CD23-targeting compositions and uses thereof for improved immunotherapies

Abstract

Immunotherapeutic compositions comprising CD23-binding moieties and uses of the same for treatment of conditions such as autoimmunity and cancer are provided. Bispecific molecules are provided.

Description

[0001] CD23 -TARGETING COMPOSITIONS AND USES THEREOF

[0002] FOR IMPROVED IMMUNOTHERAPIES

[0003] BACKGROUND CD23, known as FceRII, is the low-affinity receptor for immunoglobulin E (IgE) and plays a critical role in regulating IgE responses. As both a membrane-bound glycoprotein and a soluble protein, CD23 is involved in various regulatory processes, including cell-cell interactions and feedback mechanisms within immune responses. The receptor's ability to be cleaved into soluble forms adds to its functional diversity, impacting both cytokine activity and immune regulation, which underscores its potential as a target for therapeutic intervention. CD23 expressed primarily on B cells, monocytes, and follicular dendritic cells. It plays a crucial role in regulating IgE-mediated immune responses, antigen presentation, and B cell activation.

[0004] What is needed is improved compositions and methods for treatment of conditions such as cancer and autoimmunity.

[0005] SUMMARY OF THE INVENTION

[0006] In a first aspect, a bispecific molecule is provided. The molecule includes a) a first binding moiety capable of binding CD23, and b) a second binding moiety capable of binding a molecule expressed on a cell surface, wherein the first binding moiety and second binding moiety are joined by a linker. In certain embodiments, the second binding moiety is capable of binding CD3, optionally wherein the second binding moiety is antibody or fragment thereof. In certain embodiments, first binding moiety is an antibody or fragment thereof comprising six complementarity -determining regions of a) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 20: c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22; d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising tire amino acid sequence of SEQ ID NO: 24; e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; f) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 28; g) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30; or h) an antibodycomprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0007] In another aspect, a CD23-binding molecule is provided. In certain embodiments, the CD23-binding molecule is an antibody or fragment thereof comprising six complementaritydetermining regions of a) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22; d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising tire amino acid sequence of SEQ ID NO: 24; e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; f) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 28; g) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30; or h) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0008] In another aspect, a chimeric antigen receptor (CAR) comprising a) an extracellular domain comprising the CD23-binding molecule as described herein, b) a transmembrane domain; and c) an intracellular signaling domain.

[0009] In another aspect, a method of treating an autoimmune condition in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.

[0010] In another aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.

[0011] In another aspect, a method of treating rheumatoid arthritis in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.

[0012] In another aspect, a method of treating lupus nephritis in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.In another aspect, a method of treating atopic dermatitis in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.

[0013] In another aspect, a method of treating a B-cell malignancy in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of the bispecific molecule as described herein.

[0014] Other aspects and advantages of tire invention will be readily apparent from the following detailed description of the invention.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides an overview of the CD23 targeting strategies.

[0016] FIG. 2A-2C demonstrates functional activity of CD23xCD3 bispecific antibodies (CD23 protein binder-based) in human PBMCs. (a) Dose-dependent depletion of CD23+ target cells within human PBMCs following treatment with CD23 / CD3 bispecific antibodies (0-1 pg / mL). (b) Corresponding concentration-dependent T-cell activation measured by CD69 expression on CD3+T cells. (c) Effect of soluble CD23 (100 U / mL) on antibody activity. The lumiliximab analogue (AB0018) exhibited reduced functional activity in the presence of soluble CD23. whereas AB0170. AB0171. and AB0175 retained activity, indicating resistance to soluble antigen-mediated neutralization.

[0017] FIG. 3 provides a schematic overviews for “protein binder” constructs AB170, AB171, and AB 175 identified in Table 2.

[0018] FIG. 4 depicts the difference between prior art CD23 targeting approaches and tire novel approach described herein.

[0019] FIG. 5 is a binding comparison of CD23 antibodies to full-length, truncated, and soluble / decoy CD23 proteins. Antibody binding was assessed across three CD23 antigen formats: full-length membrane-associated protein, truncated protein, and soluble / decoy CD23. The data show robust binding to full-length and truncated CD23, while binding to the soluble / decoy form is reduced or minimal for select constructs, consistent with epitope recognition that favors membrane-associated CD23 over soluble CD23.

[0020] FIG. 6 shows the results of a flow cytometric analysis of anti-CD23 monoclonal antibody binding to CD23 extracellular domain and full-length protein overexpressed in Jurkat cells.

[0021] FIG. 7 shows CD23 monoclonal antibody clones binding to CD23 receptor on JVM2 cells.

[0022] FIG. 8 shows CD23xCD3 IgG antibody binding to CD23 receptor on JVM2 cells.

[0023] FIG. 9A is a cartoon showing the CD23xCD3 bispecific antibodies (BiTE format)described herein. FIG. 9B is a heatmap depicting dose-dependent depletion of CD23xCD3 constructs. The x-axis denotes individual antibody constructs and the y-axis denotes increasing dose. Signal intensity reflects the percentage of viable CD23+cells remaining, with black indicating 100% viable cells and white indicating 0%.

[0024] FIG. 10A is a cartoon showing the 2+1 IgG architecture of the bi-specific antibodies described herein. FIG. 10B shows dose-response heatmap of CD23+cell depletion across CD23*CD3 constructs. Columns represent individual constructs and rows represent increasing dose. Signal intensity reflects the percentage of viable CD23+ cells remaining, with black indicating 100% viability and white indicating 0%. FIG. 10C shows dose-response heatmap of T-cell activation measured by CD69 expression on CD3+cells in the same wells. Columns represent individual constructs and rows represent increasing dose. Signal intensity reflects the level of CD69 expression, with black indicating maximal activation and white indicating minimal activation.

[0025] FIG. 11A-D are heatmaps depicting dose-dependent cytokine release (IL-1β (FIG. 11A), IFN-γ (FIG. 11B), TNF-α (FIG. 11C), IL-6 (FIG. 11D)) in human PBMCs treated with CD23*CD3 antibodies. Columns represent constructs; rows represent dose. Darker shading indicates higher cytokine levels.

[0026] FIG. 12A is a dose-response heatmap of CD23+cell depletion across CD23xCD3 constructs. Columns represent individual constructs and rows represent increasing dose. Signal intensity reflects the percentage of viable CD23+cells remaining, with black indicating 100% viability and white indicating 0%. FIG. 12B is a dose-response heatmap of T-cell activation measured by CD69 expression on CD3+cells in the same wells. Columns represent individual constructs and rows represent increasing dose. Signal intensity’ reflects the level of CD69 expression, with black indicating maximal activation and white indicating minimal activation.

[0027] FIG. 13A and 13B demonstrate the killing activity of CD23xCD3 antibodies in CLL cell lines in the presence of soluble CD23. FIG. 13A shows JVM2 cells and FIG. 13B shows HG3 cells treated at 5 nM antibody concentration with or without soluble CD23 (1 pg / mL). All eight of our CD23*CD3 constructs retain cytotoxic activity in the presence of soluble CD23, indicating resistance to soluble CD23-mediated neutralization. In contrast, a prior CD23 antibody-based comparator shows reduced killing under the same conditions, consistent with neutralization by soluble CD23.

[0028] FIG. 14A and 14B demonstrate dose-dependent T-cell activation and target cell depletion by CD23*CD3 bispecific antibodies (2+1 IgG format) in Cynomolgus PBMCs. FIG. 18A - T-cell activation. Dose-response heatmap of T-cell activation in cynomolgus PBMCs. Columns represent individual CD23*CD3 antibody constructs and rows represent increasing dose. Signalintensity reflects the percentage of CD69+CD3+T cells, with darker shading indicating higher levels of T-cell activation. FIG. 18B - Target cell depletion. Dose-response heatmap of CD23+target cell depletion in cynomolgus PBMCs. Columns represent antibody constructs and rows represent increasing dose. Signal intensity reflects the percentage of CD23+cells depleted relative to untreated controls, with darker shading indicating greater target cell depletion.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Provided herein are compositions that include CD23-binding moieties with enhanced stability and targeting capabilities. The CD23-biding moieties include antibodies that bind membrane-proximal CD23 epitopes, including the neck and stalk regions, and exhibit reduced binding to soluble CD23. In some embodiments, the CD23-binding moieties include antibodies or fragments thereof, as well as polypeptides capable of binding one or more CD23 domain. The CD23-binding moieties are useful in the context of bispecific molecules that, in addition to a CD23-binding moiety, include a second binding moiety capable of binding a molecule expressed on a cell surface. The CD23-binding moieties are also useful for targeting CD23 expressing cells in the context of engineered chimeric antigen receptors. The CD23-binding moieties are useful in therapeutic applications, including treatment of cancer and autoimmune conditions.

[0031] The development of improved composition for targeting CD23 is informed by past clinical and allergy research outcomes. Traditional antibodies have shown safety and partial efficacy in early-phase trials but encountered challenges in later stages, such as Phase 3 trials for chronic lymphocytic leukemia (CLL). Hurdles to targeting CD23 include: a) neutralization by soluble CD23 proteins, reducing therapeutic efficacy, and b) resistance due to variable antigen expression on B cell clones. Additionally, studies in allergy research have highlighted the role of CD23 in modulating immune responses, further complicating the targeting of this molecule in a clinical setting, as CD23-mediated cell signaling pathways in B cell activation could be altered by other factors, such as a cytokine milieu. The CD23 targeting compositions described herein are engineered to address these issues by simultaneously targeting multiple epitopes or pathways associated with CD23. This approach not only circumvents the effects of soluble CD23 but also leverages synergistic targeting to enhance overall efficacy and reduce the likelihood of cancer cell escape. The features of the CD23-targeting constructs (e.g., antibodies that incorporate advanced scFv or polypeptides that include CD21 sushi domain or a CD23 stalk (or “helical'’) domain, ensure precise and robust engagement with membrane -bound CD23, avoiding the pitfalls associated with its soluble fonn, thus providing improved clinical outcomes in the context of conditions such as cancer and autoimmune-directed therapies. (See FIG. 1)The compositions, including bispecific antibodies described herein, have overcome the problems associated with prior approaches to targeting CD23. Namely, by targeting the nearmembrane portion of CD23, tire antibodies are not neutralized by soluble CD23. Furthermore, as is demonstrated herein, that the therapeutic efficacy associated with CD23 reduction or knockdown, is not diminished due to the novel targeting strategy.

[0032] CD23 Structure and Function

[0033] CD23, also known as FcεRII, is the low-affinity’ receptor for IgE and plays a critical role in the regulation of IgE responses. The CD23 molecule is a type II transmembrane glycoprotein that consists of a C-terminal extracellular domain resembling C-type lectins, a stalk region with multiple leucine zipper repeats crucial for oligomerization, and a short cytoplasmic domain.

[0034] a) Role in Allergy Indications

[0035] CD23 is pivotal in the regulation of IgE responses, which are central to allergic reactions. As the low-affinity receptor for IgE, CD23 modulates the levels of serum IgE and the sensitivity of cells to allergens. Its expression on B cells and its ability’ to bind and stabilize IgE enhances allergen presentation and facilitates allergic inflammation. The cleavage of CD23 leads to the formation of soluble CD23, which can act in a cytokine-like manner, further promoting IgE synthesis and sustaining tire allergic response. Therapeutically, targeting CD23 with antibodies or inhibitors has been explored to reduce IgE levels and mitigate allergic symptoms.

[0036] b) Role in Autoimmune Diseases

[0037] In autoimmune conditions, CD23 contributes to the disease pathology through its regulatory effects on immune responses. Elevated levels of soluble CD23 are observed in several autoimmune diseases, suggesting its involvement in the immune dysregulation characteristic of these conditions. Soluble CD23 can affect the proliferation of B cells and tire secretion of autoantibodies, exacerbating autoimmune reactions. Therapeutic interventions targeting CD23 have focused on modulating its expression or function to dampen inappropriate immune responses and reduce the severity of autoimmune symptoms.

[0038] c) Role in Chronic Lymphocytic Leukemia (CLL)

[0039] CD23 is highly expressed in CLL, where it serves as a marker for disease progression and prognosis. The expression of CD23 on CLL cells is associated with the survival and proliferation of these leukemic cells. Elevated levels of soluble CD23 in the plasma of CLL patients correlate with a more aggressive disease course and poorer outcomes. Targeting CD23, particularly using monoclonal antibodies like lumiliximab, has been explored in clinical trials. However, these has been limited success. A clinical trial evaluating the use of lumiliximab to treat relapsed orrefractory CLL was terminated in 2010 after failing to meet primary endpoints.

[0040] Unless defined otherw ise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, w hich provide one skilled in the art with a general guide to many of the terms used in the present application.

[0041] As used throughout this specification and the claims, the terms “comprising”, “containing”, “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.

[0042] It is to be noted that the term “a” or “an”, refers to one or more, for example. “T cell”, is understood to represent one or more T cell(s). As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0043] As used herein, the term “about” means a variability of plus or minus 10% from the reference given, unless otherwise specified.

[0044] Furthermore, “and / or” w here used herein is to be taken as specific disclosure of each of the tw o specified features or components with or without the other. Thus, the tenn “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C: A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which tire third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991): Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0046] The terms “nucleic acid sequence,” “nucleotide sequence,” or "polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can beeither single stranded or double stranded DNA or RNA, e.g., an mRNA.

[0047] Nucleic acids described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and / or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding, for example, the bispecific proteins, CARs, and expression cassettes described herein can be assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and / or for delivery to a host cells in a subject. In certain embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0048] “Variants'’ of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s). such as one or more substituted, inserted and / or deleted amino acid(s).

[0049] Preferably, these fragments and / or variants have the same biological function or specific activity compared to the full-length native protein, e.g., its specific inhibitory property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e., non-mutated physiological, sequence. Substitutions in which amino acids, which originate from the same class, arc exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bonds, e.g., side chains which have a hydroxyl function. This means that e.g., an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g., using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry,Neuberger et al. (ed.), Elsevier, Amsterdam). A variant may also include a non-natural amino acid.

[0050] A ‘ variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75, 100 or more amino acids of such protein or peptide, or over the full length of the protein or peptide.

[0051] The terms “substitution” “or “change” with respect to an amino acid sequence are intended to encompass modifications of an amino acid sequence by replacement of an amino acid with another, substituting, amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, in referring to two amino acids, is intended to mean that the amino acids share a common property recognized by one of skill in the art. For example, amino acids having hydrophobic nonacidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic nonacidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties may also be amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments. Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence.

[0052] The term “gene” can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) betw een individual coding segments (exons). As used herein, the terms “coding region” and “region encoding” and grammatical variants thereof, refer to an open reading frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.

[0053] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, tire terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0054] The term “domain” refers to a region of the protein's polypeptide chain that is selfstabilizing and that folds independently from the rest of the protein. The protein domain need not be identical to the native protein from which it is derived, but may be a variant thereof, including a variant that has a deletion, truncation, etc.The term ‘‘encoding” refers to tire inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0055] Unless otherw ise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. A nucleic acid sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0056] The term “expression” is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. Expression may be transient or may be stable.

[0057] The terms “expressing” and “overexpression” refer to increasing the expression of a gene or protein. The terms refer to an increase in expression, for example, in increase in the amount of mRNA or protein expressed in a T cell, other lymphocyte or host cell, of at least 10%, as compared to a reference control level, or an increase of least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300% or at least about 400%. Various methods for expression and / or overexpression are known to those of skill in the art. and include, but are not limited to, stably or transiently introducing an exogenous polynucleotide encoding a fusion protein, TCR, or CAR to be expressed and / or overexpressed in the cell or inducing expression or overexpression of an endogenous gene encoding the protein in the cell.

[0058] The term “autologous” refers to any material derived from the same subject to whom it is later to be re-introduced.

[0059] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0060] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to beexpressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses. retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0061] As used herein, the term “binding moiety” refers to the portion of a molecule that is capable of selectively binding to a target molecule of interest (e.g., CD23). Exemplary binding domains include an antibody variable domain (e.g., a VL or VH domain, or combination thereof), a receptor binding domain of a ligand, a polypeptide that binds a domain of a receptor.

[0062] CD23

[0063] Provided herein are compositions including nucleic acids and vectors for delivery of the same having nucleotide sequences that encode CD23 -binding moieties, as well as the encoded polypeptides / proteins, which are useful in the treatment of conditions such as autoimmunity and cancer. In certain embodiments, the antibodies bind membrane -proximal CD23 epitopes, including the neck and stalk regions, and exhibit reduced binding to soluble CD23.

[0064] CD23 is a type II transmembrane glycoprotein of approximately 45 kDa molecular weight comprising a large C-terminal globular extracellular domain that is strikingly similar to C-type lectins, followed by a stalk region bearing several repeats that serve as a putative leucine zipper that are important in CD23 oligomerization; the stalk region is followed by a short extracellular sequence (in human CD23), a single hydrophobic membrane-spanning region and a short N-terminal cytoplasmic domain. CD23 is expressed in T and B lymphocytes, polymorphonuclear leucocytes, monocytes, follicular dendritic cells, intestinal epithelial cells, and bone marrow stromal cells, and its expression to subject to regulation by a number of stimuli. In humans, CD23 is encoded by an 11-exon gene, FCER2, located at chromosome 19p 13.3, in a cluster with the DC-SIGN and DC-SIGNR genes. CD23 is also known as CD23A, CLEC4J. FCE2, IGEBF, CD23, Low affinity immunoglobulin epsilon Fc receptor, BLAST-2, C-type lectin domain family 4 member J, Fc-epsilon-RII, Immunoglobulin E-binding factor, Lymphocyte IgE receptor. See also, Jegouzo SAF, et al. J Biol Chem. 2019 Oct 11;294(41): 14845-14859, which is incorporated herein by reference.

[0065] The full-length amino acid sequence for human CD23 is:

[0066] MEEGQYSEIEELPRRRCCRRGTQIVLLGLVTAALWAGLLTLLLLWHWDTTQSLKQLEER AARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGFVCNTCPEKWINFQRKCY YFGKGTKQWVHARYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNLDLKGEFIW VDGSHVDYSNWAPGEPTSRSQGEDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPP ASEGSAESMGPDSRPDPDGRLPTPSAPLHS (SEQ ID NO: 1) -Unitprot: P06734 • FCER2 HUMAN (neck region bolded, stalk region underlined)The neck region of CD23 includes amino acids 48 to 89 of SEQ ID NO: 1.

[0067] The stalk domain of CD23 includes amino acids 90 to 156 of SEQ ID NO: 1.

[0068] CD23 Antibodies and Fragments Thereof

[0069] In certain embodiments, the CD23-binding moiety provided herein an antibody or fragment thereof that specifically binds CD23. In certain embodiments, the antibody or fragment thereof specifically binds an extracellular domain of CD23. In certain embodiments, tire antibody or fragment thereof specifically binds a CD23 head domain. In certain embodiments, the antibody or fragment thereof specifically binds a CD23 stalk domain. In certain embodiments, the antibody or fragment thereof specifically binds a polypeptide comprising amino acids 48 to 150 of SEQ ID NO: 1. In certain embodiments, the antibody or fragment thereof exhibits reduced binding to soluble CD23, as compared to lumiliximab or an analog thereof.

[0070] As used herein, an “antibody” is a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multispecific binding construct that can bind two or more epitopes, a dual specific antibody, a bi-specific antibody, a multi-specific antibody, an affinity matured antibody, a single antibody chain or an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab' construct, a F(ab')2 construct, an Fc construct, a monovalent or bivalent construct from which domains non-essential to monoclonal antibody function have been removed, a single-chain molecule containing one VL (variable region of light chain), one VH (variable region of heavy chain) antigen-binding domain, and one or tw o constant “effector” domains optionally connected by linker domains, a univalent antibody lacking a hinge region, a single domain antibody, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, or any recombinant versions thereof. Definitions and examples of these types of structures are found in the art and in, e.g., US Patent No. 9,902,772, incorporated by reference herein.

[0071] An antibody (e.g., an antibody, an antibody heavy chain, an antibody light chain, or any fragment or modification thereof) comprises three Complementarity-Determining Regions (CDRs, also known as HV, hypervariable regions, namely CDR1, CDR2, CDR3, from N-terminal to C-terminal, or 5’ to 3’ when corresponding nucleic acid sequence is referred to), and four framework regions (FRs. namely FR1, FR2, FR3 and FR4, from N-terminal to C-terminal, or 5’ to 3' when corresponding nucleic acid sequence is referred to). See, e.g., Janeway, Charles A Jr; Travers, Paul; Walport, Mark; Shlomchik, Mark J (2001). Immunobiology: The Immune System in Health and Disease (5 ed ). New York: Garland Science. ISBN 0-8153-3642-X, whichis incorporated herein by its entirety. It would be understood that in the antibody construct, CDRs are arranged non-consecutively. not immediately adjacent to each other, and may be separated by an FR. As part of the variable chain in an antibody construct and T cell receptors generated by B-cells and T-cells respectively, CDRs are where an antigen specifically binds.

[0072] As used herein, the complementarity-determining region (CDR) refers to part of the variable chains in antibodies, which bind to the corresponding epitope. Such CDRs may be determined via experiments or via various predicating tools, such as www.imgt.org / IMGT_vquest / analysis. Also provided herein is a nucleic acid sequence encoding an antibody, or a variant thereof, or an epitope binding fragment thereof as described herein. As used herein, an epitope binding fragment refers to a fragment of an antibody which is determined to be bound to an epitope. Such determination may be performed experimentally using for example ELISA or other methods discussed herein or via various predicating tools such as IMGT.org.

[0073] The term “antibody fragment’’ as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as e.g. single chain Fab, scFv, and multispecific antibodies formed from antibody fragments. The “single chain Fab” format is described, e.g., in Hust M. et al. BMC Biotechnol. 2007 Mar 8;7: 14. scFvV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs. The term “antibody fragment” is also used to refer to an antibody heavy chain or antibody light chain. In certain embodiments, the antibody or fragment thereof is a dAb, Fab, Fab’, F(ab’)2, Fv, scFv, scFv2, scFv-Fc, minibody, diabody, triabody, or tetrabody.

[0074] An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2. IgG3. IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.

[0075] Methods for producing such antibodies and antibody fragments are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment. In a particular embodiment, the antibody comprises an Fc region.

[0076] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin covalently linked to form a VH::VLheterodimer. The VH and VL are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects tire N-terminus of tire VH with the C-temiinus of the VL, or the C-temiinus of the VH with the N-tenninus of the VL.

[0077] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid including VH- and VLencoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U. S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U. S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al.. Thromb Haemost 2007 97(6):955-63: Fife eta., J Clin Invst2006 116(8):2252-61; Brocks et al., Immunotechnology 19973(3): 173-84; Moosmayer etal., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 2003 278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66). As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g.. a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0078] As used herein, '‘F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A " F(ab')2" fragment can be split into two individual Fab' fragments.

[0079] As used herein, “specifically binding ’ “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. For example, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, ty pically, more noncovalent bonds between the binding molecules or binding partners.

[0080] Provided herein are improved CD23 -binding antibodies and fragments thereof. In certain embodiments, provided herein is an anti-CD23 antibody or fragment thereof, wherein the antibody or fragment thereof. Amino acid sequences corresponding to the anti-CD23 antibodyvariable heavy chain and variable light chain are provided in the table below, with the respective complementarity -determining regions (CDRs) underlined. In certain embodiments, tire anti-CD23 antibody or fragment provided includes heavy chain and / or light chain variable domain sequences identified in these sequences. In certain embodiments, provided herein is an antibody, or a variant thereof, or an epitope binding fragment thereof comprising at least 1, at least 2, at least 3, at least 4, at least 5, or 6 CDR(s) as identified for the heavy chain and / or light chain sequences below. Table 1

[0081] S S C E E

[0082] 1

[0083] I Q Q

[0084] VLVH

[0085] n D D e N N 0 0 L

[0086] u

[0087] A m EVQLVESGGGLAKPGGSLRLSCAASG DIQMTQSPSSLSASVGDRVTITCRA

[0088] B i FR FT FNNYYMDWVRQAPGQGLE WVS R SQDIRYYLNWYQQKPGKAPKLLIYV

[0089] 0 1 I SSSGDPTWYADSVKGRFT I S RENAN ASSLQSGVPSRFSGSGSGTEFTLTV

[0090] 0 a 0 NTLFLQMNSLRAEDTAVYYCASLTTG SSLQPEDFATYYCLQVYSTPRTFGQ

[0091] 1 n SDSWGQGVLVTVSS (SEQ ID NO:

[0092] GTKVEIK (SEQ ID NO: 10 )

[0093] 8 a ID

[0094] 1

[0095] o 1 g 1 MDTRAPTQLLGLLLLWLPGATFAQV A LTQT PS PVSVAVGGTVT INCQASQS METGLRWLLLVAVLKGVQCQSLEESG B VYNNKNLAWFQQKPGQPPKQLIYVA 1 GRLVKPDETLTITCTVSGIDLDRFAM 0 SKLASGVPSRFSGSGSGTQFTLTIS 8 S WVRQAPGKGLE W I GV I YGS GNAYYA 2 5 GVQCDDAATYYCLGEFSCSSADCNA SWAKGRFTISKTSTTVDLKMTSLTTE 9 A FGGGTEWVK DTATYFCGRAVADYSTLNLWGPGTLV 1 2 4 TVSS 9 A MDTRAPTQLLGLLLLWLPGAICDPV B LTQTPSSASEPVGGTVTIKCQASES METGLRWLLLVAVLKGVQCQSVEESG ISSRLAWYQQKPGQPPKLLIYSAST 2

[0096] 0 GRLVTPGTPLTLTCTVSGIDLSSYAM LESGVPSRFKGSGSGTEFTLTISDL

[0097] 2 7 0 GWVRQAPGKGLEYIGI ISSSGSTYYA E CADAAT YYCQNNYVT S YGFGGGTE

[0098] 9 H SWAKGRFTISKTSTTVDLKITSPTTE 2 VWK

[0099] 3 6 DTATYFCARAWDLWGPGTLVTVSS 1 MDTRAPTQLLGLLLLWLPGARCAFE A LTQT PS SVEAAVGGTVT I KCQASQS METGLRWLLLVAVLKGVQCQSVEESG B INSWLAWYQQKPGQPPKLLIYKAST 2 GRLVTPGTPLTLTCTVSGIDLSTYPI 0 1 LASGVSSRFKGSGSGTEFTLTISDL 2 TWVRQAPGKGLEYVGYIHSGGSAYYA 2 3 E CADAAT YYCQNYYDT I TNT FGGGT GWAKGRFTISKTSTTVDLKITSPTTE 9 D KVWE DTATYFCARGSSWGNLWGPGTLVTVS 2

[0100]

[0101] 4 4 S 3MDTRAPTQLLGLLLLWLPGARCADV

[0102] A VMTQT PAS VEAAVGGTVT I KCQASQ METGLRWLLLVAVLKGVQCQSVEESG B S I SNLLAWYQQKPGQP PKLL I YS AS 2 GRLVTPGTPLTLTCTVSGFSLSSNAV 0 1 TLASGVPSRFKGSESGTEFTLTISD 4 SWVRQAPGKGLEHIGFIGDTGNTYYA 2 5 LE CADAAT YYCQS YYGS S S S S YGDA SWAKGRFTISKTSTTVDLKITSPTTE 9 A FGGGTEVWK DTATYFCVRAGITNLWGPGTLVTVSS 2 5 1 5 MDTRAPTQLLGLLLLWLPGATFAQV A LTQT PS PVS VWGGTVT INCQASQS METGLRWLLLVAVLKGVQCQSLEESG B VYNNKNLAWFQQKPGQPPKQLIYVA 2 GRLVKPDETLTITCTVSGIDLDRFAM 0 1 SKLASGVPSRFSGSGSGTQFTLTIS 6 S WVRQAPGKGLE W I GV I YGS GNVYYA 2 7 GVQCDDAATYYCLGEFSCSSADCNA SWAKGRFTISKASTTVDLKMTSLTTE 9 E FGGGTEVWK DTATYFCGRAVADYSTLNLWGPGTLV 2 6 1 TVSS 7 MDTRAPTQLLGLLLLWLPGATFAQV A LTQT PS PVSVAVGGAVT INCQASQS METGLRWLLLVAVLKGVQCQSLEESG B VYNYKNLAWFQQKPGQPPKQLIYTA 2 GRLVKPDETLTLTCTVSGIDLSRFAM 0 2 SSLASGVSSRFKGSGSGTQFTLAIS 8 SWVRQAPGKGLEWIGVIYGSGNSYYA 2 0 DVQCDDAATYYCLGEFSCSSADCNA SWAKGRFTISKTSTTVDLKMTSLTTE 9 G FGGGTEVWK DTATYFCGRAVADYSTLNLWGPGTLV 2 7 4 TVSS 9 MDTRAPTQLLGLLLLWLPGATFAQV A LTQT PS PVSAAVGGTVT INCQASQS METGLRWLLLVAVLKGVQCQSVEESG B VYNNKNLAWFQQKPGQPPKQLIYAA 3 GRLVTPGTPLTLTCTVSGMDLSRYAM 0 STLASGVSSRFKGSGSGTQFTLTIS 0 S WVRQAPGKGLE W I GV I YAS GNAYYA 2 4 DVQCDDAATYYCLGEFSCSSADCNA SWAKGRFTISKTSTTVYLKIASPTTE 9 FGGGTEVWK DTATYFCGRYVADYGAVGRVWGPGTL 3 8 6 VTVSS 1 MDTRAPTQLLGLLLLWLPGATFAAV A LTQTPSPVSAAVGGTVSISCQASQS METGLRWLLLVAVLKGVQCQSVEESG

[0103] B 1 VYEDNWLAWYQQKPGQRPKLLIYLA 3 GRLVTPGTPLTLTCTVSGIDLSRYNM 0 9 SNLASGVPSRFKGSGSGTQFTLTIN 2 AWVRQAPGKGLEYIGI IYSSAATYYA 2 E GVQCDDAATYYCQGVDSSSGETTFG SWAKGRFTISKTSTTVDLKMTSLTTE 9 1 GGTEVWK DTATYFCARRDVGSSGYTGAFDPWGP 3

[0104]

[0105] 9 2 GTLVTVSS 3

[0106] Table 1 A - CDRs of monoclonal antibodies of Table 1

[0107] SE SE SE SE AB Clo Q Q Q £

[0108] TD CDR1 ID CDR2 ID CDR3 ID ID ne

[0109] N N N N

[0110] 0 0 0 0 ABO 4G V VIYASGNAYYAS YVADYGAV 31 RYAMS 40 41

[0111] 298 6 H WAKG GRV

[0112]

[0113] 42V OASOSVYN LGEFSCSSA 30 43 AASTLAS 44

[0114] L NKNLA DCNA

[0115] 45 V VIYGSGNAYYAS AVADYSTL

[0116] 19 REAMS 46 47

[0117] H WAKG NL ABO 5A 48 292 4

[0118] V 18 OASOSVYN LGEFSCSSA 49 VASKLAS

[0119] L NKNLA 50 DCNA

[0120] 51 V IISSSGSTYYASW

[0121] 21 SYAMG 52 53 AWDL ABO 7H H AKG

[0122] 293 6 54

[0123] V OASESISSR QNNYVTSY

[0124] 20 55 SASTLES 56

[0125] L LA G 57 y VGYIHSGGSAYY

[0126] 23 TYPIT 58 59 GSSWGNL ABO 13 H AGWAKG

[0127] 294 D4 60

[0128] V OASOSINSW ONYYDTITN

[0129] 22 KASTLAS 62

[0130] L LA 61

[0131] I 63 V FIGDTGNTYYAS

[0132] 25 SSNAV 64 65 VRAGITNL H WAKG ABO 15 66 295 Al

[0133] V OASOSISNL OSYYGSSSS

[0134] 24 67 SASTLAS 68 L LA SYGDA

[0135] 69 y VIYGSGNVYYAS AVADYSTL 27 REAMS 70 21 H WAKG NL ABO 17E 72 296 1

[0136] y OASOSVYN LGEFSCSSA 26 73 VASKLAS 74

[0137] L NKNLA DCNA

[0138] 75 y IIYSSAATYYAS RDVGSSGY 33 RYNMA 76 77 H WAKG TGAFDP ABO 19E 78 299 2

[0139] y OASOSVYE OGVDSSSGE 32 79 LASNLAS 80 L DNWLA TT

[0140] 81 y VIYGSGNSYYAS AVADYSTL 29 REAMS 82 83

[0141] H WAKG NL ABO 20 84 297 G4

[0142] y OASOSVYN LGEFSCSSA 28 85 TASSLAS 86 L YKNLA DCNA

[0143]

[0144] 87In certain embodiments, provided are antibodies and antigen-binding fragments thereof comprising defined complementarity determining regions (CDRs). In particular, the disclosure provides antibodies comprising heavy chain CDR1, CDR2, and CDR3 sequences selected from SEQ ID NOs: 40–42, 46–48, 52–54, 58–60, 64–66, 70–72, 76–78, 82–84, and variants thereof. In addition, the disclosure provides antibodies comprising light chain CDR1, CDR2, and CDR3 sequences selected from SEQ ID NOs: 43-45, 49-51, 55-57, 61-63, 67-69, 73-75, 79-81, and 85-87, and variants thereof. In each triplet the first SEQ ID NO corresponds to CDR1, the second SEQ ID NO corresponds to CDR2, and the third SEQ ID NO corresponds to CDR3, for the respective heavy or light chain. In certain embodiments, there is provided an antibody or fragment thereof comprising a modification to one or more of the CDRs shown above, but which comprises sufficient of the amino acid sequence of each CDR shown above that the antibody is capable of binding to CD23. In certain embodiments, one or more heavy chain and / or light chain CDR sequences comprises a variant of the recited SEQ ID NO, provided that the resulting antibody retains specific binding to the target antigen. Variants may include substitutions, insertions, deletions, or combinations thereof, as described elsewhere herein. For example, variants may comprise: 1-10 amino acid substitutions; conservative amino acid substitutions; 1-5 amino acid insertions; 1-5 amino acid deletions; or combinations thereof.

[0145] In certain embodiments, an antibody comprises: a heavy chain variable region comprising:

[0146] CDR1 comprising SEQ ID NO: 40,

[0147] CDR2 comprising SEQ ID NO: 41, and

[0148] CDR3 comprising SEQ ID NO: 42;

[0149] and a light chain variable region comprising:

[0150] CDR1 comprising SEQ ID NO: 43,

[0151] CDR2 comprising SEQ ID NO: 44, and

[0152] CDR3 comprising SEQ ID NO: 45.

[0153] In further embodiments, antibodies comprise paired heavy and light chain CDR groupings selected from:

[0154] (40–42) paired with (43–45);

[0155] (46-48) paired with (49-51);

[0156] (52-54) paired with (55-57);

[0157] (58-60) paired with (61-63);

[0158] (64-66) paired with (67-69);

[0159] (70-72) paired with (73-75);

[0160] (76-78) paired with (79-81); or(82-84) paired with (85-87).

[0161] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 11 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In certain embodiments, tire antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 10 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10.

[0162] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain having the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 13 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identify with SEQ ID NO: 13. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 13 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12.

[0163] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 15 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 15 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identify with SEQ ID NO: 15. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 14 or an amino acid sequence sharing at least 90%. 95%, 96%. 97%. 98%, or 99% identity with SEQ ID NO: 14.In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 16 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 16 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 14 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14.

[0164] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain having the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain having tire amino acid sequence of SEQ ID NO: 16 and a light chain having the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 16 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In certain embodiments, tire antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 17 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.

[0165] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 19 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 18 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%. or 99% identity with SEQ ID NO: 18. In certain embodiments, the antibody or fragment thereof comprises SEQ ID NO: 40, SEQ ID NO: 41, andSEQ ID NO: 42, or a sequence having 1, 2, 3, 4, or 5 substitutions from SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42.

[0166] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 21 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 20 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20.

[0167] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 23 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. In certain embodiments, the antibody light chain or fragment thereof comprises tire light chain comprises SEQ ID NO: 22 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22.

[0168] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 25 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 24 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24.In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 27 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 26 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%. or 99% identity with SEQ ID NO: 26.

[0169] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 29 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 29. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 28 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 28.

[0170] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 31 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 31. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 30 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%. or 99% identity with SEQ ID NO: 30.

[0171] In certain embodiments, the antibody or fragment thereof comprises the three CDRs of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In certainembodiments, the antibody or fragment thereof comprises the three CDRs of a light chain comprising the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the antibody or fragment thereof comprises the six CDRs of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the antibody heavy chain or fragment thereof comprises SEQ ID NO: 33 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33. In certain embodiments, the antibody light chain or fragment thereof comprises the light chain comprises SEQ ID NO: 32 or an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32.

[0172] In certain embodiments, the antibodies described herein are humanized antibodies comprising variable regions derived from a non-human antibody and constant regions derived from a human immunoglobulin. In some embodiments, the constant region comprises a human heavy chain constant region and a human light chain constant region. The human constant regions may be selected to reduce immunogenicity in human subjects while maintaining desired effector function, stability, and pharmacokinetic properties.

[0173] The heavy and light chain constant regions may be derived from any suitable sequence, such as a human IgGl framework. In certain embodiments, the heavy chain constant region is derived from a human IgG isotype, including IgGl, IgG2. IgG3. or IgG4. In some embodiments, the heavy chain constant region is IgGl. In other embodiments, the heavy chain constant region is IgG2 or IgG4. In certain embodiments, the IgG4 constant region comprises a substitution at position S228, such as S228P, to reduce Fab-arm exchange. In some embodiments, the heavy chain constant region comprises CHI, hinge, CH2, and CH3 domains of a human IgG.

[0174] In certain embodiments, the light chain constant region is derived from a human kappa (CK) or human lambda (CX) constant region. In some embodiments, the light chain constant region is human kappa. In other embodiments, the light chain constant region is human lambda.

[0175] In certain embodiments, the constant region is selected to confer a desired effector profile. For example, a human IgGl constant region may be selected to provide antibodydependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In other embodiments, a human IgG2 or IgG4 constant region is selected to reduce effector function. In certain embodiments, the constant region comprises one or more amino acid substitutions that reduce Fc receptor binding, reduce complement activation, enhance stability, improve solubility, reduce aggregation, or extend serum half-life.

[0176] In certain embodiments, the human constant region is at least 90%, at least 95%. at least 98%, or 100% identical to a naturally occurring human germline constant region sequence. Insome embodiments, the constant region comprises allelic variants of human immunoglobulin constant regions.

[0177] In certain embodiments, the humanized antibody comprises a human constant region operably linked to a humanized variable region in which one or more complementarity determining regions (CDRs) from a non-human antibody are grafted onto a human framework region. In some embodiments, framework back-mutations are included to restore antigen binding affinity, stability, or expression. In certain embodiments, the human constant region and the humanized variable region are expressed as a full-length immunoglobulin molecule.

[0178] In certain embodiments, the constant region may be engineered to improve manufacturability, including modifications that enhance expression yield in mammalian host cells, improve folding efficiency, or reduce proteolytic susceptibility. In some embodiments, the constant region is optimized for expression in Chinese Hamster Ovary (CHO) cells or other mammalian expression systems.

[0179] In certain embodiments, the humanized antibody comprising a human constant region exhibits reduced immunogenicity compared to a corresponding chimeric or non-human antibody when administered to a human subject. In some embodiments, the antibody retains substantially the same antigen-binding specificity and affinity as the parental non-human antibody.

[0180] In certain embodiments, the human constant regions described herein may be combined with Fc engineering modifications, glycoengineering modifications, or other constant region alterations to tailor effector function, pharmacokinetics, and therapeutic performance, provided that the antibody retains desired antigen-binding properties.

[0181] In certain embodiments, the antibodies described herein comprise an engineered Fc region. The Fc region may be modified to alter one or more functional properties including, but not limited to, Fc gamma receptor (FcyR) binding, complement component Clq binding, antibody-dependent cellular cytotoxicity’ (ADCC), antibody -dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), serum half-life, neonatal Fc receptor (FcRn) binding, stability, solubility, aggregation resistance, heterodimerization, effector silencing, immune activation, and glycosylation profile. The Fc region may be derived from any immunoglobulin isotype including IgGl, IgG2, IgG3, IgG4, IgA, IgM, or hybrid isotypes.

[0182] In certain embodiments, the Fc region is engineered to reduce or eliminate Fey receptor binding and / or complement activation. In some embodiments, the Fc region comprises one or more amino acid substitutions selected from L234A, L235A, P329G, L234A / L235A (LALA), L234A / L235A / P329G (LALAPG), D265A. and N297A. In certain embodiments, the engineered Fc exhibits reduced FcyR binding, reduced Clq binding, reduced ADCC, and / or reduced CDC. Insome embodiments, effector function is reduced by at least 50%, at least 75%, at least 90%, at least 95%, or at least 99% relative to a wild-type Fc region.

[0183] In certain embodiments, the Fc region is engineered to enhance binding to neonatal Fc receptor (FcRn), thereby extending serum half-life. Exemplary substitutions include M252Y, S254T, T256E, T307Q, E380A, N434A, N434S, M428L, and combinations thereof. In some embodiments, the Fc region comprises M252Y / S254T / T256E. In certain embodiments, the Fc region comprises M428L / N434S. In some embodiments, serum half-life is extended by at least 1.2-fold, at least 1.5-fold, at least 2-fold, or at least 3-fold relative to a wild-type IgGl Fc region.

[0184] In certain embodiments, the antibody is a bispecific or multispecific antibody comprising two different heavy chains, and the Fc region is engineered to promote heterodimer formation and reduce homodimer formation. In some embodiments, one heavy chain Fc comprises a "knob" mutation and the other heavy chain Fc comprises a complementary ‘'hole” mutation. Exemplary substitutions include T366W, Y349C, T366S, L368A, Y407V, F405A, and combinations thereof. In certain embodiments, heterodimer formation exceeds 80%, 90%, or 95%.

[0185] In certain embodiments, Fc glycosylation is modified to alter effector function. In some embodiments, the Fc glycan lacks core fucose, resulting in enhanced FcyRIIIa binding and increased ADCC. In certain embodiments, glycosylation is modified by altering host cell expression systems, by enzymatic remodeling, or by site-directed mutagenesis. In some embodiments, N297 is retained to permit glycosylation, while in other embodiments N297 is substituted to eliminate glycosylation.

[0186] In certain embodiments, Fc mutations are introduced to improve thermal stability, increase melting temperature (Tm), reduce aggregation, and / or enhance manufacturability. In some embodiments, the Fc region exhibits an increased Tm of at least 1°C, at least 2°C, at least 5°C, or at least 10°C relative to a corresponding wild-type Fc region. In certain embodiments, the engineered Fc exhibits reduced formation of high molecular weight species.

[0187] In certain embodiments, the Fc region is fused to peptides, cytokines, receptor domains, enzymes, toxin moieties, drug-linker payloads, or other functional domains. In some embodiments, the antibody is an antibody-drug conjugate comprising an Fc-engineered antibody conjugated to a cytotoxic agent. In certain embodiments, conjugation occurs via engineered cysteine residues, lysine residues, enzymatic conjugation sites, or site-specific incorporation of non-natural amino acids.

[0188] In certain embodiments, the Fc region comprises combinations of FcRn-enhancing mutations, effector-silencing mutations, heterodimerization mutations, stability mutations, or glycoengineering modifications. In some embodiments, the Fc region comprises at least 2, at least 3, at least 4, at least 5, or more engineered substitutions.Fc function may be evaluated using Fey receptor binding assays, surface plasmon resonance, ADCC reporter assays, CDC assays, FcRn binding assays, and pharmacokinetic studies in vivo.

[0189] CD23 stalk domain-binding molecules

[0190] Given the CD23 stalk domain's role in trimerization and stabilization, targeting this region can modulate CD23 function. Disruption of the oligomerization process can, for example, decrease the effective clustering of IgE on B cells and thereby dampen the activation response.

[0191] Provided herein are CD23-binding moieties that include one or more domains or sequences of a CD23 protein. In certain embodiments, the one or more domains of a CD23 protein are domains or sequences that comprise a sequence of SEQ ID NO: 1 corresponding to the stalk region of CD23 (amino acids 90 to 156 of SEQ ID NO: 1). In certain embodiments, the CD23-binding moiety comprises amino acids 90 to 156 of SEQ ID NO: 1. In certain embodiments, the CD23-binding moiety comprises tandem repeats of amino acids 90 to 156 of SEQ ID NO: 1, for example 1, or at least 2, or at least 3 repeats of amino acids 90 to 156 of SEQ ID NO: 1 linked by intervening sequences, which may include, for example, sequences of the native stalk region of CD23, sequences corresponding to another domain of CD23, such as the neck domain, and / or linker sequences. In certain embodiments, the CD23-binding moiety includes up to 10 amino acid changes as compared to the native protein. In certain embodiments, the CD23-binding moiety is a sequence corresponding to amino acids 90 to 156 of SEQ ID NO: 1 that is truncated so that it has a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus and / or at the C-terminus. In certain embodiments, CD23-binding moiety includes a sequence that shares at least 90%, at least 95%, or at least 99% identity’ with amino acids 90 to 156 of SEQ ID NO: 1.

[0192] In certain embodiments, the one or more domains of a CD23 protein are domains or sequences that comprise a sequence of SEQ ID NO: 1 corresponding to amino acids 48 to 150 of SEQ ID NO: 1. In certain embodiments, the CD23-binding moiety comprises tandem repeats of amino acids 48 to 150 of SEQ ID NO: 1, for example 1, or at least 2, or at least 3 repeats of amino acids 48 to 150 of SEQ ID NO: 1 linked by intervening sequences, which may include, for example, sequences of the native stalk region of CD23, sequences corresponding to another domain of CD23, such as the neck domain, and / or linker sequences. In certain embodiments, the CD23-binding moiety includes up to 10 amino acid changes as compared to the native protein. In certain embodiments, the CD23-binding moiety is a sequence corresponding to amino acids 48 to 150 of SEQ ID NO: 1 that is truncated so that it has a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus and / or at the C-terminus. In certain embodiments, CD23-binding moiety includes a sequence that shares at least 90%, at least 95%, or at least 99% identity with amino acids 48 to 150 of SEQ ID NO: 1.

[0193] In certain embodiments, the CD23-binding moiety is a polypeptide that includes a sequence of the neck region of CD23 (amino acids 48 to 89 of SEQ ID NO: 1). In certain embodiments, the sequence of the neck region is amino acids 48 to 89 of SEQ ID NO: 1. In certain embodiments, the CD23-binding moiety comprises amino acids 48 to 89 of SEQ ID NO: 1. In certain embodiments, the CD23-binding moiety comprises a neck region sequence that is immediately N-terminal to a sequence corresponding to the stalk region of CD23 (i.e., no intervening amino acid residues). In other embodiments, CD23 -binding moiety comprises a neck region sequence that separated from the stalk region sequence of the polypeptide is at least 1, 2, 3, 4, 5, 6. 7, 8, 9, or 10 intervening amino residues, including, for example a linker sequence. In certain embodiments, the CD23-binding moiety includes up to 10 amino acid changes as compared to the native neck region sequence. In certain embodiments, the CD23-binding moiety includes a neck region sequence corresponding to amino acids 48 to 89 of SEQ ID NO: 1 that is truncated so that it has a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at tire N-terminus and / or at the C-terminus. In certain embodiments, CD23-binding moiety includes a sequence that shares at least 90%, at least 95%, or at least 99% identity with amino acids 48 to 89 of SEQ ID NO: 1.

[0194] In certain embodiments, the CD23-binding moiety is a polypeptide that includes SEQ ID NO: 34. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 34 but includes up to 10 amino acid changes as compared to the native sequence. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 34 but includes up to a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-tcnninus and / or at the C-terminus of SEQ ID NO: 34. In certain, embodiments the at least one domain has a sequence that shares at least 90%. at least 95%, or at least 99% identity with SEQ ID NO: 34.

[0195] In certain embodiments, the CD23-binding moiety is a polypeptide that includes SEQ ID NO: 35. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 35 but includes up to 10 amino acid changes as compared to the native sequence. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 35 but includes up to a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-tenninus and / or at the C-terminus of SEQ ID NO: 35. In certain, embodiments the at least one domain has a sequence that shares at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 35.

[0196] In certain embodiments, the CD23-binding moiety is a polypeptide that includes SEQ ID NO: 36. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 36 but includes up to 10 amino acid changes as compared to the native sequence. In certainembodiments, the CD23-binding moiety corresponds to SEQ ID NO: 36 but includes up to a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-temiinus and / or at tire C-terminus of SEQ ID NO: 36. In certain embodiments the at least one domain has a sequence that shares at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 36.

[0197] DTTQSLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLN GLQADLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSS (SEQ ID NO: 34) DTTQSLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLN GLQADLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGSSSSDTTQSLKQLEERAARNVSQVSK NLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADLSSFKSQELNERNEASD LLERLREEVTKLRMELQVS (SEQ ID NO: 35) (linker sequence underlined) DTTQSLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLN GLOADLSSFKSQELNERNEASDLLERLREEVTKLRMELOVSSGGGGSGGGGSGGGGSGGGGSDTTO SLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQA DLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGSSSSDTTQSLKQLEERAARNVSQVSKNLES HHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADLSSFKSQELNERNEASDLLER LREEVTKLRMELQV (SEQ ID NO: 36) (linker sequences underlined)

[0198] Examples of constructs provided herein that include a CD23-binding moieties comprising a CD23 stalk-binding polypeptide include AB0166, AB0167, AB0168, AB0169, AB0170, ABO 172, ABO 174, and ABO 175.

[0199] Utilizing CD21 Sushi Domains

[0200] CD21 (complement receptor type 2, CR2) is a transmembrane protein that

[0201] contains fifteen tandem sushi repeats in its extracellular domain. The sushi domains (also referred to as ’SC Rs") of CD21 interact with CD23 and can be useful for therapeutic targeting of CD23. As opposed to an antibody that specifically binds CD23, tire natural interaction between CD21 sushi domains and CD23 is leveraged to mimic physiological interactions. The advantages of this approach can include, for example, better stability and reduced immunogenicity of the composition. Accordingly, provide herein are CD23-binding moieties that include sushi domains of CD21.

[0202] The full-length amino acid sequence for human CD21 is:

[0203] MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGEKSLLCITKDKV DGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQAN NMWGPTRLPTCVSVFPLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSSGK WSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVAWTK MPVCEEIFCPSPPPILNGRHIGNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTW SGPAPRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEP SAPVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCPPPPVI YNGAHTGSSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAV QCSHVHIANGYKISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKETCQHVR QSLQELPAGSRVELVNTSCQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCHPPPVIVNGKHT GMMAENFLYGNEVSYECDQGFYLLGEKKLQCRSDSKGHGSWSGPSPQCLRSPPVTRCPNPEVK HGYKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIGCPPPPKTPNG NHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTWSQPAPHCKEVNCSSPADMDGIQKGL EPRKMYQYGAWTLECEDGYMLEGSPQSQCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLI VITLYVISKHRARNYYTDTSQKEAFHLEAREVYSVDPYNPAS (SEQ ID NO: 37) – P20023 · CR2_HUMAN (20 amino acid signal peptide underlined)

[0204] Domain amino acid positions of SEQ ID NO: 37

[0205] Sushi 1 21-84

[0206] Sushi 2 89-148

[0207] Sushi 3 152-212

[0208] Sushi 4 213-273

[0209] Sushi 5 274-34415In certain embodiments, Sushi 6 349-408 the CD23-binding moiety one Sushi 7 409-468

[0210] or more sushi (or “SCR”) Sushi 8 469-524

[0211] Sushi 9 525-595 domains of CD21. The sushi Sushi 10 600-659 domains of can be selected in a Sushi 11 660-716

[0212] Sushi 12 717-781 20 manner that excludes an Sushi 13 786-845 intervening domain or Sushi 14 549-909

[0213] sequences from the native

[0214]

[0215] Sushi 15 910-970

[0216] protein and, be arranged in an order that is different from their relative position in the native protein. In certain embodiments, the CD23-binding moiety includes multiple (1, 2, or 3 or more) of the sushi domain (or variants thereof) from CD21. In certain embodiments, the CD23-binding moiety includes at least 1, 2, 3, 4, 5, 6, 7, 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein. In certain embodiments, the CD23-binding moiety includes at least 1, 2, 3, 4, 5, 6, 7, 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein. In certain embodiments, the CD23-binding moiety includes less than 15, 14, 13, 12, 11, 10. 9, or 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein. The sushi domains of the CD23-binding moiety may be separated by intervening sequences that are native CD21 sequences and / or linker sequences. In certain embodiments, the CD23 -binding moiety includes sushi domains 1-8 (inclusive) of the CD21. In certain embodiments, the CD23-binding moiety includes sushi domains 2-7 (inclusive) of the CD21. In certain embodiments, the CD23-binding moiety includes at least sushi domains 1-8, at least sushi domains 2-7, at least sushi domains 2-8, at least sushi domains 1-7, at least at least sushi domains 1-3, at least sushi domains 1-4, at least sushidomains 1-5. at least sushi domains 1-6. at least sushi domains 2-5, at least sushi domains 2-4, at least sushi domains 2-5, at least sushi domains 2-6 of CD21.

[0217] In certain embodiments, the CD23-binding moiety is a polypeptide that includes SEQ ID NO: 38. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 38 but includes up to 10 amino acid changes as compared to the native sequence. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 38 but includes up to a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus and / or at the C-terminus of SEQ ID NO: 38. In certain, embodiments the at least one domain has a sequence that shares at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 38.

[0218] In certain embodiments, the CD23-binding moiety is a polypeptide that includes SEQ ID NO: 39. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 39 but includes up to 10 amino acid changes as compared to the native sequence. In certain embodiments, the CD23-binding moiety corresponds to SEQ ID NO: 39 but includes up to a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus and / or at the C-terminus of SEQ ID NO: 39. In certain, embodiments the at least one domain has a sequence that shares at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 39.

[0219] GISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSC PEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLPTCVSVFPLECPALP MIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSSGKWSAVPPTCEEARCKSLGRFPNGKVKE PPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHIGNSLANVSY GSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWSGPAPRCELSTSAVQCPHPQILRGRMVSGQK DRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEPSAPVCEKECQAPPNILNGQKEDRHMVRFDPGTS IKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYKLS GSVYQECQGTIPWFMEIRLCKE (SEQ ID NO: 38) (includes elements of sushi domains 1-8) GEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGN KSVWCQANNMWGPTRLPTCVSVFPLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIIN CLSSGKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVA WTKMPVCEEIFCPSPPPILNGRHIGNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTW SGPAPRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEPSA PVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPP (SEQ ID NO: 39) (includes elements of sushi domains 2-7)

[0220] Examples of constructs provided herein that include a CD23-binding moieties having one or more CD21 sushi domains include AB0171 and AB0171b.

[0221] The term "linker ’ as used herein, is a polypeptide that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. Linkers are usually rich in glycine for flexibility, as well as serine and / or threonine for solubility. The bispecific constructs described herein include a linker joining a first binding moiety and a second binding moiety. Incertain embodiments, the first and second linker are the same. In certain embodiments, the linker is an amino acid sequence that is about 1 to about 5 residues, about 5 to about 10 residues, about 5 to about 15 residues, about 10 to about 20 residues, about 1 to about 25 residues, or about 15 to about 30 residues. In certain embodiments, the linker is at least 5, at least 10, at least 15, at least 20, or at least 25 residues. Suitable linkers include, for example, the sequences identified in Table 2.

[0222] In certain embodiments, the linker is a multiple of GGGGS (or variants thereof), for example, having 1, 2, 3, 4, 5, 6, 7, 8 or more copies of GGGGS. In certain embodiments, the linkers are selected from amino acids sequences that comprise the following sequences, or variants thereof: a) GGGGSGGGGSGGGGSGGGGS; b) SPNSASHSGSAPQTSSAPGSQ; c) GGGAGGGKGGGAGGGEGGGAGGGKGE; d) GPLGIAGQGGGGSGGGGSGGGGSGGGGS; e) GPLGIAGQGGGGSGGGGSGGGGS; or f) GGGGS GGGGS GGGGS. In certain embodiments, the first linker comprises GGGGSGGGGSGGGGSGGGGS, or a variant thereof, and the second linker comprises GGGGSGGGGSGGGGS, or a variant thereof.

[0223] Exemplary CD23 -binding moieties for various constructs are identified in Table 2 below. Table 2 identifies features, including the CD23-binding moiety (e.g., an antibody sequence or a polypeptide that includes CD23 stalk domain or a CD21 sushi domain) and linkers for each of the constructs, as well as additional components where indicated.

[0224] TABLE 2

[0225]

[0226]

[0227]

[0228] Bispecific Molecules (Fusion Proteins)

[0229] The present disclosure provides bispecific fusion proteins that incorporate a CD23- binding moiety described herein, as well as nucleic acid sequences and immune cells that have been modified to express the bispecific fusion proteins expressing the same. Components of the bispecific fusion proteins are further described herein.

[0230] The term “bispccific” as used herein refers to a fusion protein that includes a CD23- binding moiety described herein in addition to a second binding moiety, such as an antibody or antibody fragment, that is capable of binding a molecule expressed on a cell surface. The fusion proteins are referred to herein as "bispcci fic fusion proteins / ’ “bispecific molecules,” “chimeric fusion proteins,” “bispecific constructs,” “bispecifics,” “bispecific proteins,” and the like.

[0231] In certain embodiments, the bispecific molecule includes a CD23 -binding moiety described herein joined to an antibody or antibody fragment by a linker. Suitable linkers are identified herein. In certain embodiments, the linker is chosen from GGGGS or GGGGSGPLGIAGQ, SGGGGS, or variants thereof. In certain embodiments, the linker is a multiple of GGGGS (or variants thereof), for example, having 1, 2, 3, 4, 5, 6, 7, 8 or more copies of GGGGS. In certain embodiments, the linkers are selected from amino acids sequences that comprise the following sequences, or variants thereof: a) GGGGS GGGGS GGGGS GGGGS; b) SPNSASHSGSAPQTSSAPGSQ; c) GGGAGGGKGGGAGGGEGGGAGGGKGE; d) GPLGIAGQGGGGSGGGGSGGGGSGGGGS; e) GPLGIAGQGGGGSGGGGSGGGGS; or f) GGGGS GGGGS GGGGS. In certain embodiments, the first linker comprises GGGGSGGGGSGGGGSGGGGS, or a variant thereof, and the second linker comprises GGGGSGGGGSGGGGS, or a variant thereof.

[0232] In certain embodiments, a first binding moiety capable of binding CD23 and a second binding moiety are joined by a linker composed of 1 up to 2, 3, 4, 5. 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In certain embodiments, a fusion protein includes more than one linker separating one or more polypeptides or domains of the fusion protein. In certain embodiments, where the fusion protein contains multiple linkers, each of the linkers may have the same sequence or a different sequence. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.e., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). Examples of suitable linkers are known in the artand include, e.g., poly Gly linkers and other linkers providing suitable flexibility (e.g., / / parts. igem.org / Protein_domains / Linker), which is incorporated herein by reference.

[0233] In certain embodiments, the second binding moiety is antibody or fragment thereof that is capable of specifically binding CD3. In certain embodiments, the antibody is an scFV that binds CD3.

[0234] Constructs identified in Table 2 and 3 include bispecifics having an anti-CD3 scFV. While suitable anti-CD3 antibodies sequences are known in the art, an exemplary sequence for an anti-CD3 scFV is:

[0235] METDTLLLWVLLLWVPGSTGOVOLOQSGAELARPGASVKMSCKTSGYTFTRYTMHWV KQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASP GEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (signal peptide underlined).

[0236] Another exemplary sequence for an anti-CD3 scFV is:

[0237] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTG AVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAI YFCALWYSNLWVFGGGTKLTVLGG.

[0238] In certain embodiments, the bispecific antibodies take the format of a 2+1 IgG-scFv heterodimer. A 2+1 IgG-scFv antibody is a bispecific, trifunctional antibody format (often used in T-cell engagers) comprising a full-length immunoglobulin G (IgG) against CD23 and one attached single-chain variable fragment (scFv) against CD3. See, e.g., Santich BH, Park JA, Tran H, Guo HF, Huse M, Cheung NV. Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies. Sci Transl Med. 2020 Mar

[0239] 11;12(534):eaax1315. doi: 10.1126 / scitranslmed.aax1315. PMID: 32161106, incorporated herein by reference. For example, in generating a 2+1 IgG-scFv antibody, 2 sequences are used. The first includes the variable heavy chain from an anti-CD23 antibody, such as those identified in Table 1, an IgG heavy chain constant region, such as the human IgGl heavy chain constant region, and an anti-CD3 scFv, which may comprise an anti-CD3 variable heavy chain, a linker, and an anti-CD23 variable light chain.

[0240] In certain embodiments, the bispecific antibodies take the format of BiTE antibodies. BiTE antibodies (Bispecific T-cell engagers) are a type of immunotherapy designed to treat cancer by bridging immune system T-cells directly to cancer cells. These engineered antibodies feature two binding sites: one attaches to CD23 as described herein, while the other binds to CD3on T-cells, triggering immune -mediated cell destruction. See, e.g., Huehls AM, Coupet TA, Sentman CL. Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol. 2015 Mar;93(3):290-6. doi: 10.1038 / icb.2014.93. Epub 2014 Nov 4. PMID: 25367186, which is incorporated herein by reference.

[0241] Specific examples of BiTE and 1+2 IgG-ScFv antibodies are provided in Table 3. Other BiTE and 1+2 IgG-ScFv antibodies are contemplated using the novel antibody sequences provided herein, e.g., in Table 1.

[0242] Chimeric Antigen Receptors

[0243] The present disclosure provides chimeric antigen receptors that incorporate a CD23-binding moiety described herein, as well nucleic acid sequences and immune cells that have been modified to express the chimeric antigen receptors expressing the same. Components of CARs are further described herein.

[0244] The term “chimeric antigen receptor” or “CAR” as used herein refers to a molecule (e.g., a synthetic receptor) comprising an extracellular antigen-binding domain fused to an intracellular signaling domain that is capable of activating or stimulating an immunoresponsive cell. There are three generations of CARs. “First generation” CARs are typically composed of an extracellular antigen-binding domain (e.g.. an scFv), which is fused to a transmembrane domain, which is fused to cytoplasmic / intracellular signaling domain. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA -mediated antigen presentation. “Second generation” CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. “Second generation'’ CARs comprise those that provide both co- stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen-recognizing receptor is a first-generation CAR. In certain embodiments, the antigen-recognizing receptor is a CAR that does not comprise an intracellular signaling domain of a co-stimulatory molecule or a fragment thereof. In certain embodiments, the antigen-recognizing receptor is a second-generation CAR.

[0245] In accordance with the presently disclosed subject matter, a CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is a CD23-binding moiety as provided herein. In certain embodiments, the CD23-binding moiety is fused to the transmembrane domain and then to the intracellular signaling domain.In certain embodiments, the CAR comprises a transmembrane domain. In certain embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the antigen- recognizing receptor can comprise a native or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof.

[0246] In certain embodiments, the CAR comprises a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. In certain embodiments, tire hinge / spacer region of the CAR can comprise a native or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof. The hinge / spacer region can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, a variation of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%. at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0247] In certain embodiments, the antigen-recognizing receptor is a CAR that further comprises a hinge / spacer region comprising a native or modified hinge region of a CD28 polypeptide. In certain embodiments, the hinge / spacer region of tire antigen-recognizing receptor (e.g., a CAR) comprises a CD28 polypeptide.

[0248] In certain embodiments, the hinge / spacer region is positioned between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof.

[0249] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD84 polypeptide and the transmembrane domain comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD 166 polypeptide and the transmembrane domain comprises a CD 166 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8a polypeptide and the transmembrane domain comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8b polypeptide and the transmembrane domain comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide.

[0250] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g.. a T-cell). Wild type (“native’7) CD3C comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3). CD3ζ transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T-cell) after antigen is bound. The intracellular signaling domain of the CD3ζ-chain is the primary transmitter of signals from endogenous TCRs.

[0251] In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor comprises a native CD3ζ. In certain embodiments, the native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence having a NCBI Reference No: NP_932170 or a fragment thereof, and / or may optionally compriseup to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of the full-length CD3ζ, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, the native CD3ζ comprises or consists of the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of full-length CD3ζ.

[0252] In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one co-stimulatory region comprises a co-stimulatory molecule or a portion thereof. In certain embodiments, the at least one co-stimulatory region comprises at least an intracellular domain of at least one co-stimulatory molecule or a portion thereof. Non-limiting examples of costimulatory molecules include CD28, 4- IBB, 0X40, CD27, CD40, CD 154, CD97,

[0253] CD11a / CD18, ICOS, DAP-10, CD2, and NKG2D.

[0254] In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor (e.g., a CAR) comprises a co-stimulatory signaling region that comprises a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of human CD28 or a portion thereof.

[0255] In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor comprises a co-stimulatory signaling region that comprises a 4-1BB polypeptide, e.g., an intracellular domain of 4- IBB or a portion thereof. In certain embodiments, the co-stimulatory signaling region comprises an intracellular domain of human 4- IBB or a portion thereof.

[0256] In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor comprises two co-stimulatory signaling regions, wherein the first co-stimulatory signaling region comprises an intracellular domain of a first co-stimulatory molecule or a portion thereof, and the second co-stimulatory signaling region comprises an intracellular domain of a second co-stimulatory molecule or a portion thereof. The first and second co-stimulatory molecules are independently selected from the group consisting of CD28, 4- IBB, 0X40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, and NKG2D. In certain embodiments, the intracellular signaling domain of the antigen-recognizing receptor comprises two co-stimulatory signaling regions, wherein the first co-stimulatory signaling region comprises an intracellular domain of CD28 or a portion thereof and the second co-stimulatory signaling region comprises an intracellular domain of 4-1BB or a portion thereof.

[0257] Nucleic Acids / Expression CassettesProvided herein are nucleic acids and expression cassettes that include sequences that encode a CD23-binding moiety polypeptide and proteins that include tire same (e.g., a bispecific protein that includes a CD23-binding moiety, or a chimeric antigen receptor (CAR) that includes a CD23-binding moiety). The nucleic acids include RNA species as well as DNA encoding the RNA species. In certain embodiments, the nucleic acids are included in expression cassettes, wherein the coding sequences are operably linked to expression control sequences. In certain, embodiments the nucleic acids are included in a vector or composition for delivery' to a target cell or for administration to a subject.

[0258] The terms “nucleic acid,” “nucleotide sequence,” and “nucleic acid molecule,” as used herein interchangeably, refer to a compound comprising a nucleobase and an acidic moiety, e.g.. a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or including non-naturally occurring nucleotides or nucleosides.

[0259] Furthermore, the tenns “nucleic acid,” “DNA.” “RNA,” and / or similar terms include nucleic acid analogs, e.g.. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5’ to 3 ' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxy thymidine, deoxy guanosine, and deoxy cytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine. inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5 -methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine): chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose): and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0260] As used herein, an “expression cassette” refers to a nucleic acid molecule encoding a bispecific protein or CAR that includes regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequences. Such an expression cassette may be administered to a subject for therapeutic purposes. Expression cassettes can also be used for generating a viral vector for therapeutic delivery of the nucleotide sequences described.

[0261] As used herein, the term “regulatory sequence”, or “expression control sequence” refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, and promoter sequences, which induce, repress, or otherwise control the transcription of nucleic acid sequences to which they are operably linked.

[0262] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0263] The term “operably linked” refers to functional linkage between one or more regulatory sequences and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein coding regions, are in tire same reading frame.

[0264] Suitable promoters include, e.g., constitutive promoters, regulatable promoters [see, e.g., WO 2011 / 126808 and WO 2013 / 04943], or a promoter responsive to physiologic cues. The promoter can be selected from different sources, e.g., human cytomegalovirus (CMV) immediate-early enhancer / promoter, the SV40 early enhancer / promoter, the JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, herpes simplexvirus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet derived growth factor (PDGF) promoter, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter.

[0265] In addition to a promoter, an expression cassette may contain one or more other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA for example WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. As described herein, regulatory elements comprise but not limited to: promoter: enhancer: transcription factor: transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence).

[0266] Expression cassettes can be delivered via any suitable delivery system. Suitable non-viral delivery systems are known in the art (see, e.g., Ramamoorth and Narvekar. J Clin Diagn Res. 2015 Jan: 9(l): GE01-GE06, which is incorporated herein by reference) and can be readily selected by one of skill in the art and may include, e.g., naked DNA, naked RNA, dendrimers, PLGA, polymethacrylate, an inorganic particle, a lipid particle (e.g., a lipid nanoparticle or LNP), or a chitosan-based formulation. In certain embodiments, the nucleic acids or expression cassettes provided are in a plasmid.

[0267] A “viral vector’' refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of interest is packaged in a viral capsid or envelope. Examples of viral vectors include but are not limited to lentivirus, adenoviruses, retroviruses (y-retroviruses and lentiviruses). poxviruses, adeno-associated viruses (AAVs). baculoviruses, herpes simplex viruses. In one embodiment, the viral vector is replication defective. A “replication-defective virus” refers to a viral vector, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient, i.e., they cannot generate progeny virions but retain tire ability to infect cells.

[0268] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al.. Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in tire clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like.

[0269] Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0270] In certain embodiments, the vector is a non-viral plasmid that comprises an expression cassette described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid -nucleic acid compositions, poly-glycan compositions and other polymers, lipid and / or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774–787; web publication: March 21, 2011; WO2013 / 182683, WO 2010 / 053572 and WO 2012 / 170930, all of which are incorporated herein by reference.

[0271] Plasmids, other cloning and expression vectors, properties thereof, and constructing / manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In one embodiment, an expression cassette as described herein is engineered into a suitable genetic element (a vector) useful for generating viral vectors and / or for introduction to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity’ DNA-coatcd pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See. e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0272] The term “transfected” refers to a process by which exogenous nucleic acid is transferred or introduced into tire host cell. A “transfected” cell is one which has been transfected with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0273] As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.RNA or DNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861-70 (2001).

[0274] Nucleoside-Modified mRNA

[0275] In certain embodiments, the compositions provided include a nucleic acid molecule that is a nucleoside-modified mRNA. Nucleoside-modified mRNA have advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modifications useful in the present invention is further described in U. S. Patent No. 8,278,036, which is incorporated by reference herein in its entirety.

[0276] An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA in tire compositions comprise at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. An mRNA may include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherw ise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosine in an mRNA may be 5-methylcytosine.

[0277] As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the mRNA. As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly w ith respect to the mRNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such mRNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such mRNA in the target cell, tissue, subject and / or cytoplasm. Also contemplated by the terms “modification” and “modified” as such terms related to the mRNA of the present invention are alterations w hich improve or enhancetranslation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence).

[0278] In certain embodiments, the non-viral delivery system comprises an RNA molecule which is a messenger RNA (mRNA) molecule. In certain embodiments, the mRNA molecule is an in vitro transcribed (IVT) mRNA molecule. In certain embodiments, the IVT mRNA molecule is a nucleoside-modified mRNA molecule. An mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translating sequence.

[0279] Lipid Nanoparticles

[0280] In certain embodiments, the compositions and methods provided herein require a nucleic acid molecule, wherein the nucleic acid molecule is encapsulated in a lipid nanoparticle (LNP). The encapsulated nucleic acids include DNA and / or RNA sequences that encode a bispecific protein or CAR construct as provided herein.

[0281] The term “lipid nanoparticle”, also referred to as LNP, refers to a particle having at least one dimension on tire order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids). In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In some embodiments, the DNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells. In some embodiments, the DNA or a portion thereof is associated with the lipid nanoparticles. Preferably, the lipid nanoparticles are formulated to deliver one or more DNA to one or more target cells (e.g., tumor cells).

[0282] In the context of the present disclosure, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and tire like are within the scope of a lipid nanoparticle.

[0283] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm,90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the DNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the z-average as determined by dynamic light scattering.

[0284] Viral Vectors

[0285] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to tire nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.

[0286] Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of tire inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0287] Retroviral vectors comprise cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (sec, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700).

[0288] In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.

[0289] Adeno-associated virus (“AAV’) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures. Several different AAV serotypes have been used to advantage for transduction of mammalian cells, these include, for example AAV 1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 that have different tropisms for cell types of interest.Construction of recombinant AAV vectors is described in a number of publications, including U. S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al.. J. Virol. 63:03822-3828 (1989).

[0290] In certain embodiments, one or more nucleic acid sequences provided herein are delivered to target cells by a vector or a viral vector, of which many are known and available in the art. In one embodiment, provided is a vector comprising an expression cassette as described herein. In one embodiment, the vector is a non-viral vector. In a further embodiment, the non-viral vector is a plasmid. In another embodiment, the vector is a viral vector. Viral vectors include any virus suitable for gene therapy, including but not limited to a bocavirus, adenovirus, adeno-associated virus (AAV), herpes virus, lentivirus, retrovirus, or parvovirus. However, for ease of understanding, the adeno-associated virus is referenced herein as an exemplary viral vector.

[0291] ■‘Plasmid” or “plasmid vector” generally is designated herein by a lower-case p preceded and / or followed by a vector name. Plasmids, other cloning and expression vectors, properties thereof, and constructing / manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In certain embodiments, the expression cassettes described herein are engineered into a suitable genetic element (a vector) useful for generating viral vectors and / or for delivery to a host cell, e.g.. naked DNA, phage, transposon, cosmid. episome, etc., which transfers the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor. NY.

[0292] As used herein, the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid. In the alternative, the term “host cell” may refer to any target cell for expression of a bispecif protein or CAR as provided herein. Thus, a “host cell,” refers to a prokaryotic or eukaryotic cell that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. In certain embodiments herein, the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositionsdescribed herein. In other embodiments herein, the term “host cell” refers to tire cells employed to generate and package the viral vector or recombinant virus.

[0293] Methods of making modified host cells

[0294] Also provided herein are methods of making the modified cells and compositions containing modified cells as described herein. Methods of modifying cells, e.g., immune cells or tumor cells, to introduce an exogenous sequence, such as an expression cassette or expression vector comprising a coding sequence for a bispecific protein or a CAR are known in the art. For example, see, e.g., WO 2016 / 109410 A2, which is incorporated herein by reference.

[0295] By the tenn “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. Modifying can refer to altering expression of a gene in a lymphocyte, for example, by introducing an exogeneous nucleic acid that encodes the gene.

[0296] The lymphocytes provided herein can be genetically modified, e.g., by transfection, transduction, or electroporation, to express a nucleic acid sequence encoding a fusion protein or CAR as described herein. Depending on the clinical context, e.g., patient's condition or condition to be treated, prolonged or permanent expression of the gene and / or. e.g., for robust and long-lasting CAR activity, e.g., anti-tumor activity, may be desirable. In such embodiments, die lymphocytes are genetically modified, e.g., transduced, e.g., virally transduced, using vectors comprising nucleic acid sequences encoding a gene disclosed herein to confer a desired effector function. In other embodiments, transient expression of the gene is desirable. In such embodiments, the use of, e.g., mRNA or a regulatable promoter to express the effector-enhancing gene, may be used.

[0297] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0298] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al.. 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press. NY). A suitable method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, and the like.

[0299] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable submicron sized delivery system. In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Also contemplated are lipofectamine-nucleic acid complexes.

[0300] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant nucleic acid sequence in the host cell, a variety of assays may be performed. Such assays include, for example, Southern and Northern blotting. RT-PCR and PCR, biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots).Methods of Treatment

[0301] In another aspect, methods of treating subjects having certain conditions are provided, wherein a therapeutically effective amount of the CD23 -binding moiety, or a fusion protein, a composition, or a cell comprising the same, as described herein, is administered. In certain embodiments, the CD23-binding moiety or a bispecific protein or CAR containing the same is provided via a plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein. It is contemplated that for each embodiment wherein a CD23-binding moiety polypeptide, or a fusion protein, a composition, or a cell comprising the same is administered, another embodiment is contemplated wherein a plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein is administered.

[0302] As used herein, the term “subject” means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable subjects include, without limitation, murine, rat, canine, feline, porcine, bovine, ovine, non-human primate and others. As used herein, the term “subject” is used interchangeably with “patient”.

[0303] In humans a variety of hematopoietic cell types express CD23 on their surface, including follicular dendritic cells, B cells, T cells and macrophages. CD23 molecules are also found in soluble forms in biological fluids. Soluble CD23 (sCD23) molecules are formed by proteolytic cleavage of transmembrane receptors. CD23 has pleiotropic activities including mediation of cell adhesion, regulation of IgE and histamine release, rescue of B cells from apoptosis and regulation of myeloid cell growth.

[0304] Increased expression of CD23 has been observed in a number of inflammatory diseases. CD23 has been identified in synovial biopsies from patients with chronic synovitis, and sCD23 can be measured at concentrations exceeding the normal range in the serum and synovial fluid of patients with rheumatoid arthritis (Bansal, A. S., Oliver. W., Marsh. M. N., Pumphrey, R. S., and Wilson, P. B., Immunology 79, 285-289 (1993); Hellen, E. A., Rowlands, D. C., Hansel, T. T., Kitas, G. D., and Crocker, J. J., Clin Pathol 44, 293-296 (1991); Chomarat, P., Brioloay, J., Banchereau, J., & Miossec, P., Arthritis Rheum 86, 234-242 (1993); Bansal, A., et al., Clin Exp Immunol 89, 452 455 (1992); Rezonzew, R., & Newkirk, M. N., Clin Immunol Immunopathol 71, 156-163 (1994). In addition, levels of serum sCD23 in rheumatoid arthritis patients are related to disease status and correlate with serum rheumatoid factor (Bansal, A. S., et al., Clin Exp Rheumatol 12, 281–285 (1994). Pro-inflammatory cytokines appear to be particularly important in rheumatoid arthritis, and a central role for TNF-a and IL- lb in the destruction of arthritic joints has been postulatedIn one embodiment, provided herein is a method of treating an autoimmune disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of tire CD23-binding moiety polypeptide described herein, or a bispecific protein, a composition, or cell comprising the same, as described herein. In certain embodiments, a plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein is administered to the patient. In certain embodiments, the autoimmune disorder is myasthenia gravis. In other embodiments, the autoimmune disorder is multiple sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, vitiligo, or lupus. In other embodiments, the disorder is synovitis.

[0305] The binding agents of the present invention are useful in the treatment or prophylaxis of human diseases including arthritis, lupus erythematosus, Mashimoto’s thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, Sjogren's syndrome, allergies, asthma, rhinitis, eczema, GVH, COPD, insulitis, bronchitis (particularly chronic bronchitis) or diabetes (particularly Type 1 diabetes). In certain embodiments, the disease is rheumatoid arthritis.

[0306] Autoimmune diseases are conditions arising from abnormal immune attack to the body, and they substantially increase the morbidity, mortality and healthcare costs worldwide. As T cells play a key role in the process of autoimmune diseases, engineered T-cell therapy has emerged and is also regarded as a potential approach to overcome current roadblocks in the treatment of autoimmune diseases. Either self-reactive or autoantibodies play a key role in the process of autoimmune diseases. Thus, engineering T cells to express a chimeric autoantibody receptor (CAAR) is a strategy for treatment for autoimmune disease. In one embodiment, the CAR comprises a CAAR. Sec, e.g., Zhang ct al, Chimeric antigen receptor T-cell therapy beyond cancer: current practice and future prospects, Immunotherapy, 2020 Sep; 12(13): 1021- 1034. doi: 10.2217 / imt-2020-0009. Epub 2020 Jul 30, which is incorporated herein by reference.

[0307] Autoimmune diseases include Pemphigus vulgaris (PV) (e.g., DSG3-CAAR-T) and lupus (e.g., MuSK-CAAR-T)). Other autoimmune diseases include type 1 diabetes, autoimmune thyroid disease, rheumatoid arthritis (RA), inflammatory bowel disease, colitis, systemic lupus erythematosus, and multiple sclerosis (MS). See, e.g., Chen et al, Immunotherapy Deriving from CAR-T Cell Treatment in Autoimmune Diseases, Journal of Immunology’ Research Volume 2019, December 31, 2019, which is incorporated herein by reference. Other conditions treatable with tire compositions described herein include graft-versus-host disease (GVHD) and transplant rejection.

[0308] In certain aspects, provided herein is the use of a CD23 -binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same described herein for themanufacture of a medicament for the treatment of a condition described herein. In certain embodiments, provided herein is the use of a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same described herein for the manufacture of a medicament for the treatment of arthritis, lupus erythematosus, systemic lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis.diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Sjogren's syndrome, allergies, asthma, eczema, GVH, COPD, bronchitis, insulitis, rhinitis or diabetes.

[0309] In another embodiment, provided herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same, as described herein. In certain embodiments, a plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein is administered to the patient. In certain embodiments, provided herein is the use of a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same described herein for the manufacture of a medicament for the treatment of cancer.

[0310] The term ‘‘cancer’ as used herein refers to any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication. In certain embodiments, administration of the compositions disclosed herein, e.g., according to the methods disclosed herein, treats a cancer. In certain embodiments, the cancer is selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease. Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymuscancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, secondary cancers caused by cancer treatment, and any combination thereof. In certain embodiments, the cancer is a hematologic cancer including but not limited to leukemia (such as acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoid leukemia and myelodysplastic syndrome) and malignant lymphoproliferative conditions, including lymphoma (such as multiple myeloma, nonHodgkin's lymphoma, Burkitt's lymphoma, and small cell- and large cell-follicular lymphoma). In other embodiments, a hematologic cancer can include minimal residual disease, MRD, e.g., of a leukemia, e g., of AML or MDS. Other cancers include breast cancer, lung cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, cervical cancer, liver cancer, ovarian cancer, and testicular cancer. In other embodiments, the cancer is a solid tumor cancer.

[0311] In certain embodiments, as noted, the CD23 -binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same, is provided via a nucleic acid comprising a sequence that encodes the polypeptide or fusion protein. Various methods may be employed to deliver the nucleic acid including, without limitation, via plasmid, non-viral vector, viral vector, including AAV, adenovirus, lentivirus, etc, mRNA. etc.

[0312] In one embodiment, the methods comprise administering to the subject in need thereof an effective amount of a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same described herein in combination with an effective amount of another therapy. In certain embodiments, a plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein is administered to the subject in combination with an effective amount of another therapy. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0313] A CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same as described herein, or plasmid, vector, or nucleic acid that includes a sequence encoding the polypeptide or fusion protein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[0314] In further aspects, a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506. antibodies, or other immunoablative agents such as CAMPATH. anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, or a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0315] The binding agents of the present invention may be used alone or in combination with immunosuppressive agents such as steroids, cyclosporin, or antibodies such as an antilymphocyte antibody or more preferably with a tolerance-inducing, anti- autoimmune or antiinflammatory agent such as a CD4+T cell inhibiting agent e g. an anti-CD4 antibody (preferably a blocking or non-depleting antibody), an anti-CD8 antibody, a TNF antagonist e.g. an anti-TNF antibody or TNF inhibitor e.g. soluble TNF receptor, or agents such as NSAIDs.

[0316] In certain instances, a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same is combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.

[0317] In one embodiment, a CD23 -binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine.vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide), or a checkpoint inhibitor (e.g., a PD-1 or PD-L1 inhibitor, e.g., Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq). Avelumab (Bavencio), Durvalumab (Imfinzi)).

[0318] General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine). hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol® ), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0319] In an embodiment, the chemotherapeutic agent is administered prior to administration of the CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising the same. In chemotherapeutic regimens where more than one administration of the chemotherapeutic agent is desired, the chemotherapeutic regimen is initiated or completed prior to administration of the CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same. In embodiments, thechemotherapeutic agent is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, or 30 days prior to administration of the CD23 -binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same. In embodiments, the chemotherapeutic regimen is initiated or completed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, or 30 days prior to administration of the CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same.

[0320] For the methods described herein, the composition is administered in a therapeutically or prophylactically effective amount. The term “therapeutically effective amount” refers to the amount of the composition needed to treat or ameliorate a targeted disease. The term “prophylactically effective amount” used herein refers to the amount of the composition needed to prevent a targeted disease.

[0321] The frequency with which the dose needs to be administered will depend on the half-life of the agent involved. For example, the dose may be administered on a daily basis, a twice daily basis, or every two, three, four days, five, six, seven, 10, 15 or 20 days or more.

[0322] The exact dosage and the frequency of doses may also be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician.

[0323] The agent will generally be administered as part of a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier”, as used herein, includes genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. The pharmaceutical carrier employed will thus vary depending on the route of administration. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).The agent may be delivered by any known route of administration. The agent may be delivered by a parenteral route (e.g. by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue). The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications, needles, and hyposprays. In certain embodiments, a composition is administered to the thymus, including administration to one or both lobes of the thymus.

[0324] The CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same may be administered alone or as part of a treatment regimen also involving the administration of other drugs currently used in the treatment of patients with, for example, myasthenia gravis. For example, the agent may be administered in combination with anticholinesterase agents, such as neostigmine and pyridostigmine, or immunosuppressive drugs, such as prednisone, cyclosporine, and azathioprine. Combinations of drug treatments may have an additive or synergistic effect on treatment of the disease.

[0325] In one aspect, tire methods comprise administering to the subject in need thereof an effective amount of a CD23-binding moiety described herein, or a bispecific protein, a composition, or a cell or comprising or encoding the same described herein in combination with an effective amount of another therapy. Administered “in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery’ of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous’' or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.In certain embodiments, the method includes administering to the subject a cell that expresses a CD23-binding moiety described herein, or a bispecific protein, or CAR as described herein such that the disease is treated in the subject. In one embodiment, the method includes obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient. In another embodiment, tire cells are sourced from a different subject than the patient receiving the cells.

[0326] Specific embodiments

[0327] 1. A bispecific molecule comprising: a) a first binding moiety capable of binding CD23, and b) a second binding moiety capable of binding a molecule expressed on a cell surface, wherein the first binding moiety and second binding moiety are joined by a linker.

[0328] 2. The bispecific molecule of embodiment I, wherein the second binding moiety is capable of binding CD3, optionally wherein the second binding moiety is antibody or fragment thereof.

[0329] 3. The bispecific molecule of embodiment 1, wherein the first binding moiety is an antibody or fragment thereof, optionally wherein the antibody or fragment thereof is a dAb, Fab, Fab’, F(ab’)2, Fv, scFv, scFv2, scFv-Fc, minibody, diabody, triabody, or tetrabody.

[0330] 4. The bispecific molecule of any one of embodiments 1 to 3, wherein the first binding moiety is an antibody or fragment thereof comprising six complementarity-determining regions of

[0331] a) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0332] b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0333] c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 14;

[0334] d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 14; or

[0335] e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0336] 5. The bispecific molecule of any one of embodiment 1 to 3, wherein the first binding moiety comprises a polypeptide capable of binding a CD23 stalk region.

[0337] 6. The bispecific molecule of embodiment 5, wherein the polypeptide capable of binding a CD23 stalk region comprises a sequence of amino acids 90 to 156 of SEQ ID NO: 1.7. The bispecific molecule of embodiment 6, wherein the first binding moiety further comprises a sequence corresponding to amino acids 48 to 89 of SEQ ID NO: 1.

[0338] 8. The bispecific molecule of embodiment 5. wherein the polypeptide capable of binding a CD23 stalk region comprises any one of SEQ ID NOs: 18-20, or a variant thereof.

[0339] 9. The bispecific molecule of any one of embodiments 1 to 3, wherein the polypeptide capable of binding a CD23 stalk region comprising one or more sushi domains of CD21, optionally wherein the polypeptide comprises at least sushi domains 2-7 of CD21.

[0340] 10. The bispecific molecule of embodiment 9. wherein the polypeptide capable of binding a CD23 stalk region comprises at least 1, 2, 3, 4, 5, 6, 7, 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein.

[0341] 11. The bispecific molecule of embodiment 9 or 10, wherein the polypeptide capable of binding a CD23 stalk region comprises less than 15, 14, 13, 12, 11, 10, 9, or 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein.

[0342] 12. A CD23-binding molecule, wherein the CD23-binding molecule is an antibody or fragment thereof comprising six complementarity-determining regions of

[0343] a) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0344] b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0345] c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 14;

[0346] d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 14; or

[0347] e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0348] 13. A CD23-binding molecule comprising a polypeptide capable of binding a CD23 stalk region.

[0349] 14. The CD23-binding molecule of embodiment 13, wherein the polypeptide capable of binding a CD23 stalk region comprises a sequence of amino acids 90 to 156 of SEQ ID NO: 1.

[0350] 15. The CD23-binding molecule of embodiment 13, wherein the polypeptide further comprises a sequence corresponding to amino acids 48 to 89 of SEQ ID NO: 1.16. The CD23-binding molecule of embodiment 13, wherein the polypeptide capable of binding a CD23 stalk region comprises any one of SEQ ID NOs: 18-20, or a variant thereof. 17. A CD23-binding molecule comprising a polypeptide capable of binding a CD23 stalk region comprises one or more sushi domains of CD21, optionally wherein the polypeptide comprises at least sushi domains 2-7 of CD21.

[0351] 18. The CD23-binding molecule of embodiment 17, wherein the polypeptide capable of binding a CD23 stalk region comprises at least 1, 2, 3, 4, 5, 6, 7, 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein.

[0352] 19. The CD23-binding molecule of embodiment 17 or 18, wherein the polypeptide capable of binding a CD23 stalk region comprises less than 15, 14, 13. 12, 11, 10, 9, or 8 sushi domains of CD21, which may be consecutive or non-consecutive in the native CD21 protein.

[0353] 20. A chimeric antigen receptor (CAR) comprising:

[0354] a) an extracellular domain comprising tire CD23-binding molecule of any one of embodiments 12 to 19;

[0355] b) a transmembrane domain; and

[0356] c) an intracellular signaling domain.

[0357] 21. The CAR of embodiment 20, further comprising one or more costimulatory signaling regions, optionally wherein the costimulatory signaling regions comprise a functional signaling domain of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278), or 4-1BB (CD137).

[0358] 22. The CAR of any one of embodiments 20 or 21, wherein the transmembrane domain comprises the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45. CD4. CD5. CD8. CD9, CD 16, CD22. CD33, CD37. CD64, CD80, CD86, CD134, CD137, or CD154.

[0359] 23. The CAR of any one of embodiments 20 to 33, wherein the intracellular signaling domain comprises a functional signaling domain of 4- IBB or a functional signaling domain of CD3 zeta.

[0360] 24. The CAR of any one of embodiments 20 to 23, wherein the extracellular domain is linked to the transmembrane domain by a hinge region, optionally wherein the hinge region is from CD8.25. A nucleic acid comprising a nucleotide sequence that encodes the bispecific molecule of any one of embodiments 1 to 11, the CD23-binding molecule of any one of embodiments 12 to 19, or the CAR of any one of embodiments 20 to 24.

[0361] 26. An expression cassette comprising the nucleic acid of embodiment 25.

[0362] 27. A plasmid comprising the nucleic acid of embodiment 25 or the expression cassette of embodiment 26.

[0363] 28. A host cell comprising the nucleic acid of embodiment 25, the expression cassette of embodiment 26, or the plasmid of embodiment 27, optionally wherein the host cell is a lymphocyte.

[0364] 29. A lymphocyte that has been modified to express the bispecific molecule of any one of embodiments 1 to 8, the CD23 -binding molecule of any one of embodiments 12 to 19, or the CAR of any one of embodiments 20 to 24.

[0365] 30. A recombinant viral vector comprising a nucleic acid comprising the expression cassette of embodiment 26.

[0366] 31. The recombinant viral vector of embodiment 30, wherein the recombinant viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, or a lentiviral vector.

[0367] 32. A recombinant AAV vector comprising an AAV capsid and having a vector genome packaged therein, the vector genome comprising the expression cassette of embodiment 26. 33. A lipid nanoparticle (LNP) comprising the nucleic acid of embodiment 25.

[0368] 34. The LNP of embodiment 33, wherein the nucleic acid is a nucleoside modified mRNA.

[0369] 35. A pharmaceutical composition comprising the bispecific molecule of any one of embodiments 1 to 11, the CD23-binding molecule of any one of embodiments 12 to 19, the CAR of any one of embodiments 20 to 24, the nucleic acid of embodiment 25, the expression cassette of embodiment 26, the plasmid of embodiment 27, the host cell of embodiment 28, the lymphocyte of embodiment 29, the recombinant viral vector of embodiment 30, the recombinant AAV vector of embodiment 31 or 32, or the LNP of embodiment 33 or 34, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0370] 36. A method of treating an autoimmune condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecificmolecule of any one of embodiments 1 to 11, the CD23-binding molecule of any one of embodiments 12 to 19, the CAR of any one of embodiments 20 to 24, the nucleic acid of embodiment 25, the expression cassette of embodiment 26, the plasmid of embodiment 27, the host cell of embodiment 28, the lymphocyte of embodiment 29, the recombinant viral vector of embodiment 30, the recombinant AAV vector of embodiment 31 or 32, the LNP of embodiment 33 or 34, or the pharmaceutical composition of embodiment 35.

[0371] 37. A method of treating cancer in a subject in a subject in need thereof, tire method comprising administering to the subject the method comprising administering to tire subject a therapeutically effective amount of the bispecific molecule of any one of embodiments 1 to 11, the CD23 -binding molecule of any one of embodiments 12 to 19, the CAR of any one of embodiments 20 to 24, the nucleic acid of embodiment 25, the expression cassette of embodiment 26, the plasmid of embodiment 27, the host cell of embodiment 28, the lymphocyte of embodiment 29, the recombinant viral vector of embodiment 30, the recombinant AAV vector of embodiment 31 or 32, the LNP of embodiment 33 or 34, or the pharmaceutical composition of embodiment 35.

[0372] EXAMPLES

[0373] Example 1: Generation and testing of an anti-CD23:anti-CD3 bispecific construct (AB0018) Methods: Following sequence verification, the AB0018 bispecific was produced in 293T cells. Supernatant was collected and clarified. HuPBMC from two donors were treated w ith the bispecific (AB0018) and analyzed post 24 hrs using flow cytometry to measure targeting of CD23+ B cells by T cells.

[0374] Results: Flow cytometry analysis demonstrated selective depletion of CD23+CD19+activated B cells following treatment with the CD23-targeting bispecific (data not shown). The left panel shows a significant presence of CD23+CD19+B cells no-treatment, while the right panel indicates a marked reduction post-treatment. The quantification bar graph confirms this targeted depletion with a statistically significant decrease in the CD23+CD19+subset (p-value shown in the graph).

[0375] Significance: The findings indicate that CD23 -depleting antibodies selectively target activated CD23+CD19+B cells, which are disease-driving, and highlighting their potential as a therapeutic target for autoimmune diseases and B cell-mediated disorders.

[0376] Example 2: Dose-dependent T-cell activation and target cell depletion by CD23 / CD3bispecific antibodies (CD23 binder based) in human PBMCs

[0377] Experimental Aim: To evaluate binding-driven functional activity of

[0378] CD23xCD3 bispecific antibodies (CD23 binder based) in human PBMCs by assessing T-cell activation, depletion of CD23+target cells and susceptibility to soluble CD23-mediated neutralization.

[0379] Methods: Human PBMCs were treated with a panel of CD23*CD3 bispecific antibodies (AB0170, AB0171, AB0175, and AB0018 lumiliximab analogue) across a concentration range of 0–1 µg / mL. Following incubation, flow cytometry was performed to measure CD69 expression on CD3+T cells as a marker of activation and to quantify depletion of CD23+target cells within the PBMC population. To assess soluble antigen interference, selected conditions were repeated in tire presence of soluble CD23 (100 U / mL). Data were normalized to untreated controls, and dose-response relationships were evaluated across constructs.

[0380] Results: All CD23*CD3 bispecific antibodies induced concentration-dependent T-cell activation and progressive depletion of CD23+target cells. Distinct activity profiles were observed between constructs, with several Ajaya CD23 binders demonstrating

[0381] robust activation and target elimination at higher concentrations. In the presence of soluble CD23, the lumiliximab analogue (AB0018) showed reduced functional activity, consistent with soluble antigen-mediated neutralization. In contrast, AB0170, AB0171, and AB0175 retained activity with no significant loss of function, supporting resistance to soluble CD23 interference and consistent with differential epitope engagement.

[0382] Example 3: Generation of monoclonal antibodies

[0383] Anti-CD23 monoclonal antibody, lumiliximab. demonstrated acceptable safety and target engagement with early signs of clinical activity in Phase 1 / 2 allergy studies, but did not achieve sufficient efficacy to support advancement in later-stage development. Our approach targets the human CD23 stalk region, antibodies to which avoid neutralization by soluble CD23. FIG. 4.

[0384] Monoclonal antibodies were generated according to established protocols, and humanized. Briefly, there are four general steps to humanize a monoclonal antibody. These are:

[0385] • (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains;

[0386] • (2) designing the humanized antibody, i.e. deciding which antibody framework region to use during the humanizing process;

[0387] • (3) the actual humanizing methodologies / techniques; and

[0388] • (4) the transfection and expression of the humanized antibody.4.1 Affinity analysis of ligand to analyte

[0389] Table 6. Affinity data of ligand to analyte

[0390] Ligand Analyte Chi² (RU²) ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU)

[0391]

[0392] 5A4-1 human CD23 (48-150) 1.87E-02 3.32E+05 2.27E-02 6.83E-08 37.1 7H6-1 human CD23 (48-150) 1.53E-02 3.16E+05 9.52E-03 3.02E-08 45.9 13D4-1 human CD23 (48-150) 6.31E-02 1.40E+06 3.65E-02 2.61E-08 45.9 15A1-1 human CD23 (48-150) 1.96E-02 2.37E+05 2.04E-03 8.62E-09 37.8 17E1-1 human CD23 (48-150) 8.57E-02 1.59E+05 2.74E-02 1.72E-07 102.6 20G4-3 human CD23 (48-150) 5.87E-02 3.18E+05 6.63E-02 2.09E-07 109.3 4G6-1 human CD23 (48-150) 5.51E-01 3.08E+04 1.10E-01 3.56E-06 1275.3 19E12-1 human CD23 (48-150) 6.76E-02 8.13E+05 1.00E-02 1.23E-08 50.2

[0393]

[0394] Example 4: ELISA to determine anti-CD23 monoclonal antibody binding to truncated and full-length CD23 protein, but not soluble protein

[0395] Experimental Aim: To evaluate the binding of multiple anti-CD23 antibody clones to full-length, truncated, and soluble CD23 proteins using an ELISA-based format.

[0396] Methods: Binding was assessed by indirect ELISA. Microplates were coated with full-length, truncated, or soluble CD23 proteins (1-10 pg / mL, 100 pL / well) and incubated to allow antigen adsorption. Wells were washed and blocked to prevent nonspecific binding, followed by incubation with serially diluted test antibody clones or lumiliximab-analog control. After washing, HRP-conjugated secondary antibody was added, and signal was developed using TMB substrate. The reaction was stopped and absorbance was recorded at 450 nm to quantify relative binding responses. Each condition was run with appropriate blanks / controls and replicates to ensure assay consistency, following a standard indirect ELISA workflow.

[0397] Results: Compared to lumiliximab-analog antibody clones showed differential binding profiles across antigen formats, with several clones displaying strong reactivity' to full-length CD23 and truncated forms, but no or minimal binding to soluble forms, indicating epitope-dependent recognition and clone-specific specificity (FIG. 5

[0398] Example 4: Flow cytometric analysis of anti-CD23 monoclonal antibody binding to CD23 extracellular domain and full-length protein overexpressed in Jurkat cellsExperimental Aim: To evaluate binding of anti-CD23 monoclonal antibody clones to the CD23 extracellular domain (ECD) and full-length (FL) protein overexpressed in Jurkat cells, using CD23 WT Jurkat cells as negative controls and comparing performance with lumiliximab analog.

[0399] Methods: Jurkat cell lines expressing either wild-type (CD23⁻), CD23 extracellular domain (ECD mutant), or full-length CD23 were incubated with individual anti-CD23 monoclonal antibodies. Following primary staining, bound antibodies were detected using an FITC-conjugated anti-IgG Fc secondary antibody and analyzed by flow cytometry. Binding was assessed by comparing fluorescence shifts relative to WT control cells.

[0400] Results: No binding was observed in WT Jurkat cells, confirming assay specificity and validating their use as CD23⁻ controls. Our antibody clones demonstrated clear binding to both ECD and full-length CD23 -expressing cells, indicating recognition of epitopes accessible within the extracellular region. In contrast, lumiliximab analog failed to bind the ECD mutant construct while retaining binding to full-length CD23, suggesting altered epitope accessibility and supporting differential epitope recognition between lumiliximab analog and our clones (FIG. 6).

[0401] Example 5: CD23 monoclonal antibody clones binding to CD23 receptor on JVM2 cells Experimental Aim: To assess binding of human anti-CD23 monoclonal antibody clones raised in rabbit to CD23 expressed on JVM2 cells by flow cytometry.

[0402] Methods: JVM2 cells were stained for viability using LIVE / DEAD Fixable Near-IR Dead Cell Stain. Cells were incubated with human anti-CD23 monoclonal antibody clones (7.6 pg per well) for 15 min at 4 °C. Following washes, cells were stained with rabbit anti-IgG Fc-FITC secondary antibody, washed again, and analyzed by flow cytometry. Data were gated on singlets and live cells, and fluorescence distribution shifts were evaluated relative to control conditions.

[0403] Results: Flow plots show a rightward shift in fluorescence relative to controls, indicating binding of the monoclonal antibody clones to CD23 on JVM2 cells. The observed increase in the CD23-positive population confirms engagement of cell-surface CD23 under the tested conditions and supports retention of CD23 target recognition by the rabbit-derived human anti-CD23 monoclonal antibodies. FIG. 7.

[0404] Example 6: CD23xCD3 IgG antibody binding to CD23 receptor on JVM2 cellsExperimental Aim: To evaluate binding of a CD23xCD3 bispecific antibody in IgG (2+1 format) to the CD23 receptor on JVM2 cells using flow cytometry’.

[0405] Methods: JVM2 cells were first stained with LIVE / DEAD Fixable Near-IR Dead Cell Stain to exclude nonviable cells. Cells were plated in a 96-well format and incubated with the CD23*CD3 IgG (2+2 format) antibody for 15 min at 37 °C and 4 °C. Following washing, bound antibody was detected using a human anti-IgG Fc-FITC secondary’ antibody. Cells were washed again and analyzed by flow cytometry, gating on singlets and live cells to assess fluorescence shifts relative to controls.

[0406] Results: Flow cytometry’ analysis showed a clear rightward fluorescence shift compared with baseline controls, indicating specific binding of the CD23*CD3 IgG (2+2 format) antibody to CD23 expressed on JVM2 cells. The observed signal confirms that the IgG-based bispecific construct retains functional CD23-binding capability and effectively recognizes native receptor on the cell surface (FIG. 8).

[0407] Example 7: CD23 bispecific Antibodies show potent in vitro killing (Bite format) Experimental Aim: To evaluate the cytotoxic activity of CD23xCD3 bispecific antibodies (BiTE format)(FIG. 9A) against endogenous CD23+B cells within human PBMC populations.

[0408] Methods: Human PBMCs were used as a mixed effector-target system (6:1 ratio), where the CD23+B-cell subset served as target cells and T cells acted as effectors. PBMCs were incubated with a panel of CD23xCD3 bispecific antibodies across a dose range of 0-10 ng / mL (serial dilutions). Following treatment, the frequency / viability of CD23 B cells was quantified by flow cytometry' and normalized to untreated controls. Data were visualized as a heatmap showing remaining viable CD23+cells across antibody conditions and concentrations.

[0409] Results: Bispecific antibody treatment resulted in dose-dependent depletion of CD23+B cells within PBMC cultures. Several constructs demonstrated potent cytotoxic activity at sub-ng / mL concentrations, indicating efficient T-cell redirection and target engagement, while others displayed lower efficacy. Minimal depletion occurred in untreated samples, confirming antibody-mediated killing. These results show robust functional activity of CD23xCD3 BiTE molecules in a physiologically relevant hPBMC system. FIG. 9B.

[0410] Example 8: CD23 bispecific Antibodies show potent in vitro CD23+ cells killing in PBMC (2+1 IgG format)Experimental Aim: To evaluate the cytotoxic activity and T-cell activation induced by CD23xCD3 bispecific antibodies in 2+1 IgG format (FIG. 10A) using human PBMCs. where endogenous CD23+B cells served as targets.

[0411] Methods: Human PBMCs were incubated with a panel of CD23xCD3 bispecific antibodies across a concentration range (nM serial dilutions). The CD23+B-cell subset within PBMCs acted as target cells, while T cells functioned as effectors. After incubation, flow cytometry was used to quantify remaining viable CD23+cells to assess target depletion (FIG.

[0412] 10B) and CD69 expression on T cells as a marker of activation (FIG. 10C). Data were normalized to untreated controls and visualized as heatmaps.

[0413] Results: Treatment with CD23xCD3 IgG bispecifics resulted in dose-dependent depletion of CD23 B cells across multiple constructs, demonstrating effective target engagement and cytotoxic activity in PBMC-only conditions. Concurrently, increased CD69 expression indicated robust T-cell activation, with activation levels generally correlating with target-cell depletion. Variability between constructs suggests differences in potency and effector engagement, supporting functional activity of the 2+1 IgG architecture (FIG. 10A) in mediating immune redirection and target elimination.

[0414] Example 9: CD23 bispecific Antibodies are less pro-inflammatory (2+2 IgG format) Experimental Aim: To assess the pro-inflammatory cytokine response induced by CD23*CD3 bispecific antibodies in 2+2 IgG format in human PBMCs and compare responses to a lumiliximab analog-based CD23*CD3 IgG (2+2) control antibody (AB0229).

[0415] Methods: Human PBMCs were incubated with a panel of CD23xCD3 bispecific antibodies across a concentration range (nM serial dilutions). Culture supernatants were collected and cytokine levels were quantified using the LEGENDplex™ Human Inflammation Panel 1 (13-plex, flow-based multiplex assay) following manufacturers protocol. Key cytokines analyzed included IL-13, IFNy, TNFa, and IL-6. Cytokine production induced by CD23 CD3 antibodies was compared against responses generated by the reference lumiliximab analog-based CD23xCD3 IgG (2+2) antibody (AB0229).

[0416] Results: Across the tested panel, CD23xCD3 bispecific antibodies antibodies unexpectedly reduced cytokine release relative to the lumiliximab analog-based CD23xCD3 comparator. Elevated cytokine release (IL-ip, IFNy, TNFa, and IL-6 - FIG. 11 A-l ID) was primarily associated with antibody AB0229, particularly at higher concentrations, whereas CD23 -targeting constructs produced minimal to moderate responses across doses. These findings indicate that CD23xCD3 IgG bispecifics maintain functional activity whileexhibiting a reduced inflammatory cytokine signature, suggesting a potentially improved safety’ profile with respect to systemic immune activation.

[0417] Example 10: CD23 bispecific Antibodies show potent in vitro CLL tumor cell killing (2+1 IgG format)

[0418] Experimental Aim: To evaluate cytotoxic activity and T-cell activation mediated by CD23*CD3 bispecific antibodies in 2+2 IgG format using human PBMCs co-cultured with JVM cells as CD23+target cells.

[0419] Methods: Human PBMCs were co-incubated with CD23-expressing JVM cells (6:1 ratio) and treated with a panel of CD23xCD3 bispecific antibodies across a concentration range (nM serial dilutions). In this system, JVM cells served as targets and PBMC-derived T cells functioned as effectors. Following incubation, flow cytometry was used to quantify remaining viable CD23+target cells and CD69 expression on CD3+T cells as a measure of activation. Data were normalized to untreated controls and visualized as heatmaps.

[0420] Results: Bispecific antibody treatment resulted in dose-dependent depletion of CD23+JVM target cells across multiple constructs, demonstrating effective T-cell-mediated cytotoxic activity'. Concurrent increases in CD69 expression confirmed robust T-cell activation, generally correlating with target-cell killing. Differences in response magnitude between antibodies indicate variability in potency and effector engagement. Overall, the data show that CD23xCD3 IgG bispecifics effectively redirect PBMC-derived T cells to eliminate CD23 tumor targets. FIG. 12A and 12B.

[0421] Example 11: CD23 Antibodies Retain killing potency in the Presence of Soluble CD23 Experimental Aim: To determine whether soluble CD23 (sCD23) neutralizes or inhibits the activity of CD23 antibody clones by assessing their ability to mediate target-cell killing in its presence, compared with AB0229 (lumiliximab analog) as a reference control antibody.

[0422] Methods: CD23xCD3 bispecific constructs incorporating different CD23 -binding clones were evaluated in cytotoxicity assays using JVM2 (FIG. 13 A) and HG3 CD23+(FIG.

[0423] 13B) target cell lines. Killing activity was measured under two conditions: in the absence and presence of exogenous soluble CD23. Percent CD23+cell depletion was quantified and plotted to compare functional activity with and without sCD23 exposure. AB0229 (lumiliximab analog-derived CD23xCD3) served as the benchmark comparator for sensitivity to soluble antigen interference.Results: Most of our CD23 antibody clones retained cytotoxic activity' in the presence of soluble CD23. indicating resistance to neutralization and preservation of target engagement. In contrast, the lumiliximab analog control (AB0229) showed marked loss of killing activity when soluble CD23 was present, demonstrating functional neutralization by soluble antigen. This differential behavior supports that our CD23 binders recognize epitopes less susceptible to soluble CD23 interference, consistent with targeting a distinct epitope region.

[0424] Example 12: Dose-dependent T-cell activation and target cell depletion by CD23xCD3 bispecific antibodies (2+1 IgG format) in Cynomolgus PBMCs

[0425] Experimental Aim: To evaluate binding-driven functional activity of CD23*CD3 bispecific antibodies (2+1 IgG format) in cynomolgus PBMCs by assessing T-cell activation and depletion of CD23+target cells.

[0426] Methods: Cynomolgus monkey PBMCs were treated with a panel of CD23*CD3 bispecific antibodies across a concentration range (0-10 nM). Following incubation, flow cytometry was performed to measure CD69 expression on T cells as an activation marker and to quantify depletion of CD23+target cells within the PBMC population. Data were normalized to untreated controls and summarized as heatmaps.

[0427] Results: Antibody treatment induced concentration-dependent T-cell activation and measurable depletion of CD23+cells across multiple clones, demonstrating functional engagement in cynomolgus immune cells. Activity profiles varied between antibodies, with several constructs showing stronger activation and target depletion at higher concentrations. These findings indicate cross-reactive binding and effector function in cynomolgus PBMCs, supporting translational relevance for nonclinical evaluation. FIG. 14A and 14B.

[0428] References

[0429] 1. Ludin, C., H. Hofstetter, M. Sarfati, et al. " Cloning and Expression of the cDNA Coding for a Human Lymphocyte IgE Receptor." EMBO Journal 6 (1987): 109-114.

[0430] 2. Suter. U., R. Bastos, H. Hofstetter. " Molecular Structure of the Gene and tire 5'-Flanking Region of the Human Lymphocyte Immunoglobulin E Receptor." Nucleic Acids Research 15 (1987): 7295-7308.

[0431] 3. Conrad. D. H., J. W. Ford, J. L. Sturgill, et al. " CD23: An Overlooked Regulator of Allergic Disease." Current Allergy' and Asthma Reports 7 (2007): 331-337.4. Bonnefoy, J. Y., S. Lecoanet-Henchoz, J. F. Gauchat, et al. " Structure and Functions of CD23." International Review of Immunology 16 (1997): 113-128.

[0432] 5. Gould, H. J., and B. J. Sutton. " IgE in Allergy and Asthma Today." Nature Reviews Immunology 8 (2008): 205-217.

[0433] 6. Sarfati, M., S. Fournier, C. Y. Wu, et al. " Expression, Regulation, and Function of Human Fc Epsilon RII (CD23) Antigen." Immunological Research 11 (1992): 260-272.

[0434] 7. Armitage, R. J., L. K. Goff, P. C. Beverley. " Expression and Functional Role of CD23 on T Cells." European Journal of Immunology 19 (1989): 31-35.

[0435] 8. Yamaoka, K. A., M. Arock. F. Issaly, et al. " Granulocyte Macrophage Colony Stimulating Factor Induces Fc Epsilon RII / CD23 Expression on Normal Human Polymorphonuclear Neutrophils." International Immunology 8 (1996): 479-490.

[0436] 9. Vercelli, D., H. H. Jabara. B. W. Lee, et al. " Human Recombinant Interleukin 4 Induces Fc Epsilon R2 / CD23 on Normal Human Monocytes." Journal of Experimental Medicine 167 (1988): 1406-1416.

[0437] 10. Rieber, E. P., G. Rank, I. Kohler, et al. " Membrane Expression of Fc Epsilon RII / CD23 and Release of Soluble CD23 by Follicular Dendritic Cells." Advances in Experimental Medicine and Biology 329 (1993): 393-398.

[0438] All publications cited in this specification are incorporated herein by reference. US Provisional Patent Application No. 63 / 759,760 filed February 18, 2025 is incorporated herein by reference in its entirety. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.Table 3

[0439] between Linker CD23 VhVL

[0440] Antbociy Ab CD23 CD23 linker sequence 2 (light chain) Ma me clone no. Format CD3 VhVL sequence sequence I DIQMTQSPSSLSASVGDRVTITCRASQDIRYYLNWYQQKP GKAPKLLIYVASSLQSGVPSRFSGSGSGTEFTLTVSSLQPE DFATYYCLQVYSTPRTFGQGTKVEIKGSHQSSPNTASGPN SQHAPSGSSEVQLVESGGGLAKPGGSLRLSCAASGFRFT FNNYYMDWVRQAPGQGLEWVSRISSSGDPTWYADSVKG RFTISRENANNTLFLQMNSLRAEDTAVYYCASLTTGSDSW GQGVLVTVSSGGGGSEVQLVESGGGLVQPKGSLKLSCAA SGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGN FGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYA GSHQSSPN NWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAAL

[0441] Lumi TASGPNSQ TITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHH

[0442]

[0443] AB0225 analogue BiTE G4S X1 Non rep HAPSGSS HHH*EVQLVESGGGLAKPGGSLRLSCAASGFRFTFNNYYMDWV RQAPGQGLEWVSRISSSGDPTWYADSVKGRFTISRENAN NTLFLQM NSLRAEDTAVYYCAS LTTGS DSWGQGVLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGP MGWSCIILFLVATATGVHSGDIQMTQSPS SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV SLSASVGDRVTITCRASQDIRYYLNWYQQ DGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE KPGKAPKLLIYVASSLQSGVPSRFSGSGS YKCKVSNKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTK GTEFTLTVSSLQPEDFATYYCLQVYSTPRT NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS FGQGTKVEIKRTVAAPSVFIFPPSDEQLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ GTASWCLLNNFYPREAKVQWKVDNALQ KSLSLSPGKGGGGSGGGGSEVQLVESGGGLVQPKGSLK SGNSQESVTEQDSKDSTYSLSSTLTLSKAD LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY YEKHKVYACEVTHQGLSSPVTKSFNRGEC ATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCV ** RHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSG GGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTT SNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGD AB_0229 + Lumi KAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGS

[0444] AB_0230 analogue 2+1 IgG G4SX4 **

[0445] EVQLVESGGGLAKPGGSLRLSCAASGFRFTFNNYYMDWV RQAPGQGLEWVSRISSSGDPTWYADSVKGRFTISRENAN MGWSCIILFLVATATGVHSGDIQMTQSPS NTLFLQM NSLRAEDTAVYYCAS LTTGS DSWGQGVLVTVSS SLSASVGDRVTITCRASQDIRYYLNWYQQ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW KPGKAPKLLIYVASSLQSGVPSRFSGSGS NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI GTEFTLTVSSLQPEDFATYYCLQVYSTPRT CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGP FGQGTKVEIKRTVAAPSVFIFPPSDEQLKS SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV GTASWCLLNNFYPREAKVQWKVDNALQ DGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE SGNSQESVTEQDSKDSTYSLSSTLTLSKAD YKCKVSNKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTK YEKHKVYACEVTHQGLSSPVTKSFNRGEC NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS ** AB_0230 + Lumi DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[0446]

[0447] AB_0231 analogue igG NA KSLSLSPGKS**AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYAASTLASGVSSRFKGSGSGTQFTLTISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCT VSGMDLSRYAMSWVRQAPGKGLEWIGVIYASGNAYYAS WAKGRFTISKTSTTVYLKIASPTTEDTATYFCGRYVADYGAV GRVWGPGTLVTVSSGGGGSEVQLVESGGGLVQPKGSLKL SCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYA TYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGG GGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTS GSHQSSPN NYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDK TASGPNSQ AALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGS AB0236 4G6 BiTE G4S X1 Non rep HAPSGSS HHHHHH*

[0448] AQVLTQTPSPVSVAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNAYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN LWGPGTLVTVSSGGGGSEVQLVESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRH GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSN GSHQSSPN YANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKA TASGPNSQ ALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSH

[0449]

[0450] AB0238 5A4 BiTE G4S X1 Non rep HAPSGSS HHHHH*DPVLTQTPSSASEPVGGTVTIKCQASESISSRLAWYQQKPG QPPKLLIYSASTLESGVPSRFKGSGSGTEFTLTISDLECADA ATYYCQNNYVTSYGFGGGTEVWKGSHQSSPNTASGPNS QHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDLSSYA MGWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTST TVDLKITSPTTEDTATYFCARAWDLWGPGTLVTVSSGGGG SEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQ SILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAWTQES GSHQSSPN ALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL TASGPNSQ IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA AB0240 7H6 BiTE G4S X1 Non rep HAPSGSS LWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0451] DWMTQTPASVEAAVGGTVTIKCQASQSISNLLAWYQQKP GQPPKLLIYSASTLASGVPSRFKGSESGTEFTLTISDLECAD AATYYCQSYYGSSSSSYGDAFGGGTEVWKGSHQSSPNT ASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGF SLSSNAVSWVRQAPGKGLEHIGFIGDTGNTYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCVRAGITNLWGPGTLVT VSSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNT YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ GSHQSSPN AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP TASGPNSQ DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE

[0452]

[0453] AB0244 15A1 BiTE G4S X1 Non rep HAPSGSS DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*AQVLTQTPSPVSWVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNVYYASW AKGRFTISKASTTVDLKMTSLTTEDTATYFCGRAVADYSTLN LWGPGTLVTVSSGGGGSEVQLVESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRH GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSN GSHQSSPN YANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKA TASGPNSQ ALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSH AB0247 17E1 BiTE G4S X1 Non rep HAPSGSS HHHHH*

[0454] AAVLTQTPSPVSAAVGGTVSISCQASQSVYEDNWLAWYQ QKPGQRPKLLIYLASNLASGVPSRFKGSGSGTQFTLTINGV QCDDAATYYCQGVDSSSGETTFGGGTEVWKGSHQSSP NTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVS GIDLSRYNMAWVRQAPGKGLEYIGIIYSSAATYYASWAKGR FTISKTSTTVDLKMTSLTTEDTATYFCARRDVGSSGYTGAFD PWGPGTLVTVSSGGGGSEVQLVESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRH GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSN GSHQSSPN YANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKA TASGPNSQ ALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSH

[0455]

[0456] AB0248 9E12 BiTE G4S X1 Non rep HAPSGSS HHHHH*AQVLTQTPSPVSVAVGGAVTINCQASQSVYNYKNLAWFQ QKPGQPPKQLIYTASSLASGVSSRFKGSGSGTQFTLAISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTLTCT VSGIDLSRFAMSWVRQAPGKGLEWIGVIYGSGNSYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN LWGPGTLVTVSSGGGGSEVQLVESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRH GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSN GSHQSSPN YANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKA TASGPNSQ ALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSH AB0251 20G4 BiTE G4S X1 Non rep HAPSGSS HHHHH*

[0457] AFELTQTPSSVEAAVGGTVTIKCQASQSINSWLAWYQQKP GQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLECAD AATYYCQNYYDTITNTFGGGTKVWEGSHQSSPNTASGPN SQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDLSTYPI TWVRQAPGKGLEYVGYIHSGGSAYYAGWAKGRFTISKTST TVDLKITSPTTEDTATYFCARGSSWGNLWGPGTLVTVSSG GGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD DSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAWT GSHQSSPN QESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHL TASGPNSQ FTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAI

[0458]

[0459] AB0243 13D4 BiTE G4S X1 Non rep HAPSGSS YFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*QSVEESGGRLVTPGTPLTLTCTVSGMDLSRYAMSWVRQA PGKGLEWIGVIYASGNAYYASWAKGRFTISKTSTTVYLKIAS PTTEDTATYFCGRYVADYGAVGRVWGPGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTKNQV

[0460] 4G6 SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS MGWSCIILFLVATATGVHSGAQVLTQTPS LSPGKGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCA PVSAAVGGTVTINCQASQSVYNNKNLAW ASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYY FQQKPGQPPKQLIYAASTLASGVSSRFKG ADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHG SGSGTQFTLTISDVQCDDAATYYCLGEFS NFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGG CSSADCNAFGGGTEVWKRTVAAPSVFIF SGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYA PPSDEQLKSGTASWCLLNNFYPREAKVQ NWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAAL WKVDNALQSGNSQESVTEQDSKDSTYSL AB_259+26 TITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHH SSTLTLSKADYEKHKVYACEVTHQGLSSPV

[0461]

[0462] 0 2+1 IgG N / A NA HHH* TKSFNRGEC*QSLEESGGRLVKPDETLTITCTVSGIDLDRFAMSWVRQAP GKGLEWIGVIYGSGNAYYASWAKGRFTISKTSTTVDLKMTS LTTEDTATYFCGRAVADYSTLNLWGPGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTKNQVSL

[0463] 5A4 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS MGWSCIILFLVATATGVHSGAQVLTQTPS PGKGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAAS PVSVAVGGTVTINCQASQSVYNNKNLAW GFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD FQQKPGQPPKQLIYVASKLASGVPSRFSG SVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNF SGSGTQFTLTISGVQCDDAATYYCLGEFS GNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSG CSSADCNAFGGGTEVWKRTVAAPSVFIF GGGSQAWTQESALTTSPGETVFLTCRSSTGAVTTSNYAN PPSDEQLKSGTASWCLLNNFYPREAKVQ WVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTI WKVDNALQSGNSQESVTEQDSKDSTYSL AB_261+26 TGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHH SSTLTLSKADYEKHKVYACEVTHQGLSSPV

[0464]

[0465] 2 2+1 IgG N / A NA HH* TKSFNRGEC*QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAP GKGLEYIGIISSSGSTYYASWAKGRFTISKTSTTVDLKITSPTT EDTATYFCARAWDLWGPGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREGQVYTLPPSRDELTKNQVSLTCLVKGFY

[0466] 7H6

[0467] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGG MGWSCIILFLVATATGVHSGDPVLTQTPSS GSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTY ASEPVGGTVTIKCQASESISSRLAWYQQK AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT PGQPPKLLIYSASTLESGVPSRFKGSGSGT ISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS EFTLTISDLECADAATYYCQNNYVTSYGFG WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ GGTEWVKRTVAAPSVFIFPPSDEQLKSGT AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP ASWCLLNNFYPREAKVQWKVDNALQSG DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE AB_263+26 NSQESVTEQDSKDSTYSLSSTLTLSKADYE

[0468] DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0469]

[0470] 4 2+1 IgG N / A NA KHKVYACEVTHQGLSSPVTKSFNRGEC*QSVEESGGRLVTPGTPLTLTCTVSGIDLSTYPITVWRQAPG KGLEYVGYIHSGGSAYYAGWAKGRFTISKTSTTVDLKITSPT TEDTATYFCARGSSWGNLWGPGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREGQVYTLPPSRDELTKNQVSLTCLVK

[0471] 13D4

[0472] GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG MGWSCIILFLVATATGVHSGAFELTQTPSS GGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTF VEAAVGGTVTI KCQASQSI NSWLAWYQQ NTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD KPGQPPKLLIYKASTLASGVSSRFKGSGSG RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSY TEFTLTISDLECADAATYYCQNYYDTITNTF VSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG GGGTKVWERTVAAPSVFIFPPSDEQLKSG SQAWTQESALTTSPGETVFLTCRSSTGAVTTSNYANWVQE TASWCLLNNFYPREAKVQWKVDNALQS KPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQ AB_265+26 GNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0473] TEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0474]

[0475] 6 2+1 IgG N / A NA EKHKVYACEVTHQGLSSPVTKSFNRGEC*QSVEESGGRLVTPGTPLTLTCTVSGFSLSSNAVSWVRQAP GKGLEHIGFIGDTGNTYYASWAKGRFTISKTSTTVDLKITSP TTEDTATYFCVRAGITNLWGPGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREGQVYTLPPSRDELTKNQVSLTCLVK

[0476] 15A1

[0477] GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL MGWSCIILFLVATATGVHSGDWMTQTPA TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG SVEAAVGGTVTIKCQASQSISNLLAWYQQ GGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTF KPGQPPKLLIYSASTLASGVPSRFKGSESG NTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD TEFTLTISDLECADAATYYCQSYYGSSSSS RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSY YGDAFGGGTEVWKRTVAAPSVFIFPPSDE VSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG QLKSGTASWCLLNNFYPREAKVQWKVD SQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQE NALQSGNSQESVTEQDSKDSTYSLSSTLT KPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQ AB_267+26 LSKADYEKHKVYACEVTHQGLSSPVTKSF TEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0478] 8 2+1 IgG N / A NA NRGEC*

[0479] MGWSCIILFLVATATGVHSGAQVLTQTPS PVSVWGGTVTINCQASQSVYNNKNLAW FQQKPGQPPKQLIYVASKLASGVPSRFSG GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAWTQE SGSGTQFTLTISGVQCDDAATYYCLGEFS SALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTG CSSADCNAFGGGTEWVKRTVAAPSVFIF LGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC PPSDEQLKSGTASWCLLNNFYPREAKVQ ALWYSN LWVFGGGTKLTVLGGS HHHHH H *

[0480] WKVDNALQSGNSQESVTEQDSKDSTYSL AB_269+27 SSTLTLSKADYEKHKVYACEVTHQGLSSPV

[0481]

[0482] 0 17E1 2+1 IgG N / A NA TKSFNRGEC*QSVEESGGRLVTPGTPLTLTCTVSGIDLSRYNMAWVRQAP GKGLEYIGIIYSSAATYYASWAKGRFTISKTSTTVDLKMTSLT TEDTATYFCARRDVGSSGYTGAFDPWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTKN

[0483] 19E12 QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS MGWSCIILFLVATATGVHSGAAVLTQTPSP LSLSPGKGGGGSGGGGSEVQLVESGGGLVQPKGSLKLS VSAAVGGTVSISCQASQSVYEDNWLAWY CAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT QQKPGQRPKLLIYLASNLASGVPSRFKGS YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR GSGTQFTLTI NGVQCDDAATYYCQGVDS HGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGG SSGETTFGGGTEVWKRTVAAPSVFIFPPS GGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTS DEQLKSGTASWCLLNNFYPREAKVQWK NYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDK VDNALQSGNSQESVTEQDSKDSTYSLSST AB_271+27 AALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGS LTLSKADYEKHKVYACEVTHQGLSSPVTKS

[0484]

[0485] 2 2+1 IgG N / A NA HHHHHH* FNRGEC*QSLEESGGRLVKPDETLTLTCTVSGIDLSRFAMSWVRQAP GKGLEWIGVIYGSGNSYYASWAKGRFTISKTSTTVDLKMTS LTTEDTATYFCGRAVADYSTLNLWGPGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREGQVYTLPPSRDELTKNQVSL

[0486] 20G4 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS MGWSCIILFLVATATGVHSGSQVLTQTPS PGKGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAAS PVSVAVGGAVTINCQASQSVYNYKNLAW GFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD FQQKPGQPPKQLIYTASSLASGVSSRFKG SVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNF SGSGTQFTLAISDVQCDDAATYYCLGEFS GNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSG CSSADCNAFGGGTEVWKRTVAAPSVFIF GGGSQAWTQESALTTSPGETVFLTCRSSTGAVTTSNYAN PPSDEQLKSGTASWCLLNNFYPREAKVQ WVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTI WKVDNALQSGNSQESVTEQDSKDSTYSL AB_273+27 TGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHH SSTLTLSKADYEKHKVYACEVTHQGLSSPV 4 2+1 IgG N / A NA HH* TKSFNRGEC*

[0487] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYAASTLASGVSSRFKGSGSGTQFTLTISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCT VSGMDLSRYAMSWVRQAPGKGLEWIGVIYASGNAYYAS WAKGRFTISKTSTTVYLKIASPTTEDTATYFCGRYVADYGAV GSHQSSPNT GRVWGPGTLVTVSSGGGGSGGGGSGGGGSEVQLVESG AB0237 4G6 G4SX3 ASGPNSQHA

[0488] GGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLE PSGSS WVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMN NLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSQAWTQESALTTSPGET VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRA PGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLW

[0489]

[0490] BiTE Non rep VFGGGTKLTVLGGSHHHHHH*AQVLTQTPSPVSVAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNAYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGG AB0239 5A4 G4SX3 ASGPNSQHA LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA PSGSS RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAWTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGG

[0491] BiTE Non rep TKLTVLGGSHHHHHH*

[0492] DPVLTQTPSSASEPVGGTVTIKCQASESISSRLAWYQQKPG QPPKLLIYSASTLESGVPSRFKGSGSGTEFTLTISDLECADA ATYYCQNNYVTSYGFGGGTEWVKGSHQSSPNTASGPNS QHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDLSSYA MGWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTST TVDLKITSPTTEDTATYFCARAWDLWGPGTLVTVSSGGGG GSHQSSPNT SGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGF AB0241 7H6 G4SX3 ASGPNSQHA TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSV PSGSS KDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFG NSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHH

[0493]

[0494] BiTE Non rep HH*ARCAFELTQTPSSVEAAVGGTVTIKCQASQSINSWLAWYQ QKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLE CADAATYYCQNYYDTITNTFGGGTKVWEGSHQSSPNTAS GPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDL STYPITWVRQAPGKGLEYVGYIHSGGSAYYAGWAKGRFTI SKTSTTVDLKITSPTTEDTATYFCARGSSWGNLWGPGTLVT GSHQSSPNT VSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKL AB0242 13D4 G4SX3 ASGPNSQHA SCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYA PSGSS TYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGG GGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDK AALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGS

[0495] BiTE Non rep HHHHHH*

[0496] DWMTQTPASVEAAVGGTVTIKCQASQSISNLLAWYQQKP GQPPKLLIYSASTLASGVPSRFKGSESGTEFTLTISDLECAD AATYYCQSYYGSSSSSYGDAFGGGTEVWKGSHQSSPNT ASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGF SLSSNAVSWVRQAPGKGLEHIGFIGDTGNTYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCVRAGITNLWGPGTLVT GSHQSSPNT VSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKL AB0245 15A1 G4SX3 ASGPNSQHA SCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYA PSGSS TYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGG GGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTS NYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDK AALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGGS

[0497]

[0498] BiTE Non rep HHHHHH*AQVLTQTPSPVSVWGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNVYYASW AKGRFTISKASTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGG AB0246 G4SX3 ASGPNSQHA LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA PSGSS RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAWTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGG

[0499] 17E1 BiTE Non rep TKLTVLGGSHHHHHH*

[0500] AAVLTQTPSPVSAAVGGTVSISCQASQSVYEDNWLAWYQ QKPGQRPKLLIYLASNLASGVPSRFKGSGSGTQFTLTINGV QCDDAATYYCQGVDSSSGETTFGGGTEVWKGSHQSSP NTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVS GIDLSRYNMAWVRQAPGKGLEYIGIIYSSAATYYASWAKGR FTISKTSTTVDLKMTSLTTEDTATYFCARRDVGSSGYTGAFD GSHQSSPNT PWGPGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGG AB0249 19E12 G4SX3 ASGPNSQHA LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA PSGSS RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAWTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGG

[0501]

[0502] BiTE Non rep TKLTVLGGSHHHHHH*AQVLTQTPSPVSVAVGGAVTINCQASQSVYNYKNLAWFQ QKPGQPPKQLIYTASSLASGVSSRFKGSGSGTQFTLAISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTLTCT VSGIDLSRFAMSWVRQAPGKGLEWIGVIYGSGNSYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGG AB0250 20G4 G4SX3 ASGPNSQHA LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA PSGSS RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAWTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGG

[0503] BiTE Non rep TKLTVLGGSHHHHHH*

[0504] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYAASTLASGVSSRFKGSGSGTQFTLTISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCT VSGMDLSRYAMSWVRQAPGKGLEWIGVIYASGNAYYAS WAKGRFTISKTSTTVYLKIASPTTEDTATYFCGRYVADYGAV GSHQSSPNT GRVWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGG AB0280 4G6 G4SX6 ASGPNSQHA GGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNT PSGSS YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0505]

[0506] BiTE Non repAQVLTQTPSPVSVAVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNAYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG AB0281 5A4 G4SX6 ASGPNSQHA SGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYA PSGSS MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH* BiTE Non rep

[0507] DPVLTQTPSSASEPVGGTVTIKCQASESISSRLAWYQQKPG QPPKLLIYSASTLESGVPSRFKGSGSGTEFTLTISDLECADA ATYYCQNNYVTSYGFGGGTEVWKGSHQSSPNTASGPNS QHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDLSSYA MGWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTST TVDLKITSPTTEDTATYFCARAWDLWGPGTLVTVSSGGGG GSHQSSPNT SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGG AB0282 7H6 G4SX6 ASGPNSQHA LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA PSGSS RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAWTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGG

[0508]

[0509] BiTE Non rep TKLTVLGGSHHHHHH*ARCAFELTQTPSSVEAAVGGTVTIKCQASQSINSWLAWYQ QKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLE CADAATYYCQNYYDTITNTFGGGTKVWEGSHQSSPNTAS GPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGIDL STYPITWVRQAPGKGLEYVGYIHSGGSAYYAGWAKGRFTI SKTSTTVDLKITSPTTEDTATYFCARGSSWGNLWGPGTLVT GSHQSSPNT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV AB0283 13D4 G4SX6 ASGPNSQHA QLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAP PSGSS GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILY LQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSQAWTQESALTTS PGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGT NKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWY

[0510] BiTE Non rep SNLWVFGGGTKLTVLGGSHHHHHH*

[0511] DWMTQTPASVEAAVGGTVTIKCQASQSISNLLAWYQQKP GQPPKLLIYSASTLASGVPSRFKGSESGTEFTLTISDLECAD AATYYCQSYYGSSSSSYGDAFGGGTEVWKGSHQSSPNT ASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGF SLSSNAVSWVRQAPGKGLEHIGFIGDTGNTYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCVRAGITNLWGPGTLVT GSHQSSPNT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV AB0284 15A1 G4SX6 ASGPNSQHA QLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAP PSGSS GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILY LQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSQAWTQESALTTS PGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGT NKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWY

[0512]

[0513] BiTE Non rep SNLWVFGGGTKLTVLGGSHHHHHH*AQVLTQTPSPVSWVGGTVTINCQASQSVYNNKNLAWFQ QKPGQPPKQLIYVASKLASGVPSRFSGSGSGTQFTLTISGV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTITCT VSGIDLDRFAMSWVRQAPGKGLEWIGVIYGSGNVYYASW AKGRFTISKASTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG AB0285 G4SX6 ASGPNSQHA SGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYA PSGSS MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH* 17E1 BiTE Non rep

[0514] AAVLTQTPSPVSAAVGGTVSISCQASQSVYEDNWLAWYQ QKPGQRPKLLIYLASNLASGVPSRFKGSGSGTQFTLTINGV QCDDAATYYCQGVDSSSGETTFGGGTEVWKGSHQSSP NTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVS GIDLSRYNMAWVRQAPGKGLEYIGIIYSSAATYYASWAKGR FTISKTSTTVDLKMTSLTTEDTATYFCARRDVGSSGYTGAFD GSHQSSPNT PWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG AB0286 19E12 G4SX6 ASGPNSQHA SGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYA PSGSS MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0515]

[0516] BiTE Non repAQVLTQTPSPVSVAVGGAVTINCQASQSVYNYKNLAWFQ QKPGQPPKQLIYTASSLASGVSSRFKGSGSGTQFTLAISDV QCDDAATYYCLGEFSCSSADCNAFGGGTEVWKGSHQS SPNTASGPNSQHAPSGSSQSLEESGGRLVKPDETLTLTCT VSGIDLSRFAMSWVRQAPGKGLEWIGVIYGSGNSYYASW AKGRFTISKTSTTVDLKMTSLTTEDTATYFCGRAVADYSTLN GSHQSSPNT LWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG AB0287 20G4 G4SX6 ASGPNSQHA SGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYA PSGSS MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQ AWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKP DHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTE DEAIYFCALWYSNLWVFGGGTKLTVLGGSHHHHHH* BiTE Non rep

[0517] DWMTQTPASVEAAVGGTVTIKCQASQSISNLLAWYQQKP GQPPKLLIYSASTLASGVPSRFKGSESGTEFTLTISDLECAD AATYYCQSYYGSSSSSYGDAFGGGTEVWKGSHQSSPNT ASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGF SLSSNAVSWVRQAPGKGLEHIGFIGDTGNTYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCVRAGITNLWGPGTLVT GSHQSSPNT VSSGGGAGGGKGGGAGGGEGGGAGGGKGEEVQLVES AB0288 15A1 ASGPNSQHA GGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGL PSGSS EWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMN GGGAGG NLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVS GKGGGA SGGGGSGGGGSGGGGSGGGGSQAWTQESALTTSPGET GGGEGG VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRA GAGGGK PGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLW

[0518]

[0519] BiTE GE Non rep VFGGGTKLTVLGGSHHHHHH*AAVLTQTPSPVSAAVGGTVSISCQASQSVYEDNWLAWYQ QKPGQRPKLLIYLASNLASGVPSRFKGSGSGTQFTLTINGV QCDDAATYYCQGVDSSSGETTFGGGTEVWKGSHQSSP NTASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVS GIDLSRYNMAWVRQAPGKGLEYIGIIYSSAATYYASWAKGR GGGAGG FTISKTSTTVDLKMTSLTTEDTATYFCARRDVGSSGYTGAFD GKGGGA GSHQSSPNT PWGPGTLVTVSSGGGAGGGKGGGAGGGEGGGAGGGK AB0289 19E12 GGGEGG ASGPNSQHA GEEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWV GAGGGK PSGSS RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS GE QSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAWTQE SALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTG LIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC

[0520] BiTE Non rep ALWYSNLWVFGGGTKLTVLGGSHHHHHH*

[0521] DWMTQTPASVEAAVGGTVTIKCQASQSISNLLAWYQQKP GQPPKLLIYSASTLASGVPSRFKGSESGTEFTLTISDLECAD AATYYCQSYYGSSSSSYGDAFGGGTEVWKGSHQSSPNT ASGPNSQHAPSGSSQSVEESGGRLVTPGTPLTLTCTVSGF SLSSNAVSWVRQAPGKGLEHIGFIGDTGNTYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCVRAGITNLWGPGTLVT GSHQSSPNT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQA AB0290 15A1 G4SX6 ASGPNSQHA VVTQESALTTSPGETVTLTCRSSTGAVTTSNYANVWQEKPD PSGSS HLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNLWVFGGGTKLTVLGGGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTF NTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSY

[0522] BiTE Non rep VSWFAYWGQGTLVTVSSSHHHHHH*

[0523] ALWYSNLWVFGGGTKLTVLGGGGGGSGGGGSGGGGSG GSHQSSPNT GGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMN AB0291 19E12 G4SX6 ASGPNSQHA WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD PSGSS DSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY

[0524]

[0525] BiTE Non rep WGQGTLVTVSSSHHHHHH*MDTRAPTQLLGLLLLWLPGATFAQVL TQTPSPVSVAVGGTVTINCQASQSV

[0526] Monocl METGLRWLLLVAVLKGVQCQSLEESGGRLVKPDET YNNKNLAWFQQKPGQPPKQLIYVAS onal LTITCTVSGIDLDRFAMSWVRQAPGKGLEWIGVIYG KLASGVPSRFSGSGSGTQFTLTISGV Anti bod SGNAYYASWAKGRFTISKTSTTVDLKMTSLTTEDTA QCDDAATYYCLGEFSCSSADCNAFG AB0292 5A4 y NA NA NA TYFCGRAVADYSTLNLWGPGTLVTVSS GGTEVVVK MDTRAPTQLLGLLLLWLPGAICDPVL TQTPSSASEPVGGTVTIKCQASESIS

[0527] Monocl METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGT SRLAWYQQKPGQPPKLLIYSASTLES onal PLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIISS GVPSRFKGSGSGTEFTLTISDLECAD Anti bod SGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTAT AATYYCQNNYVTSYGFGGGTEVVVK AB0293 7H6 y NA NA NA YFCARAWDLWGPGTLVTVSS MDTRAPTQLLGLLLLWLPGARCAFEL TQTPSSVEAAVGGTVTIKCQASQSIN

[0528] Monocl METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGT SWLAWYQQKPGQPPKLLIYKASTLA onal PLTLTCTVSGIDLSTYPITWVRQAPGKGLEYVGYIHS SGVSSRFKGSGSGTEFTLTISDLECA Anti bod GGSAYYAGWAKGRFTISKTSTTVDLKITSPTTEDTA DAATYYCQNYYDTITNTFGGGTKVVV AB0294 13D4 y NA NA NA TYFCARGSSWGNLWGPGTLVTVSS E MDTRAPTQLLGLLLLWLPGARCADV VMTQTPASVEAAVGGTVTIKCQASQ

[0529] Monocl METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGT SISNLLAWYQQKPGQPPKLLIYSAST onal PLTLTCTVSGFSLSSNAVSWVRQAPGKGLEHIGFIG LASGVPSRFKGSESGTEFTLTISDLE Anti bod DTGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDT CADAATYYCQSYYGSSSSSYGDAFG AB0295 15A1 y NA NA NA ATYFCVRAGITNLWGPGTLVTVSS GGTEVVVK MDTRAPTQLLGLLLLWLPGATFAQVL TQTPSPVSVVVGGTVTINCQASQSV

[0530] Monocl METGLRWLLLVAVLKGVQCQSLEESGGRLVKPDET YNNKNLAWFQQKPGQPPKQLIYVAS onal LTITCTVSGIDLDRFAMSWVRQAPGKGLEWIGVIYG KLASGVPSRFSGSGSGTQFTLTISGV Anti bod SGNVYYASWAKGRFTISKASTTVDLKMTSLTTEDTA QCDDAATYYCLGEFSCSSADCNAFG AB0296 17E1 y NA NA NA TYFCGRAVADYSTLNLWGPGTLVTVSS GGTEVVVK MDTRAPTQLLGLLLLWLPGATFAQVL TQTPSPVSVAVGGAVTINCQASQSV

[0531] Monocl METGLRWLLLVAVLKGVQCQSLEESGGRLVKPDET YNYKNLAWFQQKPGQPPKQLIYTAS onal LTLTCTVSGIDLSRFAMSWVRQAPGKGLEWIGVIYG SLASGVSSRFKGSGSGTQFTLAISDV Anti bod SGNSYYASWAKGRFTISKTSTTVDLKMTSLTTEDTA QCDDAATYYCLGEFSCSSADCNAFG

[0532]

[0533] AB0297 20G4 y NA NA NA TYFCGRAVADYSTLNLWGPGTLVTVSS GGTEVVVKMDTRAPTQLLGLLLLWLPGATFAQVL TQTPSPVSAAVGGTVTINCQASQSV

[0534] Monocl METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGT YNNKNLAWFQQKPGQPPKQLIYAAS onal PLTLTCTVSGMDLSRYAMSWVRQAPGKGLEWIGVI TLASGVSSRFKGSGSGTQFTLTISDV Anti bod YASGNAYYASWAKGRFTISKTSTTVYLKIASPTTED QCDDAATYYCLGEFSCSSADCNAFG AB0298 4G6 y NA NA NA TATYFCGRYVADYGAVGRVWGPGTLVTVSS GGTEVVVK MDTRAPTQLLGLLLLWLPGATFAAVL TQTPSPVSAAVGGTVSISCQASQSV

[0535] Monocl METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGT YEDNWLAWYQQKPGQRPKLLIYLAS onal PLTLTCTVSGIDLSRYNMAWVRQAPGKGLEYIGIIYS NLASGVPSRFKGSGSGTQFTLTINGV Anti bod SAATYYASWAKGRFTISKTSTTVDLKMTSLTTEDTA QCDDAATYYCQGVDSSSGETTFGG AB0299 19E12 y NA NA NA TYFCARRDVGSSGYTGAFDPWGPGTLVTVSS GTEVVVK Humani SGIDLSTYPITWVRQAPGKGLEWVSGYIHSGGSAYYADSV SINSWLAWYQQKPGKVPKLLIYKASTLAS AB_0303+3 zed KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSSWG GVPSRFSGSGSGTDFTLTISSLQPEDVATY 04 13D4 2+llgG N / A N / A N / A NLWGPGTLVTVSS YCQNYYDTITNTFGQGTKLEIK Humani DIQMTQSPSSLSASVGDRVTITCQASQSIS zed EVQLVESGGGLVKPGGSLRLSCAASGFSLSSNAVSWVRQ NLLAWYQQKPGKVPKLLIYSASTLASGVP AB_0305+3 2+llgG APGKGLEHIGFIGDTGNTYYADSVKGRFTISRDNAKNSLYL SRFSGSGSGTDFTLTISSLQPEDVATYYCQ 06 15A1 Antibod N / A N / A N / A QM NS LRAEDTAVYYCARAGITN LWGPGTLVTVSS SYYGSSSSSYGDAFGQGTKLEIK Humani EVQLVESGGGLVKPGGSLRLSCAASGIDLSRYNMAWVRQ DIQMTQSPSSLSASVGDRVTITCQASQSV zed APGKGLEWVSIIYSSAATYYADSVKGRFTISRDNAKNSLYL YEDNWLAWYQQKPGKVPKLLIYLASNLAS AB_0307+3 2+llgG QMNSLRAEDTAVYYCARRDVGSSGYTGAFDPWGPGTLVT GVPSRFSGSGSGTDFTLTISSLQPEDVATY 08 19E12 Antibod N / A N / A N / A VSS YCQGVDSSSGETTFGQGTKLEIK Extracellu

[0536] Lar GWSCIILFLVATATGVHSHHHHHHDTTQSLKQLEERAARN

[0537] Human domain VSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRL

[0538] CD23 (48- for Ab Immuno KSQDLELSWNLNGLQADLSSFKSQELNERNEASDLLERL

[0539] 150) discovery gen N / A N / A N / A REEVTKLR MELQVSSGFVCNTCPEKWINFQRKCYYFGKGTKQWVHA RYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNL DLKGEFIWVDGSHVDYSNWAPGEPTSRSQGEDCVMMR

[0540] soluble GSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESMG

[0541]

[0542] AB0208 Cd23 SCD23 N / A N / A N / A PDSRPDPDGRLPTPSAPLHS

Claims

What is claimed is:

1. A bispecific molecule comprising: a) a first binding moiety capable of binding CD23, and b) a second binding moiety capable of binding a molecule expressed on a cell surface, wherein the first binding moiety and second binding moiety are joined by a linker.

2. The bispecific molecule of claim 1, wherein tire second binding moiety is capable of binding CD3, optionally wherein the second binding moiety is antibody or fragment thereof.

3. The bispecific molecule of claim 1, wherein the first binding moiety is an antibody or fragment thereof, optionally wherein the antibody or fragment thereof is a dAb, Fab. Fab’.F(ab’)2, Fv, scFv, scFv2, scFv-Fc, minibody, diabody, triabody, or tetrabody.

4. The bispecific molecule of any one of claims 1 to 3, wherein tire first binding moiety binds an epitope found within amino acids 48 to 150 of SEQ ID NO. 1.

5. The bispecific molecule of any one of claims 1 to 4, wherein tire first binding moiety is an antibody or fragment thereof comprising six complementarity -determining regions ofa) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22;d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising tire amino acid sequence of SEQ ID NO: 24;e) an antibody comprising a hcavv chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;f) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 28;g) an antibody comprising a heavy chain comprising tire amino acid sequence of SEQ ID NO: 31 and a light chain comprising tire amino acid sequence of SEQ ID NO: 30; orh) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

6. The bispecific molecule of any one of claims 1 to 5, wherein the second binding moiety is an anti-CD3 scFv.

7. The bispecific molecule of claim 6, wherein the anti-CD3 scFv has the sequence of METDTLLLWVLLLWVPGSTGQVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWV KQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASP GEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK or EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTG AVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAI YFCALWYSNLWVFGGGTKLTVLGG8. The bispecific molecule of any one of claims 1 to 6, wherein the molecule is a BiTE.

9. The bispecific molecule of any one of claims 1 to 6, wherein the molecule is a 2+1 IgG-ScFv heterodimer.

10. The bispecific molecule of any one of claims 1 to 8, wherein the molecule comprises a sequence of Table 3.

11. A CD23-binding molecule, wherein the CD23-binding molecule is an antibody or fragment thereof comprising six complementarity-determining regions ofa) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.b) an antibody comprising a heavy chain comprising tire amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22;d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 24;e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;f) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising tire amino acid sequence of SEQ ID NO: 28;g) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30; orh) an antibody comprising a heavy chain comprising tire amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

12. A chimeric antigen receptor (CAR) comprising:a) an extracellular domain comprising the CD23-binding molecule of claim 11 b) a transmembrane domain; andc) an intracellular signaling domain.

13. The CAR of claim 12, further comprising one or more costimulatory signaling regions, optionally wherein tire costimulatory signaling regions comprise a functional signaling domain of 0X40, CD2, CD27. CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278), or 4-1BB (CD137).

14. The CAR of any one of claims 12 or 13, wherein the transmembrane domain comprises the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

15. The CAR of any one of claims 12 to 14, wherein the intracellular signaling domain comprises a functional signaling domain of 4- IBB or a functional signaling domain of CD3 zeta.

16. The CAR of any one of claims 12 to 15, wherein the extracellular domain is linked to the transmembrane domain by a hinge region, optionally wherein the hinge region is from CD8.

17. A nucleic acid comprising a nucleotide sequence that encodes the bispecific molecule of any one of claims 1 to 10, tire CD23 -binding molecule of claim 11, or the CAR of any one of claims 12 to 16.

18. An expression cassette comprising the nucleic acid of claim 17.

19. A plasmid comprising the nucleic acid of claim 17 or the expression cassette of claim 18.

20. A host cell comprising the nucleic acid of claim 17, the expression cassette of claim 18, or the plasmid of claim 19, optionally wherein the host cell is a lymphocyte.

21. A lymphocyte that has been modified to express the bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or tire CAR of any one of claims 12 to 16.

22. A recombinant viral vector comprising a nucleic acid comprising the expression cassette of claim 18.

23. The recombinant viral vector of claim 11, wherein the recombinant viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, or a lentiviral vector.

24. A recombinant AAV vector comprising an AAV capsid and having a vector genome packaged therein, tire vector genome comprising the expression cassette of claim 18.

25. A lipid nanoparticle (LNP) comprising the nucleic acid of claim 17.

26. The LNP of claim 25, wherein tire nucleic acid is a nucleoside modified mRNA.

27. A pharmaceutical composition comprising the bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21, the recombinant viral vector of claim 22, tire recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 26, and a pharmaceutically acceptable excipient, carrier, or diluent.

28. A method of treating an autoimmune condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, tire lymphocyte of claim 21, the recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmaceutical composition of claim 27.

29. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, tire plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21, the recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmacal composition of claim 27.

30. A method of treating rheumatoid arthritis in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of claims 1 to 10, theCD23-binding molecule of claim 11, or tire CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21. the recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmaceutical composition of claim 27.

31. The method of claim 30 wherein the rheumatoid arthritis is refractory RA.

32. A method of treating lupus nephritis in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of claims 1 to 10, tire CD23-binding molecule of claim 11. or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21, the recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmaceutical composition of claim 27.

33. A method of treating atopic dermatitis or allergic disease in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of tire bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21, the recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmaceutical composition of claim 27.

34. A method of treating a B-cell malignancy in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of claims 1 to 10, the CD23-binding molecule of claim 11, or the CAR of any one of claims 12 to 16, the nucleic acid of claim 17, the expression cassette of claim 18, the plasmid of claim 19, the host cell of claim 20, the lymphocyte of claim 21, tire recombinant viral vector of claim 22, the recombinant AAV vector of claim 23 or 24, or the LNP of claim 25 or 264, or the pharmaceutical composition of claim 27.

35. The method of claim 34, wherein the B-cell malignancy is CLL.

36. The method of any of claims 28 to 35, wherein the subject has elevated soluble CD23.

37. The method of any of claims 28 to 36, wherein the subject has CD23+ pathogenic B-cell populations, as determined by flow cytometry or immunohistochemistry.

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