L478A / Y517V (also referred to elsewhere as "Y349C / T366S / L368A / Y407V") "hole" mutations) [ka] [ka]

[0293] The purity of the recombinant fusion protein was assessed by SE-UPLC chromatography, and the molecular weight was determined by reducing and non-reducing SDS gel electrophoresis. The yield, purity, molecular weight and extinction coefficient for the recombinant fusion protein are shown in Table 10.

[0294] [Table 10]

[0295] Binding assays. Binding of VLRB fusion proteins and other Ab reagents to cells was assayed by flow cytometry using a Miltenyi MACSQuant™ Analyzer 10 Flow Cytometer. Bivalent VLRB 2 Human IgG1 Fc and tetravalent VLRB 4Human IgG1 fusion proteins and daratumumab were directly labeled with fluorophores using the Zenon™ Human IgG Labeling Kit (Thermofisher, Cat. No. Z25402). Cells were incubated with blocking buffer (Southern Biotech, Cat. No. 0060-01) for 20 min on ice, strained through a snap-cap cell strainer (Vendor, Cat. No. 60819-524), transferred to wells of a 96-well microtiter plate at 400,000 cells per well, and isolated by centrifugation at 300×g for 5 min at 4° C. Cells were resuspended in 100 μL PBS-2% FBS containing the indicated concentrations of fluorophore-labeled VLRB fusion proteins, incubated in PBS-2% FBS for 30 min on ice, and washed twice with cold PBS-2% FBS before flow cytometry analysis. For other Ab reagents, cells in microtiter plate wells were resuspended in 100 μL of PBS-2% FBS containing the indicated concentrations of unlabeled Ab reagent for 30 min on ice, after which bound Ab reagent was detected with a fluorophore-labeled goat anti-mouse Ab. Dead cells were excluded by inclusion of propidium iodide (1 μg / ml). Flow cytometry data were analyzed using the FlowJo software package.

[0296] Complement-dependent cytotoxicity (CDC) assay. Activation of complement-dependent cytotoxicity (CDC) by VLRB fusion proteins and other Abs was assessed by flow cytometry with PI staining to quantify dead cells. Indicated concentrations of VLRB fusion proteins and other Abs were incubated with the indicated number of target cells in PBS-2% FBS for 30 min on ice, washed once with cold PBS-2% FBS, resuspended in RPMI 1640 containing 0%, 20% or 40% human serum as a source of complement, and incubated at 37°C and 5% CO before staining with PI and flow cytometry analysis. 2 The plates were incubated at RT in a humidified atmosphere for 4 h.

[0297] Antibody-dependent cellular cytotoxicity (ADCC) assay. Activation of antibody-dependent cellular cytotoxicity (ADCC) by VLRB fusion proteins and other Abs was assessed using human PBMC effector cells by detecting lactate dehydrogenase (LDH) released into the cell culture medium using the Promega ADCC Reporter Bioassay kit (Promega catalog no. G7015). The indicated concentrations of VLRB fusion proteins or other Abs were incubated with the indicated number of target cells in PBS-2% FBS for 30 min on ice, washed with cold PBS-2% FBS, resuspended in complete medium containing the indicated number of Jurkat cells stably expressing human FcγRIIIa V158 (high affinity) linked to NFAT-induced luciferase, and incubated at 37°C and 5% CO. 2 The plates were incubated at RT for 6 h in a humidified atmosphere. Luminescence was measured using a FLUOstar® Omega multimode microplate reader (BMG Labtech).

[0298] PBMC activation assay. Activation of human PBMC T cells by MM3 VLRB BiTe protein was assessed using Jurkat TCR / CD3 NFAT cells (Promega catalog number J1621) by flow cytometric measurement of PBMC T cell CD25 and CD69 cell surface expression. Assays using Jurkat TCR / CD3 NFAT cells were performed according to the kit instructions. For assays using human PBMC, the indicated concentrations of MM3 VLRB BiTe protein were incubated with the indicated number of target cells in PBS-2% FBS for 30 min on ice, washed once with cold PBS-2% FBS, then resuspended in complete medium containing the indicated number of PBMC and incubated at 37° C. and 5% CO 2The cells were incubated overnight at 4°C in a humidified atmosphere and then stained with Ab combinations of anti-CD25 and anti-CD4, anti-CD25 and anti-CD8, anti-CD69 and anti-CD4, and anti-CD69 and anti-CD8 to visualize activation of both CD4 and CD8 PBMC T cells as measured by both CD25 and CD69 cell surface proteins.

[0299] Activation of cytotoxic T cell killing of target cells. Activation of cytotoxic T cell killing of target cells by MM3 VLRB BiTe was assessed using human PBMCs as a source of T cells and Daudi target cells by detecting lactate dehydrogenase (LDH) released into the cell culture medium using the Promega ADCC Reporter Bioassay kit (Promega catalog no. G7015). The indicated concentrations of MM3 VLRB BiTe protein were incubated with the indicated number of target cells in PBS-2% FBS for 30 min on ice, washed with cold PBS-2% FBS, then resuspended in complete medium containing the indicated number of human PBMCs and incubated at 37° C. and 5% CO. 2 The plates were incubated at RT for 4 h in a humidified atmosphere. Luminescence was measured using a FLUOstar® Omega multimode microplate reader (BMG Labtech).

[0300] result Binding properties of VLRB Ig fusion proteins. MM3, N8 and O13 VLRB 2 Human IgG1 Fc, MM3 VLRB 4 Human IgG1 and MM3 VLRB 4The binding properties of the anti-CD3 scFv human IgG1 bispecific T cell engager (BiTe) fusion protein were evaluated by flow cytometry. The results are summarized in Table 11 and exemplary flow cytometry data are shown in Figures 10-13. The specificity of N8 VLRB for tyrosine sulfate-modified HLA class I antigens (Chan, et al. "A tyrosine sulfation-dependent HLA-I modification identifies memory B cells and plasma cells." Sci Adv. 2018;4(11):eaar7653. Epub 2018 / 11 / 13. doi: 10.1126 / sciadv.aar7653. PubMed PMID: 30417091; PMCID: PMC6221509.), and the specificity of O13 VLRB for O blood group 2 H trisaccharide antigen (Collins, et al., "Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates." Structure. 2017;25(11):1667-78 e4. Epub 2017 / 10 / 11. doi: 10.1016 / j.str.2017.09.003. PubMed PMID: 28988747; PMCID: PMC5677568.) and the specificity of MM3 VLRB for higher order structures of CD38, e.g., the tetramer (Yu, et al., "Identification of human plasma cells with a lamprey monoclonal antibody." JCI Insight. 2016;1(3). Epub 2016 / 05 / 07. doi: 10.1172 / jci.insight.84738. PubMed PMID: 27152361; PMCID: PMC4854299.) have been described. The cell line binding specificity of these VLRB fusion proteins shown in Table 11 and Figures 10-13 is consistent with the data reported for the "parent" VLRB antibodies.

[0301] All cell lines known to express CD38 are positive for binding by daratumumab and anti-CD38 HIT mAb. Daudi and Raji, cell lines that express high amounts of CD38, are associated with MM3 VLRB 2 Human IgG1 Fc and MM3 VLRB 4 Cell lines such as BJAB, which are also positive for binding by human IgG1 but express low levels of CD38, are negative for MM3 VLRB binding. 4 Binding of human IgG1 to bivalent MM3 VLRB 2 The specificity of binding is superior to that of human IgG1 Fc. All anti-CD38 reagents bind KMS-11 CD38. - The absence of binding to cells was compared with Daudi, Raji and BJAB N8 for the N8 reagent. - By the absence of binding to cells, as well as by N8 + Weak but not specific N8 VLRB expression in KMS-11 cells 2 This is supported by human IgG1 Fc binding; finally, MM3 likely recognizes a "neotope" that is created when CD38 forms a higher order structure, i.e., a tetramer, and high CD38 expression is likely required to drive the formation of MM3 VLRB-detectable CD38 tetramers via mass action.

[0302] Activation of CDC and ADCC immune effector functions. 14A-14C report the results of an assay to measure activation of CDC and ADCC immune effector functions by VLRB fusion proteins. At the protein amount (0.5 μg) and complement amount (20% human serum) tested, bivalent MM3 VLRB 2 Human IgG1 Fc also tetravalent MM3 VLRB 4The human IgG1 fusion protein also demonstrated no detectable CDC activation activity (Figure 14A). The tetravalent MM3 VLRB human IgG1 fusion protein (Figure 14B), but not the bivalent MM3 VLRB human IgG1 Fc fusion protein (Figure 14C), demonstrated activation of FcγRIII (CD16a)-mediated ADCC effector function, albeit with a much lower potency than the positive control daratumumab IgG1 antibody. 2 MM3 VLRB compared to IgG1 Fc structure 4 The increased valency and avidity-enhanced apparent affinity of the IgG1 structure, although considerably lower than that achieved with the positive control anti-CD38 IgG1 mAb daratumumab, likely results in a higher density of IgG1 Fc binding sites for stable binding and cross-linking of CD16a.

[0303] The absence or low effector activity of the MM3 VLRB fusion protein is consistent with the O13 VLRB 2 This is not an inherent property of VLRB IgG fusion proteins, as shown by the potent CDC and ADCC activation properties of the human IgG1 Fc fusion protein (Figures 15A-15B). The O13 VLRB target, blood group O2H trisaccharide glycan antigen, is the O13 VLRB target due to: 2 IgG1 Fc binding sites are present on the surface of KMS-12 and KMS-18 cells at a density sufficient to "aggregate": (a) stable binding of the C1q component of complement to initiate the antibody-mediated "classical" complement pathway for cell lysis, and (b) stable binding and cross-linking of the CD16a Fc receptor (FcγRIIIa) to activate lysis of the target cell; whereas the cell surface density of CD38 tetramers, which are the MM3 VLRB target of Daudi (and other) cells, is sufficient to "aggregate" the MM3 VLRB for stable binding or cross-linking. 2It is inappropriate for aggregating IgG1 Fc C1q or FcγRIII binding sites. Note that although present on BJAB cells, the H trisaccharide is much less abundant on BJAB cells than on KMS-12 and 18 cells (Figure 13) and its density is insufficient to fully activate ADCC by BJAB target cells (Figure 15B).

[0304] MM3 VLRB 4 : Anti-CD3 scFv human IgG1 T cell activation. Activation of T cells by MM3 VLRB BiTe was assessed using Jurkat TCR / CD3 NFAT effector cells (Promega Cat. No. J1621) (FIG. 16) and human PBMC T cells by flow cytometric measurement of CD25 and CD69 cell surface expression on PBMC T cells (FIGS. 17A-17D). Target cells utilized in the assays are as indicated. Activation of Jurkat TCR / CD3 NFAT cells with Daudi target cells by MM3 VLRB BiTe was comparable to activation with the positive control Blinatumomab anti-CD19 scFv:anti-CD3 BiTe, indicating that CD19 expression is comparable to expression of MM3 CD38 tetramer targets by Daudi cells. It should be noted that the molecular weight of MM3 VLRB BiTe is more than four times larger than that of blinatumomab, and equal weights of the two BiTes correspond to less than one-fourth the molar amount of MM3 VLRB BiTe than blinatumomab. The activation potency of MM3 VLRB BiTe with various target cell lines follows the binding strength of the tetrameric MM3 VLRB human IgG1 fusion protein to these cell lines (see Table 11), indicating that the T cell activation activity of MM3 VLRB BiTe is determined by the molecular target expression density. Human PBMC CD8 and CD4 T cells activated MM3 VLRB BiTe with MM3 VLRB BiTe. hi MM3 VLRB only in the presence of target / activator Daudi cells 4: Activated to express cell surface CD25 by anti-CD3 scFv human IgG1 BiTe, and minimal activation, if any, as measured by CD25 cell surface expression, was observed in MM3 lo MM3 VLRB detected using other target / activator cells including Raji cells, i.e., measured using CD25 cell surface expression 4 : T cell activation by anti-CD3 scFv human IgG1 is antigen-specific (Figures 17A-17D). Human PBMC CD8 and CD4 T cells were activated by MM3 hi Targeting Daudi cells and MM3 lo In the presence of target Raji cells, MM3 VLRB 4 : Activated to express cell surface CD69 by anti-CD3 scFv human IgG1 BiTe, but MM3 - This is not the case when BJAB target cells are present. Activation of human PBMC CD8 T cells, as measured by CD69 cell surface expression and to a lesser extent by CD25 cell surface expression, was significantly increased in KMS-11 MM3 - MM3 VLRB in the presence of target cells 4 : seen with anti-CD3 scFv human IgG1 BiTe, indicating that KMS-11 cells can be recognized by and activate "normal" human PBMC CD8 T cells. Cell surface expression of CD69 appears to be a more sensitive, or less specific, indicator of T cell activation than cell surface expression of CD25.

[0305] MM3 VLRB in T cell killing 4 : Anti-CD3 scFv human IgG1 activating. The results for activation of human PBMC T cells to lyse Daudi target cells are shown in Figure 18. As shown, MM3 VLRB BiTe dose-dependently "arms" PBMC T cells to kill Daudi cells.

[0306] [Table 11] summary 1) VLRB 2 IgG Fc, VLRB 4 IgG and VLRB 4 : The anti-CD3 scFv IgG fusion protein can be produced in acceptable yields using conventional recombinant protein production methods and purified to acceptable purity using Protein A affinity chromatography (Table 10). 2) The VLRB IgG fusion proteins retain the binding properties of the parent VLRB antibody (Table 11 and Figures 10A-13). 3) VLRB 4 IgG fusion protein is a VLRB 2 It demonstrates superior binding compared to an IgG Fc fusion protein (Table 11 and Figures 10A-10C). 4) VLRB 4 IgG fusion protein is a VLRB 2 Compared to IgG Fc fusion proteins, they demonstrate superior activation of immune effector functions, such as CDC and ADCC (Figures 14A-14C). 5) VLRB 4 :Anti-CD3 scFv IgG BiTe binds to the molecular target recognized by VLRB antibodies and CD3 + and T cells (Figures 11 and 12). 6) to cells expressing targets recognized by VLRB antibodies, and to CD3 + VLRB to T cells 4 : Binding of anti-CD3 scFv IgG BiTe activates T cells to lyse cells recognized by VLRB antibodies. 7) CD3 + VLRB to T cells alone 4 : Binding of anti-CD3 scFv IgG BiTe does not activate T cells to lyse other cells not recognized by the VLRB antibody, i.e., T cell activation by VLRB BiTe is antigen-specific for the antigen recognized by the VLRB antibody.

[0307] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the material for which they are cited are specifically incorporated by reference.

[0308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A heavy chain fusion protein comprising one or more variable lymphocyte receptor B (VLRB) antigen-binding domains and a CH1 immunoglobulin domain (CH1) or a CL immunoglobulin domain (CL), optionally wherein the VLRB is at the N-terminus of the fusion protein, the C-terminus of the fusion protein, or a combination thereof.

2. The heavy chain fusion protein of claim 1 further comprising an immunoglobulin hinge domain (hinge).

3. The heavy chain fusion protein of claim 1 further comprising a CH2 immunoglobulin domain (CH2).

4. The heavy chain fusion protein of claim 1 further comprising a CH3 immunoglobulin domain (CH3).

5. The heavy chain fusion protein of claim 1 further comprising a CH4 immunoglobulin domain (CH4).

6. 2. The heavy chain fusion protein of claim 1, further comprising a second (VLRB) antigen binding domain.

7. 10. The heavy chain fusion protein of claim 1, further comprising a variable region of an immunoglobulin heavy chain (VH), optionally wherein the VH is at the N-terminus of the fusion protein and the VLRB antigen-binding domain and the VH domain are fused to different ends of the fusion protein.

8. The heavy chain fusion protein of claim 1, further comprising one or more of a monovalent or multivalent single-chain variable fragment (ScFv), a VHH, a polypeptide ligand (L) or a polypeptide receptor (R), as appropriate, at the N-terminus or C-terminus of the fusion protein, wherein the VLRB antigen-binding domain and the monovalent or multivalent single-chain variable fragment (ScFv), a VHH, a polypeptide ligand (L) or a polypeptide receptor (R) are at different ends of the fusion protein.

9. 2. The heavy chain fusion protein of claim 1, comprising the domain structure of Table 1.

10. A heavy chain fusion protein comprising a VLRB antigen binding domain and the structure of Table 1.

11. 2. The heavy chain fusion protein of claim 1, wherein one or both of the VLRB antigen binding domains bind to a cancer or tumor antigen or an antigen expressed by an immune cell.

12. 9. The heavy chain fusion protein of claim 8, wherein one or more of the VH, ScFv, VHH, L, or R binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon.

13. 2. The heavy chain fusion protein of claim 1, wherein each of said immunoglobulin domains is independently selected from mammalian, optionally human, IgA, IgD, IgE, IgG and IgM, or variants thereof having 70% sequence identity thereto.

14. 14. The heavy chain fusion protein of claim 13, wherein the IgG is IgG1, IgG2, IgG3 and / or IgG4, and / or the IgA is IgA1 and / or IgA2.

15. 2. The heavy chain fusion protein of claim 1, comprising the structure CH1-hinge-CH2-CH3 or CL-hinge-CH2-CH3 with an N-terminal and / or C-terminal VLRB domain.

16. 16. The heavy chain fusion protein of any one of claims 1 to 15, wherein the CL domain is SEQ ID NO: 23 or 24, or a variant thereof having at least 70% sequence identity thereto.

17. 16. The heavy chain fusion protein of any one of claims 1 to 15, wherein the CH1, CH2 and / or CH3 comprise the CH1, CH2 and / or CH3 sequence of SEQ ID NO: 25, 26, 62, 63 or 64, or a variant thereof having at least 70% sequence identity thereto.

18. 16. The heavy chain fusion protein of any one of claims 1 to 15, comprising the amino acid sequence of SEQ ID NO: 25, 26, 62, 63, 64, 65 or 66, or a variant thereof having at least 70% sequence identity thereto.

19. 16. The heavy chain fusion protein of any one of claims 1 to 15, comprising the amino acid sequence of SEQ ID NO: 53, 55 or 56, with or without a signal sequence, or a variant thereof having at least 70% sequence identity thereto, optionally wherein the VLRB antigen-binding domain is not mutated compared to SEQ ID NO: 53, 56 or 56.

20. A heavy chain fusion protein comprising the amino acid sequence of one of SEQ ID NOs: 53, 55 or 65, with or without a signal sequence.

21. A light chain fusion protein comprising one or two variable lymphocyte receptor B (VLRB) antigen binding domains and a CL or CH1 domain, wherein the VLRB antigen binding domains are the same or different, and optionally the VLRB antigen binding domain is N-terminal to the CL or CH1 domain, C-terminal to the CL or CH1 domain, or a combination thereof.

22. The light chain fusion protein of claim 21, further comprising a variable region of an immunoglobulin light chain (VL), ScFv, VHH, L or R, as appropriate, at the N-terminus or C-terminus of the fusion protein, wherein the VLRB antigen-binding domain and the monovalent or multivalent single-chain variable fragment (ScFv), VHH, polypeptide ligand (L) or polypeptide receptor (R) are at different ends of the fusion protein.

23. 23. The light chain fusion protein of claim 22, comprising the domain structure of Table 2.

24. A light chain fusion protein comprising one or more VLRB antigen binding domains and the domain structure of Table 2.

25. 22. The light chain fusion protein of claim 21, wherein the VLRB antigen binding domain binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon.

26. 22. The light chain fusion protein of claim 21, wherein the variable region of an immunoglobulin light chain (VL), ScFv, VHH, L, or R binds to a cancer or tumor antigen or an antigen expressed by an immune cell thereon.

27. 22. The light chain fusion protein of claim 21, wherein each of said immunoglobulin domains is independently selected from mammalian, optionally human, IgA, IgD, IgE, IgG and IgM, or a variant thereof having 70% sequence identity thereto.

28. 28. The light chain fusion protein of claim 27, wherein the IgG is IgG1, IgG2, IgG3 and / or IgG4, and / or the IgA is IgA1 and / or IgA2.

29. A light chain fusion protein as described in claim 21, comprising: one or two VLRB antigen-binding domains or one VLRB antigen-binding domain and an immunoglobulin light chain (VL), ScFv, VHH, L or R (optionally at the N-terminus or C-terminus of the fusion protein) and the VLRB antigen-binding domain and a monovalent or multivalent single-chain variable fragment (ScFv), VHH, polypeptide ligand (L) or polypeptide receptor (R), fused to the N-terminus and C-terminus of the CL or CH domain.

30. 30. The light chain fusion protein of any one of claims 21 to 29, wherein the CL domain is SEQ ID NO: 23 or 24, or a variant thereof having at least 70% sequence identity thereto.

31. 30. The light chain fusion protein of any one of claims 21 to 29, wherein the CH1 comprises the sequence of CH1 of SEQ ID NO: 25, 26, 62, 63, 64, 65 or 66, or a variant thereof having at least 70% sequence identity thereto.

32. 30. The light chain fusion protein of any one of claims 21 to 29, comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least 70% sequence identity thereto.

33. 30. The light chain fusion protein of any one of claims 21 to 29, comprising the amino acid sequence of SEQ ID NO: 54, with or without a signal sequence, or a variant thereof having at least 70% sequence identity thereto, optionally wherein the VLRB antigen-binding domain is not mutated compared to SEQ ID NO:

54.

34. A light chain fusion protein comprising the amino acid sequence of SEQ ID NO: 54, with or without a signal sequence.

35. 30. A heavy chain fusion protein according to any one of claims 1 to 15 and / or a light chain fusion protein according to any one of claims 21 to 29, further comprising an active drug cargo conjugated thereto.

36. A nucleic acid encoding a heavy chain fusion protein according to any one of claims 1 to 15 and / or a light chain fusion protein according to any one of claims 21 to 29.

37. 37. The nucleic acid of claim 36, further comprising an expression control sequence.

38. A cell comprising the nucleic acid of claim 36.

39. A chimeric antibody comprising two heavy chain fusion proteins and two light chain fusion proteins, wherein each of the heavy chain fusion proteins comprises: (a) a heavy chain fusion protein comprising one or more variable lymphocyte receptor B (VLRB) antigen-binding domains and a CH1 immunoglobulin domain (CH1) or a CL immunoglobulin domain (CL), optionally wherein the VLRB is at the N-terminus of the fusion protein, the C-terminus of the fusion protein, or a combination thereof; or (b) a heavy chain fusion protein comprising a VLRB antigen-binding domain and the structure of Table 1 wherein each of said light chain fusion proteins is: (c) a light chain fusion protein comprising one or two variable lymphocyte receptor B (VLRB) antigen-binding domains and a CL or CH1 domain, wherein the VLRB antigen-binding domains are the same or different, and optionally the VLRB antigen-binding domain is N-terminal to the CL or CH1 domain, C-terminal to the CL or CH1 domain, or a combination thereof; or (d) a light chain fusion protein comprising one or more VLRB antigen-binding domains and the domain structure of Table 2. A chimeric antibody.

40. 40. The antibody of claim 39, wherein the two heavy chain fusion proteins are the same.

41. 40. The antibody of claim 39, wherein the two heavy chain fusion proteins are different.

42. 40. The antibody of claim 39, wherein the two light chain fusion proteins are the same.

43. 40. The antibody of claim 39, wherein the two light chain fusion proteins are different.

44. 40. The antibody of claim 39, which is monospecific.

45. 40. The antibody of claim 39, which is bispecific.

46. 40. The antibody of claim 39, which is multispecific.

47. 40. The antibody of claim 39, comprising a structure as set forth in any of Table 3, Table 4, or Figures 2, 3A-3D, 4, 5, or 6.

48. A chimeric antibody comprising SEQ ID NO:53, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO:54, which forms a tetrameric antibody structure with itself.

49. A chimeric antibody comprising at least one VLRB antigen binding domain, said antibody comprising SEQ ID NO: 55, optionally with an MM3 VLRB hole heavy chain, optionally SEQ ID NO: 56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO: 54, forming a tetrameric antibody.

50. A chimeric antibody comprising at least one VLRB antigen binding domain, said antibody comprising SEQ ID NO: 65, optionally with an MM3 VLRB hole heavy chain, optionally SEQ ID NO: 56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO: 54, that forms a tetrameric antibody.

51. A chimeric antibody comprising at least one VLRB antigen binding domain, said antibody comprising SEQ ID NO: 66, optionally with an MM3 VLRB hole heavy chain, optionally SEQ ID NO: 56, and / or one or more MM3 VLRB light chains, optionally SEQ ID NO: 54, that form a tetrameric antibody.

52. A chimeric antibody comprising two heavy chains independently selected from Table 1 and two light chains independently selected from Table 2, wherein the chimeric antibody comprises at least one VLRB antigen-binding domain.

53. A chimeric antibody comprising a structure as set forth in any of Table 3, Table 4, or Figures 2, 3A-3D, 4, 5, or 6, wherein the chimeric antibody comprises at least one VLRB antigen binding domain.

54. A chimeric antibody comprising a dimer of a VLRB antigen-binding domain fused to a hinge-CH2-CH3.

55. 55. The antibody of claim 54, comprising a dimer of the amino acid sequence of any one of SEQ ID NOs: 51 or 52.

56. 40. The antibody of claim 39, which is capable of binding to a cancer or tumor antigen.

57. 40. The antibody of claim 39, which is capable of binding to an immune cell.

58. 40. The antibody of claim 39, which is capable of binding to both a cancer or tumor antigen and an immune cell.

59. 40. The antibody of claim 39, wherein the immune cell is a natural killer (NK) cell or a macrophage.

60. 40. The antibody of claim 39, having antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP) activity.

61. 40. The antibody of claim 39, which is capable of inducing T cell activation, T cell proliferation, T cell killing of target cells, or a combination thereof.

62. 40. The antibody of claim 39, comprising an active drug cargo conjugated thereto.

63. 63. A composition comprising an antibody according to any one of claims 39 to 62.

64. The composition described in claim 63 in an effective amount for inducing a therapeutic or diagnostic result in a subject in need thereof.

65. 64. The composition of claim 63 in a formulation suitable for parenteral or enteral administration.

66. The composition described in claim 63 for use in a method of treating a subject in need of treatment.

67. The composition described in claim 63 for use in a method for inducing an immune response in a subject in need thereof.

68. 64. The composition of claim 63 for use in a method of treating a subject for cancer.

69. 69. The composition of claim 68, wherein the antibody binds to cells of the cancer.

70. 64. The composition of claim 63 for use in a method of treating a subject for an infectious disease.

71. 71. The composition for use of claim 70, wherein the antibody binds to infected cells.

72. 72. The composition for use of any one of claims 66 to 71, wherein the antibody binds to one or more immune cell types.

73. A composition for use according to any one of claims 66 to 71, wherein the method comprises an immune response selected from antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP) activity.

74. 72. The heavy chain fusion protein of claim 16, or the light chain fusion protein of claim 30, or the nucleic acid of claim 36, or the chimeric antibody of claim 39, or the composition of claim 63, or the composition for use of any one of claims 66 to 71, wherein the antibody comprises an anti-CD3 antigen-binding domain / antibody specific for T cells, or comprises an anti-CD8 antigen-binding domain or a CD8 ligand TL antigen and is specific for cytotoxic T cells.

75. The heavy chain fusion protein of claim 16, or the light chain fusion protein of claim 30, or the nucleic acid of claim 36, or the chimeric antibody of claim 39, or the composition of claim 63, or the composition for use of any one of claims 66 to 71, wherein the fusion protein and / or antibody, wherein one or more of the VLRB domains is an ScFv domain comprising an anti-CD3 or anti-CD8 antigen binding domain or an MM3 VLRB antigen binding domain optionally combined with a CD8 ligand, and optionally the ligand is a thymic leukemia antigen.

76. 72. A heavy chain fusion protein according to claim 16, or a light chain fusion protein according to claim 30, or a nucleic acid according to claim 36, or a chimeric antibody according to claim 39, or a composition according to claim 63, or a composition for use according to any one of claims 66 to 71, comprising an anti-CD3 binding domain of SEQ ID NO: 22 or SEQ ID NO:

34.

77. 72. A heavy chain fusion protein according to claim 16, or a light chain fusion protein according to claim 30, or a nucleic acid according to claim 36, or a chimeric antibody according to claim 39, or a composition according to claim 63, or a composition for use according to any one of claims 66 to 71, having the structure of any one of Figures 3A, 3B, 3C, 3D, 4, 5 or 6.

78. A fusion protein comprising a VLRB antigen-binding domain fused to hinge-CH2-CH3.

79. 79. The fusion protein of claim 78, comprising the amino acid sequence of any one of SEQ ID NOs: 51 or 52.

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