WO2024258610A2

WO2024258610A2 - Sstr-binding bi-specific t-cell engaging molecules

Info

Publication number: WO2024258610A2
Application number: PCT/US2024/031402
Authority: WO
Inventors: Jonathan R. STROSBERG; Elenora PELLE
Original assignee: H Lee Moffitt Cancer Center and Research Institute Inc
Current assignee: H Lee Moffitt Cancer Center and Research Institute Inc
Priority date: 2023-06-15
Filing date: 2024-05-29
Publication date: 2024-12-19
Anticipated expiration: 2025-12-15
Also published as: WO2024258610A3

Abstract

Disclosed are compositions and methods for targeted treatment of SSTR-expressing cancers. For example, disclosed herein are Bispecific T-Cell Engaging (BiTE) molecules (fusion polypeptides) (also referred to herein as bispecific molecules) that are able to crosslink CD3 complex on immune effector cells with SSTR2 on NETs. Also disclosed are chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to target and kill SSTR-expressing cancers.

Description

SSTR-BINDING BI-SPECIFIC T-CELL ENGAGING MOLECULES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/508,281 , filed June 15, 2023, which is hereby incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “320805-2120 Sequence Listing” created on May 29, 2024, and having 78,607 bytes. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

[0003] The incidence and prevalence of neuroendocrine tumors (NETs) have increased in the past 20 years. NETs are clinically and biologically heterogeneous tumors that originate from the pancreas or the intestinal tract. They can cause symptoms related to tumor burden as well as hormone hypersecretion and are typically incurable in the metastatic setting. Most NETs overexpress receptors for somatostatin. Somatostatin inhibits the release of many hormones and other secretory proteins; its effects are mediated by G protein-coupled receptors that are expressed in a tissue-specific manner.

[0004] Current management strategies for NETs include surgery, radiological intervention, cytotoxic chemotherapies, somatostatin analogs and biological agents such as sunitinib and everolimus. Immunotherapy (sometimes called biological therapy, biotherapy, or biological response modifier therapy), which uses the body's immune system, either directly or indirectly, to shrink or eradicate cancer has been studied for many years as an adjunct to conventional cancer therapy. Standard immunotherapy treatments have not yet demonstrated significant activity in well-differentiated neuroendocrine tumors.

SUMMARY OF THE INVENTION

[0005] Most NETs overexpress somatostatin receptors, particularly subtype 2 (SSTR2). Disclosed herein are Bispecific T-Cell Engaging (BiTE) molecules (fusion polypeptides) (also referred to herein as bispecific molecules) that are able to crosslink CD3 complex on immune effector cells with SSTR2 on NETs. The BiTE molecules can be engineered from fusion polypeptides comprising 1) a SSTR-binding agent and 2) variable domains of antibodies that specifically bind CD3.

[0006] Also disclosed is a method of providing an anti-tumor immunity in a subject with a SSTR-expressing cancer that involves administering to the subject an effective amount of a BiTE molecule disclosed herein. In some cases, the cancer comprises a neuroendocrine tumor (NET), such as a gastroenteropancreatic neuroendocrine tumor (GEP-NET).

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF FIGURES

[0008] FIG. 1 shows expression of optimized sequences of the disclosed BiTE subcloned into a vector (pAcGP67a) designed for protein expression in insect cells using Baculovirus. Trichoplusia-ni (High Five) cells were used to express the recombinant protein, which was isolated from the supernatant using nickel affinity chromatography. The proteins were characterized by SDS-PAGE. All the BiTE-like molecules were efficiently expressed on P0. The molecular weight was consistent with the expected one. FLP and RLP were the most abundantly secreted. More BiTE-like molecules with different linker will be produced and tested.

[0009] FIG. 2 shows flow cytometry used to detect the ability of the recombinant protein in binding the CD3. Human T cells were incubated with the anti-SSTR BiTE at different concentrations. The BiTE was stained with an anti-Myc antibody specific for a Myc-tag on the BiTE. The anti-SSTR BiTE binds the CD3 on T-cells. At 100nM, the BiTE binds more than 85% of the T-cells.

[0010] FIG. 3 shows the BiTE-like molecule binds almost the entire CD4+ subpopulation of T-cells at 105 nM.

[0011] FIG. 4 shows the BiTE-like molecule binds the majority of CD8+ T-cells at 105 nM.

[0012] FIG. 5 shows interaction of the BiTE with T cells and SSTR+ target cells by confocal microscopy. The anti SSTR-BiTE was stained with AF647 and the 293T cells were transfected with a vector encoding for a GFP-SSTR2 fusion protein. T cells are not stained. Both T-cells and 293T SSTR2+GFP+ cells were seeded together with the BiTE. After 20 min of incubation, the images were acquired by confocal microscopy. The BiTE binds the CD3+ T cells (red) as well as the SSTR2+GFP+ 293T cells, where the SSR2 (green) and the BiTE (red) are clearly co-expressed.

[0013] FIG. 6 shows BiTE mediated SSTR-specific T cell activation. Human T cells were cocultured with SSTR+ 293T cells with or without 100nM of anti-SSTR BiTE. SSTR- 293T cells were used as negative control, as well as T cells with media or with anti-SSTR BiTE only. T cells stimulated with anti CD3/CD28 beads were used as positive control. The T cells activation was evaluated measuring their IFNy secretion by enzyme-linked immunosorbent assay (ELISA). The IFN-y secretion was significantly increased when the T cells were cocultured with SSTR+ 293T and BiTE, compared with the conditions without BiTE or with SSTR- 293T cells (p < 0.0001), demonstrating that the T cell activation is specific for the SSTR and mediated by the BiTE.

[0014] FIGs. 7A and 7B show a cytotoxic effect is observed with unstimulated T cells in presence of 100nM BiTE and SSTR2 cells.

[0015] FIGs. 8A and 8B show BiTE mediates SSTR-specific T cell killing.

[0016] FIG. 9 shows the peptide-based T cell engager SEQ ID NO:70 successfully enhances the formation of the immunological synapses, resulting in a SSTR-specific T cell activation.

DETAILED DESCRIPTION

[0017] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0020] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0021] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0022] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0023] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0024] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0025] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

Definitions

[0026] The term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0027] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0028] The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0029] The term “antigen binding site” refers to a region of an antibody that specifically binds an epitope on an antigen.

[0030] The term “aptamer” refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity. A “nucleic acid aptamer” is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. A “peptide aptamer” is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.

[0031] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0032] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecules is a fusion protein.

[0033] The term “engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen binding site derived from the variable domain of the heavy chain and/or light chain of an antibody and may optionally comprise the entire or part of the variable and/or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY).

[0034] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0035] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0036] The term “Fab fragment” refers to a fragment of an antibody comprising an antigen-binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule. [0037] The term “F(ab')2 fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0038] The term “Fc fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain.

[0039] The term “Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.

[0040] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may be transfected into cells, e.g. in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0041] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0042] The term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0043] The term “multivalent antibody” refers to an antibody or engineered antibody comprising more than one antigen recognition site. For example, a “bivalent” antibody has two antigen recognition sites, whereas a “tetravalent” antibody has four antigen recognition sites. The terms “monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, a “monospecific” antibody's antigen recognition sites all bind the same epitope. A “bispecific” antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A “multivalent monospecific” antibody has multiple antigen recognition sites that all bind the same epitope. A “multivalent bispecific” antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

[0044] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0045] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0046] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. [0047] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0048] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the aminoterminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and/or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.

[0049] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0050] The term “single chain variable fragment or scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0051] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0052] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 10⁵ I H (e.g., 10⁶ M^-1, 10⁷ M“¹, 10⁸ M~¹, 10⁹ M~¹, 10¹⁰ M"¹, 10¹¹ M"¹, and 10¹² M^-1 or more) with that second molecule.

[0053] The term “specifically deliver” as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule.

[0054] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0055] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0056] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0057] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. [0058] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid subsitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0059] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

SSTR-Binding Agent

[0060] The SSTR-binding agent of the disclosed BiTE molecule, CAR, or combination thereof, is in some embodiments a natural or synthetic polypeptide that binds SSTR receptors. In some embodiments, the SSTR-binding agent is an octreotide-derived peptide. For example, the SSTR-binding agent can contain one or more octreotide-derived peptides having the amino acid sequence FCFWKTCT (SEQ ID NO:1). In some embodiments, the polypeptide contains 2, 3, 4, 5, or 6 octreotide-derived peptides, each separated by a linker.

[0061] In some embodiments, the SSTR antigen binding domain is a somatostatin-28, somatostatin-14, lanreotide, or pasireotide peptide.

[0062] In some embodiments, the SSTR antigen binding domain has the amino acid sequence: SANSNPAMAPRERKAGCKNFFWKTFTSC (Somatostatin-28, SEQ ID NO:2).

[0063] In some embodiments, the SSTR antigen binding domain has the amino acid sequence: AGCKNFFWKTFTSC (Somatostatin-14, SEQ ID NO:3).

[0064] In some embodiments, the SSTR antigen binding domain is a somatostatin analog. For example, the somatostatin analog can be octreotide having the amino acid sequence FCFWKTCT (SEQ ID NO:4) or F octreotide having the amino acid sequence FCFFKTCT (SEQ ID NO:5). In some embodiments, the somatostatin analog is Consomatin G1 having the amino acid sequence LFCFWKSCW (SEQ ID NO:6) or F-Consomatin G1 having the amino acid sequence LFCFFKSCW (SEQ ID NO:7).

[0065] In some embodiments, the SSTR antigen binding domain is 3-(2-naphthyl)-DL- alanyl-DL-cysteinyl-DL-tyrosyl-DL-tryptophyl-DL-lysyl-DL-valyl-DL-cysteinyl-DL-threoninamide (2->7)-disulfide (Lanreotide). Therefore, in some embodiments, the SSTR antigen binding domain comprises the amino acid sequence CYWKVC (SEQ ID NO:83) or CYFKVC (SEQ ID NO:84). In some embodiments, this SSTR antigen binding domain is circularized. [0066] In some embodiments, the SSTR antigen binding domain is cyclo((4R)-4-(2- aminoethylcarbamoyloxy)-L-prolyl-L-phenylglycyl-D-tryptophyl-L-lysyl-4-O-benzyl-L-tyrosyl-L- phenylalanyl-) (Pasireotide).

[0067] The SSTR-binding agent is in some embodiments an antibody fragment that specifically binds SSTR. For example, the antigen binding domain can be a Fab or a singlechain variable fragment (scFv) of an antibody that specifically binds SSTR. The anti-SSTR binding agent is in some embodiments an aptamer that specifically binds CD83. For example, the anti-SSTR binding agent can be a peptide aptamer selected from a random sequence pool based on its ability to bind SSTR. The anti-SSTR binding agent can also be a natural ligand of SSTR, or a variant and/or fragment thereof capable of binding SSTR.

[0068] Antibodies, including scFvs, that selectively bind SSTR2 are described in US 2018/0118827, which is incorporated by reference in its entirety for these antibodies.

[0069] In some embodiments, the anti-SSTR scFv can comprise a variable heavy (VH) domain having CDR1 , CDR2 and CDR3 sequences and a variable light (V ) domain having CDR1 , CDR2 and CDR3 sequences.

[0070] For example, in some embodiments, the CDR1 sequence of the VH domain comprises the amino acid sequence DYGMA (SEQ ID NO:8), CDR2 sequence of the H domain comprises the amino acid sequence FISNLGYSIYYADSVKG (SEQ ID NO:9), CDR3 sequence of the VH domain comprises the amino acid sequence APYDYDSFDPMDY (SEQ ID NQ:10), CDR1 sequence of the V_L comprises the amino acid sequence KSSQSLLNSRNRKNYLA (SEQ ID NO:11), CDR2 sequence of the V domain comprises the amino acid sequence WASTRES (SEQ ID NO: 12), and CDR3 sequence of the V_L domain comprises the amino acid sequence KQSYYLWT (SEQ ID NO: 13).

[0071] In some embodiments, the anti-SSTR scFv V_H domain comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTVS (SEQ ID NO:14).

[0072] In some embodiments, the anti-SSTR scFv VL domain comprises the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPDQSPKLLIYWASTRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEIK (SEQ ID NO:15).

[0073] The heavy and light chains are preferably separated by a linker, such as those described above. [0074] In some embodiments, the anti-SSTR scFv comprises an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTVSGGG GSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPDQS PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEIK (SEQ ID NO:16).

[0075] In some embodiments, the anti-SSTR scFv comprises an amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPDQSPKLLIYWASTRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEIKGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTVS (SEQ ID NO:17).

[0076] In some embodiments, the anti-SSTR scFv comprises an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTVSGST SGSGKPGSGEGSTKGDIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPD QSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEI K (SEQ ID NO: 18).

[0077] In some embodiments, the anti-SSTR scFv comprises an amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPDQSPKLLIYWASTRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEIKGSTSGSGKPGSGEG STKGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTV S (SEQ ID NO: 19).

BiTE molecules

[0078] Bispecific T-Cell Engaging (BiTE) molecules may contain a heavy chain comprising one or more variable regions and/or a light chain comprising one or more variable regions. Bispecific antibodies can be constructed using only antibody variable domains. A fairly efficient and relatively simple method is to make the linker sequence between the VH and VL domains so short that they cannot fold over and bind one another. Reduction of the linker length to 3-12 residues prevents the monomeric configuration of the scFv molecule and favors intermolecular VH-VL pairings with formation of a 60 kDa non-covalent scFv dimer “diabody”. The diabody format can also be used for generation of recombinant bi-specific antibodies, which are obtained by the noncovalent association of two single-chain fusion products, consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody. Reducing the linker length still further below three residues can result in the formation of trimers (“triabody”, about 90 kDa) or tetramers (“tetrabody”, about 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson, 2005, Nature Biotechnology, 23:1126-1136. All of such engineered antibodies may be used in the fusion polypeptides provided herein.

[0079] Peptide linkers suitable for production of scFv antibodies are described in Kumada Y, et al. Biochemical Engineering Journal. 2007 35(2): 158- 165; Albrecht H, et al. J Immunol Methods. 2006 310(1-2):100-16; Feng J, et al. J Immunol Methods. 2003 282(1-2):33- 43; Griffiths AD, et al. Curr Opin Biotechnol. 1998 9(1):102-8; Huston JS, et al. Methods Enzymol. 1991 203:46-88; Bird RE, et al. Science. 1988 242(4877):423-6; Takkinen K, et al. Protein Eng. 1991 4(7):837-41 ; Smallshaw JE, et al. Protein Eng. 1999 12(7):623-30; Argos P. J Mol Biol. 1990211(4):943-58; and Whitlow M, et al. Protein Eng. 19936(8):989-95, which are hereby incorporated by reference for the teachings of these linkers and methods of producing scFv antibodies against different targets using various linkers.

[0080] Tetravalent Tandab® may be prepared substantially as described in WO 1999/057150 A3 or US2006/0233787, which are incorporated by reference for the teaching of methods of making Tandab® molecules.

[0081] The antigen recognition sites or entire variable regions of the engineered antibodies may be derived from one or more parental antibodies directed against any antigen of interest (e.g., target receptor ECD or TMUL ECD). The parental antibodies can include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using sequences of antibodies or antibody fragments known to be specific for an antigen of interest. Sequences that may be derived from parental antibodies include heavy and/or light chain variable regions and/or CDRs, framework regions or other portions thereof.

[0082] In some embodiments, the Bispecific T-Cell Engaging (BiTE) molecule (fusion polypeptide) has the following formula:

SSTR — V_L3 - V_H3, SSTR — V_H3 - V_L3, V_L3 - V_H3 — SSTR, or H3 - V_L3 — SSTR, wherein “SSTR” is a SSTR-binding agent; wherein “VH3” is a heavy chain variable domain specific for CD3; wherein “VL3” is a light chain variable domain specific for the CD3; wherein consists of a peptide linker; and wherein ” consists of a peptide hinge sequence.

[0083] In some cases, the BiTE molecule has an affinity for SSTR2 and CD3 corresponding to a K_D of about 10^-7 M, 10⁸ M, 10^-9 M, or less.

[0084] In some cases, the V_L3 comprises the amino acid sequence MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKF SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRA (SEQ ID NO:20), or a fragment or variant thereof able to bind CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:20. In some cases, the V_H3 comprises the amino acid sequence EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYN QKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO:21), or a fragment or variant thereof able to bind CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:21.

[0085] Other anti-CD3 antibody sequences are known in the art, such as OKT3, which can be used in the disclosed system. OKT3 has the amino acid sequence disclosed, e.g., in U.S. Patent Nos. 4,658,019, 6,113,901 and 6,491 ,916 (each of which is incorporated herein by reference in its entirety), or the amino acid sequence of the monoclonal antibody produced by the cell line deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia 20110-2209 on July 28, 1993 as Accession Number CRL-8001. Several humanized versions of OKT3 are also reported in U.S. Patent No. 6,491 ,916.

[0086] In some cases, the V_H3 comprises the amino acid sequence DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE (SEQ ID NO:22), or a fragment or variant thereof able to bind CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:22. In some cases, the V_L3 comprises the amino acid sequence GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:23), or a fragment or variant thereof able to bind CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:23. [0087] Flexible and rigid linkers are known in the art and described, for example, in Chen, X., et al. Adv Drug Deliv Rev. 201365(10): 1357-1369, which is incorporated by reference in its entirety for the teaching of these linkers and their uses.

[0088] For example, in some embodiments, the hinge sequence is a flexible linker, such as GGS, GGSGGS ((GGS)₂, SEQ ID NO:24), GGSGGSGGS ((GGS)₃, SEQ ID NO:25), GGSGGSGGSGGS ((GGS)₄, SEQ ID NO:26), GGGS (SEQ ID NO:27), GGGSGGGS ((GGGS)₂, SEQ ID NO:28), GGGSGGGSGGGS ((GGGS)₃, SEQ ID NO:29), GGGSGGGSGGGSGGGS ((GGGS)₄, SEQ ID NQ:30), GGGGS (SEQ ID NO:31), GGGGSGGGGS ((GGGGS)₂, SEQ ID NO:32), GGGGSGGGGSGGGGS ((GGGGS)₃, SEQ ID NO:33), or GGGGSGGGGSGGGGSGGGGS ((GGGGS)₄, SEQ ID NO:34).

[0089] In some embodiments, the hinge sequence is a rigid linker such as AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (A(EAAAK)₄ALEA(EAAAK)₄A, SEQ ID NO:35) or AEAAAKEAAAKA (SEQ ID NO:36).

[0090] In some embodiments, the hinge sequence contains a Pro-rich linker, such as PAPAP (SEQ ID NO:37).

[0091] In some embodiments, the hinge sequence is a combination or hybrid of these known linkers, such as PAPAPGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:38), PAPAPAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO:39), or PAPAPGGGSEAAAKEAAAKEAAAKEAAAKGGGS (SEQ ID NO:40).

[0092] In some embodiments, the hinge sequence is APAPAPAPAP (SEQ ID NO:41), APAPAPAPAPAP (SEQ ID NO:42), APAPAPAPAPAPAP (SEQ ID NO:43), APAPAPAPAPAPAPAP (SEQ ID NO:44), APAPAPAPAPAPAPAPAP (SEQ ID NO:45), APAPAPAPAPAPAPAPAPAP (SEQ ID NO:46), APAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:47), PAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:48), PAPAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:49), PAPAPAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NQ:50), PAPAPAPAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:51), PAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:52), or PAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP SEQ ID NO:53).

[0093] In some embodiments, the hinge sequence is GGGGGG (SEQ ID NO:54), GGGGGGGG (SEQ ID NO:55), EAAAK (SEQ ID NO:56), EAAAKEAAAK (SEQ ID NO:57), EAAAKEAAAKEAAAK (SEQ ID NO:58), APAPAPAPAP (SEQ ID NO:59), VSQTSKLTRAETVFPDV (SEQ ID NQ:60), PLGLWA (SEQ ID NO:61), RVLAEA (SEQ ID NO:62), EDVVCCSMSY (SEQ ID NO:63), GGIEGRGS (SEQ ID NO:64), TRHRQPRGWE (SEQ ID NO:65), AGNRVRRSVG (SEQ ID NO:66), RRRRRRRRR (SEQ ID NO:67), GFLG (SEQ ID NO:68), or LE.

[0094] These hinges can be included in the SS14-SS14 site as well as in the SSS14- CD3, resulting in several different combinations.

[0095] The first peptide linker and second peptide linker are preferably long enough to not interfere with proper folding and association of the V_H-V_L chains but not so long as to cause added immunogenicity. They can be flexible, rigid, or Pro-rich linkers as well.

[0096] Therefore, in some embodiments, the disclosed BiTE molecules can have the amino acid sequence AGCKNFFWKTFTSCGGGGSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSG YTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSE DSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSA SPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTIS SMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:69);

FCFWKTCTGGGGSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRY TMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVY YCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEK VTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAE DAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NQ:70);

FCFFKTCTGGGGSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYT MHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYY CARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKV TMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAED AATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:71);

LFCFFKSCWGGGGSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTR YTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGE KVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEA EDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:72);

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGS GGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIY DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGS GGSGGGSGGGSGGGSAGCKNFFWKTFTSC (SEQ ID NO:73); DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGS GGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIY DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGS GGSGGGSGGGSGGGSFCFWKTCT (SEQ ID NO:74);

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGS GGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIY

DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGS GGSGGGSGGGSGGGSFCFFKTCT (SEQ ID NO:75);

DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGS GGSGGGSGGGSGGGSLFCFWKSCW (SEQ ID NO:76);

DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGS GGSGGGSGGGSGGGSLFCFFKSCW (SEQ ID NO:77);

AGCKNFFWKTFTSCGGGSGGGSGGGSGGGSGGGSDIKLQQSGAELARPGASVKMSCKTSG YTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSE DSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSA

SPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTIS SMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:78);

FCFWKTCTGGGSGGGSGGGSGGGSGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRY TMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVY YCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEK

VTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAE DAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:79);

FCFFKTCTGGGSGGGSGGGSGGGSGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYT MHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYY CARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKV

TMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAED AATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:80);

LFCFWKSCWGGGSGGGSGGGSGGGSGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTR YTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGE KVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEA EDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID N0:81); or LFCFFKSCWGGGSGGGSGGGSGGGSGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTR YTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGE KVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEA EDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:82).

[0097] Candidate engineered antibodies for inclusion in the fusion polypeptides, or the fusion polypeptides themselves, may be screened for activity using a variety of known assays. For example, screening assays to determine binding specificity are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y., 1988, Chapter 6.

[0098] In some embodiments, the BiTE molecule may be subjected to an alteration to render it less immunogenic when administered to a human. Such an alteration may comprise one or more of the techniques commonly known as chimerization, humanization, CDR-grafting, deimmunization and/or mutation of framework region amino acids to correspond to the closest human germline sequence (germlining). Bispecific antibodies which have been altered will therefore remain administrable for a longer period of time with reduced or no immune response- related side effects than corresponding bispecific antibodies which have not undergone any such alteration(s). One of ordinary skill in the art will understand how to determine whether, and to what degree an antibody must be altered in order to prevent it from eliciting an unwanted host immune response.

Therapeutic Methods

[0099] Immune effector cells expressing the disclosed CARs can elicit an anti-tumor immune response against SSTR-expressing cancer cells. The anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific to SSTR. [0100] Adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anti-cancer therapeutic. Following the collection of a patient’s immune effector cells, the cells may be genetically engineered to express the disclosed SSTR-specific CARs, then infused back into the patient.

[0101] The disclosed CAR-modified immune effector cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-15, or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodimetns formulated for intravenous administration. Pharmaceutical compositions may be administered in any manner appropriate treat MM. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0102] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0103] In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently re-draw blood (or have an apheresis performed), activate T cells therefrom according to the disclosed methods, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.

[0104] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0105] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[0106] The cancer of the disclosed methods can be any SSTR-expressing cell in a subject undergoing unregulated growth, invasion, or metastasis. In some cases, the cancer can be any SSTR-expressing malignancy. In some cases, the cancer comprises a gastroenteropancreatic neuroendocrine tumor (GEP-NET). GEP-NETs, also known as carcinoids and islet cell tumors, are tumors derived from neuroendocrine cells that can occur anywhere along the gastrointestinal tract and comprise a heterogeneous family of neoplasms with a wide and complex spectrum of clinical behavior. GEP-NETs have traditionally been divided into foregut (esophagus, stomach, proximal duodenum, liver and pancreas), midgut (distal duodenum ileum, jejunum, ascending colon and proximal two thirds of transverse colon) and hindgut tumors (distal third of transverse colon, descending colon, sigmoid colon and rectum). GEP-NETs are characterized by their ability to produce, store and secrete a large number of peptide hormones and biogenic amines which can lead to the development of distinct clinical syndromes. Based on this, GEP-NETs are broadly subdivided into “functional” or “nonfunctional” tumors (with or without a clinical syndrome attributable to hormonal hypersecretion, respectively). Among the “functional” tumors, each of these secreted substances causes a specific clinical syndrome, including carcinoid, Zollinger-Ellison, insulinoma, Verner-Morrison, and glucagonoma syndromes. Specific markers for these syndromes are basal and/or stimulated levels of urinary 5-hydroxyindoleacetic acid, serum or plasma gastrin, insulin, vasoactive intestinal polypeptide and glucagon, respectively. General markers such as chromogranin A, pancreatic polypeptide, serum neuron-specific enolase and subunit of glycoprotein hormones have been used for screening purposes in patients without distinct clinical hormone-related syndromes. The most important general circulating tumor marker is chromogranin A, expressed in 80-90% of all patients with GEP-NETs. Chromogranin A determination is also useful for staging, prognosis and follow up, since the serum concentration correlates to the tumor mass.

[0107] The disclosed CARs can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.

[0108] The disclosed CARs can be used in combination with a checkpoint inhibitor. The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1, an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK-3475), PD- L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0109] Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0110] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds PDL1 , such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds PD1 , such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0111] The disclosed CARs can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapy: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells, without necessarily initiating an immune response in the patient, while active immunotherapy actively triggers an endogenous immune response. Passive strategies include the use of the monoclonal antibodies (mAbs) produced by B cells in response to a specific antigen. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens permitted the pharmaceutical development of mAbs that could specifically target tumor cells for destruction by the immune system. Thus far, mAbs have been the biggest success story for immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein that is highly expressed on the surface of B cell malignancies such as non-Hodgkin’s lymphoma (NHL). Rituximab is approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0112] Generating optimal “killer” CD8 T cell responses also requires T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including 0X40 (CD134) and 4-1 BB (CD137). 0X40 is of particular interest as treatment with an activating (agonist) anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors.

[0113] In some embodiments, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0114] In some embodiments, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin .

[0115] In some embodiments, such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0116] In some embodiments, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0117] In some embodiments, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBI (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).

[0118] In some embodiments, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib. [0119] Therefore, in some embodiments, a disclosed antibody is used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and/or rituximab.

[0120] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0121] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a cell cycle co ntrol/apo ptosis regulator (or "regulating agent"). A cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055) . Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosisinducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

[0122] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or a hormone inhibitor (such as octreotide/sandostatin). [0123] In some embodiments, a therapeutic agent for use in combination with an CARs for treating the disorders as described above may be an anti-cancer nucleic acid or an anticancer inhibitory RNA molecule.

[0124] Combined administration, as described above, may be simultaneous, separate, or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

[0125] In some embodiments, the disclosed CARs is administered in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium- 241 , gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131 , and indium-111 .

[0126] In some embodiments, the disclosed CARs is administered in combination with surgery.

[0127] CAR-T cells may be designed in several ways that enhance tumor cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and prolong their therapeutic half-life. TRUCK (T-cells Redirected for Universal Cytokine Killing) T cells for example, possess a CAR but are also engineered to release cytokines such as IL-12 that promote tumor killing. Because these cells are designed to release a molecular payload upon activation of the CAR once localized to the tumor environment, these CAR-T cells are sometimes also referred to as ‘armored CARs’. Several cytokines as cancer therapies are being investigated both pre-clinically and clinically, and may also prove useful when similarly incorporated into a TRUCK form of CAR-T therapy. Among these include IL-2, IL-3. IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-a, IFN-y, TNF-a, TRAIL, FLT3 ligand, Lymphotactin, and TGF-p (Dranoff 2004). “Self-driving” or “homing” CAR-T cells are engineered to express a chemokine receptor in addition to their CAR. As certain chemokines can be upregulated in tumors, incorporation of a chemokine receptor aids in tumor trafficking to and infiltration by the adoptive T-cell, thereby enhancing both specificity and functionality of the CAR-T (Moon 2011). Universal CAR-T cells also possess a CAR, but are engineered such that they do not express endogenous TCR (T-cell receptor) or MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signaling repertoire of the adoptive T-cell therapy prevents graft-versus-host-disease and rejection, respectively. Armored CAR-T cells are additionally so named for their ability to evade tumor immunosuppression and tumor- induced CAR-T hypofunction. These particular CAR-Ts possess a CAR, and may be engineered to not express checkpoint inhibitors. Alternatively, these CAR-Ts can be coadministered with a monoclonal antibody (mAb) that blocks checkpoint signaling. Administration of an anti-PDL1 antibody significantly restored the killing ability of CAR TILs (tumor infiltrating lymphocytes). While PD1-PDL1 and CTLA-4-CD80/CD86 signaling pathways have been investigated, it is possible to target other immune checkpoint signaling molecules in the design of an armored CAR-T including LAG-3, Tim-3, IDO-1 , 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin-ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase) . Armored CAR-Ts may also be engineered to express proteins or receptors that protect them against or make them resistant to the effects of tumor- secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with the double negative form of the TGF- receptor are resistant to the immunosuppression by lymphoma secreted TGF-[3. These transduced cells showed notably increased antitumor activity in vivo when compared to their control counterparts.

[0128] In some embodiments, the disclosed CAR is used in combination with a CAR that specifically binds CXCR4. For example, the CAR-T cell can be engineered to have two CARs — one that binds SSTR, and one that binds CXCR4. Tandem and dual CAR-T cells are unique in that they possess two distinct antigen binding domains. A tandem CAR contains two sequential antigen binding domains facing the extracellular environment connected to the intracellular costimulatory and stimulatory domains. A dual CAR can be engineered such that one extracellular antigen binding domain is connected to the intracellular costimulatory domain and a second, distinct extracellular antigen binding domain is connected to the intracellular stimulatory domain. Because the stimulatory and costimulatory domains are split between two separate antigen binding domains, dual CARs are also referred to as “split CARs”. In both tandem and dual CAR designs, binding of both antigen binding domains is necessary to allow signaling of the CAR circuit in the T-cell. Because these two CAR designs have binding affinities for different, distinct antigens, they are also referred to as “bi-specific” CARs.

[0129] One primary concern with CAR-T cells as a form of “living therapeutic” is their manipulability in vivo and their potential immune-stimulating side effects. To better control CAR- T therapy and prevent against unwanted side effects, a variety of features have been engineered including off-switches, safety mechanisms, and conditional control mechanisms. Both self-destruct and marked/tagged CAR-T cells for example, are engineered to have an “off- switch” that promotes clearance of the CAR-expressing T-cell. A self-destruct CAR-T contains a CAR, but is also engineered to express a pro-apoptotic suicide gene or “elimination gene” inducible upon administration of an exogenous molecule. A variety of suicide genes may be employed for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC TAG, and truncated EGFR (endothelial growth factor receptor). HSK for example, will convert the prodrug ganciclovir (GCV) into GCV-triphosphate that incorporates itself into replicating DNA, ultimately leading to cell death. iCasp9 is a chimeric protein containing components of FK506-binding protein that binds the small molecule AP1903, leading to caspase 9 dimerization and apoptosis. A marked/ tagged CAR-T cell however, is one that possesses a CAR but also is engineered to express a selection marker. Administration of a mAb against this selection marker will promote clearance of the CAR-T cell. Truncated EGFR is one such targetable antigen by the anti-EGFR mAb, and administration of cetuximab works to promotes elimination of the CAR-T cell. CARs created to have these features are also referred to as sCARs for ‘switchable CARs’, and RCARs for ‘regulatable CARs’. A “safety CAR”, also known as an “inhibitory CAR” (iCAR), is engineered to express two antigen binding domains. One of these extracellular domains is directed against a tumor related antigen and bound to an intracellular costimulatory and stimulatory domain. The second extracellular antigen binding domain however is specific for normal tissue and bound to an intracellular checkpoint domain such as CTLA4, PD1, or CD45. Incorporation of multiple intracellular inhibitory domains to the iCAR is also possible. Some inhibitory molecules that may provide these inhibitory domains include B7-H1 , B7-1 , CD160, PIH, 2B4, CEACAM (CEACAM- 1. CEACAM-3, and/or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1 , and TGF -R. In the presence of normal tissue, stimulation of this second antigen binding domain will work to inhibit the CAR. It should be noted that due to this dual antigen specificity, iCARs are also a form of bi-specific CAR-T cells. The safety CAR-T engineering enhances specificity of the CAR-T cell for tumor tissue, and is advantageous in situations where certain normal tissues may express very low levels of a tumor associated antigen that would lead to off target effects with a standard CAR (Morgan 2010). A conditional CAR-T cell expresses an extracellular antigen binding domain connected to an intracellular costimulatory domain and a separate, intracellular costimulator. The costimulatory and stimulatory domain sequences are engineered in such a way that upon administration of an exogenous molecule the resultant proteins will come together intracellularly to complete the CAR circuit. In this way, CAR-T activation can be modulated, and possibly even ‘fine-tuned’ or personalized to a specific patient. Similar to a dual CAR design, the stimulatory and costimulatory domains are physically separated when inactive in the conditional CAR; for this reason these too are also referred to as a “split CAR”. [0130] In some embodiments, two or more of these engineered features may be combined to create an enhanced, multifunctional CAR-T. For example, it is possible to create a CAR-T cell with either dual- or conditional- CAR design that also releases cytokines like a TRUCK. In some embodiments, a dual-conditional CAR-T cell could be made such that it expresses two CARs with two separate antigen binding domains against two distinct cancer antigens, each bound to their respective costimulatory domains. The costimulatory domain would only become functional with the stimulatory domain after the activating molecule is administered. For this CAR-T cell to be effective the cancer must express both cancer antigens and the activating molecule must be administered to the patient; this design thereby incorporating features of both dual and conditional CAR-T cells.

[0131] Typically, CAR-T cells are created using ct-|3 T cells, however y-b T cells may also be used. In some embodiments, the described CAR constructs, domains, and engineered features used to generate CAR-T cells could similarly be employed in the generation of other types of CAR-expressing immune cells including NK (natural killer) cells, B cells, mast cells, myeloid-derived phagocytes, and NKT cells. Alternatively, a CAR-expressing cell may be created to have properties of both T-cell and NK cells. In an additional embodiment, the transduced with CARs may be autologous or allogeneic.

[0132] Several different methods for CAR expression may be used including retroviral transduction (including y-retroviral), lentiviral transduction, transposon/transposases (Sleeping Beauty and PiggyBac systems), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion/disruption) has become of increasing importance with respect to the possibility for engineering CAR-T cells as well. CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator like effector nuclease) systems are three potential methods through which CAR-T cells may be generated.

Pharmaceutical composition

[0133] Also disclosed is a pharmaceutical composition comprising a disclosed molecule in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharmaceutical ly-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutical ly-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0134] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a bispecific antibody of the present invention . Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Methods of Treatment

[0135] Adoptive transfer of the disclosed BiTE molecules and/or cells can be used to treat a neuroendocrine tumor (NET) in a subject.

[0136] The disclosed antibodies and/or cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-15, or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodimetns formulated for intravenous administration. Pharmaceutical compositions may be administered in any manner appropriate treat MM. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0137] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0138] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0139] In certain embodiments, the disclosed antibodies and/or cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[0140] The disclosed antibodies and/or cells can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.

[0141] The disclosed antibodies and/or cells can be used in combination with a checkpoint inhibitor. The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1 , an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0142] Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0143] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds PDL1 , such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds PD1 , such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0144] The disclosed antibodies and/or cells can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapy: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells, without necessarily initiating an immune response in the patient, while active immunotherapy actively triggers an endogenous immune response. Passive strategies include the use of the monoclonal antibodies (mAbs) produced by B cells in response to a specific antigen. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens permitted the pharmaceutical development of mAbs that could specifically target tumor cells for destruction by the immune system. Thus far, mAbs have been the biggest success story for immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein that is highly expressed on the surface of B cell malignancies such as non-Hodgkin’s lymphoma (NHL). Rituximab is approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0145] Generating optimal “killer” CD8 T cell responses also requires T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including 0X40 (CD134) and 4-1 BB (CD137). 0X40 is of particular interest as treatment with an activating (agonist) anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors. [0146] In some embodiments, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0147] In some embodiments, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.

[0148] In some embodiments, such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0149] In some embodiments, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0150] In some embodiments, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBI (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).

[0151] In some embodiments, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0152] Therefore, in some embodiments, a disclosed antibody is used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and/or rituximab.

[0153] In some embodiments, a therapeutic agent for use in combination with off-the- shelf T cells for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL- 29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0154] In some embodiments, a therapeutic agent for use in combination off-the-shelf T cells for treating the disorders as described above may be a cell cycle control/apoptosis regulator (or "regulating agent"). A cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01 , KW-2401), and roscovitine (R- roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055) . Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosisinducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

[0155] In some embodiments, a therapeutic agent for use in combination with off-the- shelf T cells for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or a hormone inhibitor (such as octreotide/sandostatin).

[0156] In some embodiments, a therapeutic agent for use in combination with off-the- shelf T cells for treating the disorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0157] Combined administration, as described above, may be simultaneous, separate, or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

[0158] In some embodiments, the disclosed antibodies and/or cells are administered in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide- 131 , and indium-111.

[0159] In some embodiments, the disclosed antibodies and/or cells are administered in combination with surgery.

[0160] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1 :

[0161] FIG. 1 shows expression of optimized sequences of the disclosed BiTE subcloned into a vector (pAcGP67a) designed for protein expression in insect cells using Baculovirus. Trichoplusia-ni (High Five) cells were used to express the recombinant protein, which was isolated from the supernatant using nickel affinity chromatography. The proteins were characterized by SDS-PAGE. All the BiTE-like molecules were efficiently expressed on P0. The molecular weight was consistent with the expected one. FLP and RLP were the most abundantly secreted. More BiTE-like molecules with different linker will be produced and tested.

[0162] FIG. 2 shows flow cytometry used to detect the ability of the recombinant protein in binding the CD3. Human T cells were incubated with the anti-SSTR BiTE at different concentrations. The BiTE was stained with an anti-Myc antibody specific for a Myc-tag on the BiTE. The anti-SSTR BiTE binds the CD3 on T-cells. At 100nM, the BiTE binds more than 85% of the T-cells.

[0163] FIG. 3 shows the BiTE-like molecule binds almost the entire CD4+ subpopulation of T-cells at 105 nM.

[0164] FIG. 4 shows the BiTE-like molecule binds the majority of CD8+ T-cells at 105 nM.

[0165] FIG. 5 shows interaction of the BiTE with T cells and SSTR+ target cells by confocal microscopy. The anti SSTR-BiTE was stained with AF647 and the 293T cells were transfected with a vector encoding for a GFP-SSTR2 fusion protein. T cells are not stained. Both T-cells and 293T SSTR2+GFP+ cells were seeded together with the BiTE. After 20 min of incubation, the images were acquired by confocal microscopy. The BiTE binds the CD3+ T cells (red) as well as the SSTR2+GFP+ 293T cells, where the SSR2 (green) and the BiTE (red) are clearly co-expressed.

[0166] FIG. 6 shows BiTE mediated SSTR-specific T cell activation. Human T cells were cocultured with SSTR+ 293T cells with or without 100nM of anti-SSTR BiTE. SSTR- 293T cells were used as negative control, as well as T cells with media or with anti-SSTR BiTE only. T cells stimulated with anti CD3/CD28 beads were used as positive control. The T cells activation was evaluated measuring their I FNy secretion by enzyme-linked immunosorbent assay (ELISA). The IFN-y secretion was significantly increased when the T cells were cocultured with SSTR+ 293T and BiTE, compared with the conditions without BiTE or with SSTR- 293T cells (p < 0.0001), demonstrating that the T cell activation is specific for the SSTR and mediated by the BiTE.

[0167] Despite the binding affinity of a peptide towards its ligand is usually lower than an antibody, the peptide-based T cell engager efficiently activates the T cells against SSTR- expressing T cells. Human T cells were cocultured with SSTR+ 293T cells with or without 100nM of peptide-based T cell engager. SSTR- 293T cells were used as negative control, as well as T cells with media or with anti-SSTR peptide-based T cell engager only. T cells stimulated with anti CD3/CD28 beads were used as positive control. The T cells activation was evaluated measuring their IFNy secretion by enzyme-linked immunosorbent assay (ELISA). The IFN-y secretion was significantly increased when the T cells were cocultured with SSTR+ 293T and peptide-based T cell engager, compared with the conditions without peptide-based T cell engager or with SSTR- 293T cells (Fig. 9, p < 0.0001), demonstrating that the T cell activation is specific for the SSTR and mediated by the peptide-based T cell engager.

[0168] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0169] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1 . A bispecific T-cell engaging (BiTE) molecule comprising a fusion polypeptide having the following formula:

SSTR — V_L3 - V_H3, SSTR — V_H3 - V_L3, L3 - V_H3 — SSTR, or V_H3 - V_L3 — SSTR, wherein “SSTR” is a SSTR-binding agent; wherein “VH3” is a heavy chain variable domain specific for CD3; wherein “V_L3” is a light chain variable domain specific for the CD3; wherein consists of a peptide linker; and wherein

consists of a peptide hinge sequence.

2. The BiTE molecule of claim 1 , wherein the SSTR-binding agent comprises the amino acid sequence FCFWKTCT (SEQ ID NO:1).

3. The BiTE molecule of claim 2, wherein the SSTR-binding agent comprises somatostatin- 14 having amino acid sequence AGCKNFFWKTFTSC (SEQ ID NO:3).

4. The BiTE molecule of claim 1 , wherein the SSTR-binding agent comprises a somatostatin analog.

5. The BiTE molecule of claim 4, wherein the somatostatin analog is octreotide having the amino acid sequence FCFWKTCT (SEQ ID NO:4) or F octreotide having the amino acid sequence FCFFKTCT (SEQ ID NO:5).

6. The BiTE molecule of claim 4, wherein the somatostatin analog is Consomatin G1 having the amino acid sequence LFCFWKSCW (SEQ ID NO:6) or F-Consomatin G1 having the amino acid sequence LFCFFKSCW (SEQ ID NO:7).

7. The BiTE molecule of any one of claims 1 to 6, wherein the peptide hinge comprises the amino acid sequence GGS, GGSGGS ((GGS)₂, SEQ ID NO:24), GGSGGSGGS ((GGS)₃, SEQ ID NO:25), GGSGGSGGSGGS ((GGS)₄, SEQ ID NO:26), GGGS (SEQ ID NO:27), GGGSGGGS ((GGGS)₂, SEQ ID NO:28), GGGSGGGSGGGS ((GGGS)₃, SEQ ID NO:29), GGGSGGGSGGGSGGGS ((GGGS)₄, SEQ ID NQ:30), GGGGS (SEQ ID NO:31), GGGGSGGGGS ((GGGGS)₂, SEQ ID NO:32), GGGGSGGGGSGGGGS ((GGGGS)₃, SEQ ID NO:33), or GGGGSGGGGSGGGGSGGGGS ((GGGGS)₄, SEQ ID NO:34).

8. The BiTE molecule of any one of claims 1 to 6, wherein the peptide hinge comprises the amino acid sequence AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (A(EAAAK)₄ALEA(EAAAK)₄A, SEQ ID NO:35) or AEAAAKEAAAKA (SEQ ID NO:36).

9. The BiTE molecule of any one of claims 1 to 6, wherein the peptide hinge comprises the amino acid sequence PAPAPGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:37), PAPAPAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO:38), or PAPAPGGGSEAAAKEAAAKEAAAKEAAAKGGGS (SEQ ID NO:39).

10. The BiTE molecule of any one of claims 1 to 9, wherein the V_L3 comprises the amino acid sequence MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKF SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRA (SEQ ID NQ:20) and the V_H3 comprises the amino acid sequence

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYN QKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO:21).

11. The BiTE molecule of any one of claims 1 to 9, wherein the VL3 comprises the amino acid sequence DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE (SEQ ID NO:22) and the H3 comprises the amino acid sequence

GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:23).

12. The BiTE molecule of any one of claims 1 to 11, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81, or SEQ ID NO:82.

13. An isolated nucleic acid sequence encoding the BiTE molecule of any one of claims 1 to 12.

14. A vector comprising the isolated nucleic acid sequence of claim 13.

15. A method of providing an anti-cancer immunity in a subject with a SSTR-expressing cancer, the method comprising administering to the subject an effective amount of the BiTE molecule of any one of claims 1 to 12, thereby providing an anti-tumor immunity in the subject.

16. The method of claim 15, further comprising administering to the subject a checkpoint inhibitor.

17. The method of claim 16, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

18. The method of any one of claims 15 to 17, further comprising administering to the subject an agent capable of inducing SSTR up-regulation by tumor cells.

19. The method of any one of claims 15 to 18, wherein the cancer is a neuroendocrine tumor.