Multimers of 211astatine conjugated single domain antibodies to treat solid tumors

Abstract

Certain embodiments of the invention provide multimers of 211Astatine α-emitter conjugated single domain antibodies and uses thereof to treat cancer.

Description

[0001] MULTIMERS OF211ASTATINE CONJUGATED SINGLE DOMAIN ANTIBODIES TO

[0002] TREAT SOLID TUMORS

[0003] CROSS-REFERENCE TO RELATED APPLICATION(S)

[0004] This application claims priority to United States Provisional Application Number 63 / 709,111 that was filed on October 18, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.

[0005] BACKGROUND

[0006] Current methods for treating cancer, e.g., solid tumors, often involve low therapeutic efficacies and have undesirable high toxicities. As such, improved methods of treating cancer, and in particular solid tumors, are needed.

[0007] SUMMARY

[0008] Provided herein are monomers and multimers of21'Astatine a-emitter conjugated single domain antibodies useful, e.g., to treat solid tumors.

[0009] Certain embodiments of the invention provide antibody monomers and multimers and methods and uses thereof as described herein.

[0010] DETAILED DESCRIPTION

[0011] Described herein are monomers and multimers of single domain antibodies that are conjugated to astatine emitters for the treatment of cancers, including solid tumors.

[0012] Certain embodiments provide an antibody multimer, wherein the antibody multimer comprises at least 2 single domain antibody monomers, wherein at least one of the single domain antibody monomers is conjugated to21 1Astatine.

[0013] In certain embodiments, the antibody multimer comprises 2, 3, or 4 single domain antibodies.

[0014] In certain embodiments, each single domain antibody monomer of the multimer is conjugated to21 1Astatine.

[0015] In certain embodiments, at least one of the single domain antibody monomers is a low affinity single domain antibody.

[0016] In certain embodiments, each of the single domain antibody monomers is a low affinity single domain antibody.

[0017] In certain embodiments, at least one of the monomers is targeted to HER2, EGFR, HER3, TROP2, c-MET, PD-L1, PDGFRA, VEGFR2, PSMA, GPC3, EPCAM, FAP, B7H3, or Nectin-4.

[0018] In certain embodiments, the monomers are connected with flexible protein linkers between the monomers.

[0019] In certain embodiments, each linker is 3-30 amino acids in length.

[0020] In certain embodiments, the linkers form the chain that connects individual monomers together.

[0021] Certain embodiments provide a DNA sequence encoding an antibody multimer described herein.

[0022] Certain embodiments provide a pharmaceutical composition comprising an antibody multimer described herein and a pharmaceutically acceptable carrier.

[0023] Certain embodiments provide an antibody multimer described herein for the prophylactic or therapeutic treatment of cancer in a subject.

[0024] Certain embodiments provide the use of an antibody multimer described herein to prepare a medicament for the treatment of cancer in a subject.

[0025] In certain embodiments, the cancer is a metastatic cancer, a solid tumor, a lung cancer, a breast cancer, a head and neck cancer, a GI cancer, an ovarian cancer, a prostate cancer, a bladder cancer or a glioblastoma. In certain embodiments, the cancer is a micrometastas.

[0026] The antibody multimers can also be used at multiple stages of disease treatment, and can be used in combination with other treatments for cancer.

[0027] Certain embodiments provide an antibody multimer described herein for use in medical therapy.

[0028] Certain embodiments provide an antibody monomer, wherein the antibody monomer is conjugated to21'Astatine.

[0029] 21 1Astatine as a therapeutic overcomes the following current challenges and provides the following benefits:

[0030] • Not rare in nature o Inexpensive, widely available, non-radioactive starting material o Easily produced in cyclotrons around the world o Logistics a focus of governmental initiatives • Has a half-life of about 7 hours o Efficient and stable labeling o High level of radioactivity shipped to GMP manufacturing site accounts for decay in transport with conjugation and purification near medical site o Multiple collaborative initiatives underway globally to facilitate clinical trials

[0031] • Available cyclotron currently produces up to 250 mCi (9.3 GBq) of21 1Astatine, which can be shipped to locations within 6 hours with sufficient activity for a clinical study.

[0032] 211Astatine-sdAbs are optimal radiotherapies

[0033] 211Astatine-sdAbs solve the following problems: Severe bystander damage and lower anti-tumor toxicity of P-emitters; Biologic complexities that hinder ADSs; Marked bystander damage and low therapeutic windows of other a-emitters cused by the “recoil effect”; Difficult conjugation processes and extremely difficult supply chains of other a-emitters; and Starting material of211Astatine is Bismuth, a widely available, inexpensive, and non-radioactive metal. For example,225Actinium, a popular a-emitter, has multiple highly difficult challenges. Further, the chemistry of21'Astatine overcomes chelator-based kidney toxicity.

[0034] SdAbs and their use for delivering211Astatine in therapeutic and diagnostic contexts

[0035] SdAbs have fewer off-target toxicity risks: : o Broad distribution of small molecules / peptides causes toxicity in off-target tissues such as liver, bone marrow, and heart o Long half-lives of full-length IgGs do not allow full exploitation of fast clearance of Astatine o Similar to21 1Astatine, single domain antibodies have short half-lives and are quickly cleared from blood and unbound tissues

[0036] SdAb engineering possibilities o SdAbs can be conjugated with other molecules, including other sdAbs, for increased specificity, internalization, and PK optimization o High stability of sdAbs is advantageous for manufacturing and distribution

[0037] • Interestingly, while SdAbs may be the best match for211At delivery, monomers are not necessarily the best platform design o Half-life of monomer is extremely short

[0038] ■ Very short time in circulation requires extremely fast and tight binding to tumor antigens to achieve binding before clearance from circulation

[0039] ■ Minimal penetration into tumor despite small size (influenced by size, affinity, and time in circulation)

[0040] ■ High off-tumor, on-target effects (due to extremely fast / tight binding) o Binding and biodistribution of different multimers and binding affinities provides optimized multimer platform for translation

[0041] Binding affinity impacts specificity: side effects

[0042] The binding affinities of cetuximab and nimozotumab differ by only 1 order of magnitude. (Kd cetuximab ~ 1.9 nM, Kd nimozotumab - 21 nM). However, use of cetuximab often results in severe skin toxicity, severe hypomagnesemia, or gastrointestinal adverse events, which nimozotumab does not in similar indications. It is hypothesized that this difference in toxicity profile is due to the difference in affinity of these two monoclonal Abs. When EGFR density is high, in targeted disease regions, both antibodies bind with both binding sites, and therefore have similar effect in those regions. When EGFR density is low, in regions of normal expression, the tighter binding by cetuximab results in sustained interaction, but the less intense binding by nimozotumab results in release of the mAb and removal from the region of low EGFR expression. It is proposed herein that sdAb multimers of low affinity will have improved specificity profiles compared to sdAb monomers and other targeting moi eties of high affinity.

[0043] Further, the size (degree of multimerization) and binding affinity strongly influence important therapeutic factors of sdAbs. Targets

[0044] An advantage of sdAb multimer-targeted211At is to provide improved specificity and decreased toxicity compared to mAb-targeted ADCs. In certain embodiments, the targets can be evaluated as follows.

[0045] Compositions and Administration

[0046] Certain embodiments of the invention provide a pharmaceutical composition comprising an antibody multimer described herein, and a pharmaceutically acceptable carrier.

[0047] In certain embodiments, the protein is present in a liquid composition (e.g., saline, D5W, or buffered solution).

[0048] In certain embodiments, the antibody multimer is present in a solid composition. In certain embodiments, the antibody multimer is present in a lyophilized composition, which may be reconstituted with proper solution prior to administration as a liquid. In certain embodiments, the lyophilized composition further comprises one or more excipients selected from the group consisting of a cryo-lyoprotectant (e.g., trehalose, sucrose) and a bulking agent (e.g., mannitol, glycine).

[0049] For in vivo use, an antibody multimer of the invention is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, the antibody multimer may be present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease, as measured using a representative assay). A pharmaceutical composition may comprise an antibody multimer in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition.

[0050] Compositions for injection will commonly comprise a solution of the antibody multimer dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer’s solution. Pharmaceutical compositions desirably are sterile and generally free of undesirable matter. These pharmaceutical compositions can be sterilized by conventional, well known sterilization techniques. The pharmaceutical compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The pharmaceutical compositions may contain salt, sugar, preservative, surfactant, or any other suitable excipient.

[0051] The pharmaceutical composition may contain any suitable concentration of the antibody multimer. The concentration of the antibody multimer in the pharmaceutical composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient’s needs. In certain embodiments, the concentration of antibody multimer in a solution formulation for injection will range from about 0.1% (w / w) to about 10% (w / w), or more such as 100 mg of antibody multimer per milliliter of the formulation.

[0052] In certain embodiments, the antibody of the present invention may be systemically administered, e.g., intravenously, in combination with a pharmaceutically acceptable carrier. In certain embodiments, the antibody multimer may be administered intravenously, subcutaneously, intradermally, intramuscularly, intraperitoneally, or intrathecally by infusion or injection. In certain embodiments, the antibody multimer of the present invention may be locally administered into a particular tissue, structure, or organ.

[0053] Frequency of administration can range from multiple doses to a single dose per week, or less frequently (e.g., single dose per month or every two to three months). In certain embodiments, the antibody multimer of the present invention may be administered, e.g., intravenously or subcutaneously, to a mammal in need of, for example, about once every two weeks, once every three weeks, once every month, once every five weeks, or once every six weeks. In certain embodiments, the antibody multimer may be administered about once every month or once every two or three months. In certain embodiments, the antibody multimer may be administered about once every week. In some embodiments, the antibody multimer is administered from about once per month to about five times per week.

[0054] In one embodiment, the antibody multimer is administered to the patient parenterally. Dosing of the antibody multimer can be by any suitable route, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. The antibody multimer dose can range from about 5 mg / kg (body weight) to about 50 mg / kg, from about 10 pg / kg to about 5 mg / kg, or from about 100 pg / kg to about 1 mg / kg. The antibody multimer dose can be about 100, 200, 300, 400, or 500 pg / kg. The antibody multimer dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg, or more. The antibody multimer dose can also be outside of these ranges, depending on the type and severity of the disorder being treated. The antibody multimer dose for a patient can be adjusted by physician or pharmacist.

[0055] In certain embodiments, the antibody multimer may be delivered using a device (e.g., a device comprising one or two chambers, for example, containing the antibody multimer, and / or liquid). In certain embodiments, the device is an injector (e.g., a self-injector device comprising the antibody multimer).

[0056] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of an antibody multimer either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0057] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean reducing risk of death, reducing the severity of the disorder, or prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0058] The term “Fc region” refers to a C-terminal region of an immunoglobulin heavy chain polypeptide. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The term includes native sequence of Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc region (using herein the numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The C-terminal lysine (Lys447) of the Fc region may or may not be present. One or more C-terminal residue(s) of Fc region may be absent or replaced by other amino acid substitution(s).

[0059] A “variant Fc” or “engineered Fc” refers to an Fc region that has been modified relative to a parent, native Fc region. A variant Fc may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising a modification or substitution at one or more amino acid positions in the Fc region. An engineered Fc also include modified glycosylation in the Fc region.

[0060] The terms “Fc receptor” or “FcR” refer to a receptor that binds to the Fc region. There are three main classes of Fc receptors: (1) FcyR which binds to IgG, (2) FcaR which binds to IgA, and (3) FcaR which binds to IgE. The FcyR family includes several members, such as Fcyl (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16A), and FcyRIIIB (CD16B). The Fey receptors differ in their affinity for IgG and also have different affinities for the IgG subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).

[0061] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, chimeric antibody, humanized antibody, human antibody, monoclonal antibody, single-domain antibody, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity. An antibody may include a full-length immunoglobulin molecule or a portion of a full-length immunoglobulin molecule that contains an antigen binding site that specifically binds an antigen of a target of interest. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species (e.g., human, or mouse). An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2 and scFv.

[0062] The term “antigen binding domain” or “binding domain” as used herein refers to one or more fragments of an antibody that retains the ability to specifically bind a target antigen. Examples of antigen binding domain includes, but not limited to, Fab (a monovalent fragment consisting of the VL, VH, CL and CHI domains), single-chain variable fragment (scFv), singledomain antibody (nanobody or VHH), a VL (light chain variable region), and a VH (heavy chain variable region).

[0063] A scFv is a fusion protein of the variable region of the heavy (VH) and light chains (VL) of an immunoglobulin that is connected by means of a linker peptide. The linker is usually short, about 10-25 amino acids in length. If flexibility is important, the linker will contain a significant number of glycines. If solubility is important, serines or threonines may be utilized in the linker. The linker may link the amino-terminus of the VH to the carb oxy -terminus of the VL, or the linker may link the carboxy -terminus of the VH to the amino-terminus of the VL.

[0064] A Fv fragment is an antibody fragment, and contains a complete antigen recognition and binding site. The six CDRs of both the variable regions (VH and VL) interact with each other to form an antigen-binding site. However, a variable region (or a half Fv, which contains only three antigen-specific CDRs) alone is also known to be able to recognize and bind to an antigen, although its affinity is lower than the affinity of the entire binding site.

[0065] A Fab fragment (also referred to as F(ab)) also contains a light chain constant region and heavy chain constant region CHI. For example, papain digestion of an antibody produces two kinds of fragments: an antigen-binding fragment, called a Fab fragment, containing the variable regions of a heavy chain and light chain, which serve as an antigen-binding domain; and the remaining portion, which is called an “Fc” because it is readily crystallized. A Fab' fragment is different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CHI region, which contains one or more cysteine residues from the hinge region of an antibody. A Fab' fragment is, however, structurally equivalent to Fab in that both are antigen-binding fragments which comprise the variable regions of a heavy chain and light chain. Herein, an antigen-binding fragment comprising the variable regions of a heavy chain and light chain which may serve as an antigen-binding domain, and which is equivalent to that obtained by papain digestion, is referred to as a “Fab-like antibody,” even when it is not identical to an antibody fragment produced by protease digestion. Fab'-SH is Fab' with one or more cysteine residues having free thiol groups in its constant region. A F(ab') fragment is produced by cleaving the disulfide bond between the cysteine residues in the hinge region of F(ab')2. Other chemically crosslinked antibody fragments are also known to those skilled in the art. Pepsin digestion of an antibody yields two fragments; one is a F(ab')2 fragment which comprises two antigen-binding domains, and the other is the remaining fragment (referred to as pFc'). Herein, an antibody fragment equivalent to that obtained by pepsin digestion is referred to as a “F(ab')2-like antibody” when it comprises two antigenbinding domains. Such antibody fragments can also be produced, for example, by genetic engineering.

[0066] A small, functional antibody known as a heavy-chain antibody (HCAb) was first reported in camelid serum in 1993. Unlike conventional antibodies with a heterotetrametric structure, camelid-derived HCAb is devoid of light-chain polypeptides and lacks the first constant domain (CHI) in heavy-chains. The antigen-binding fragment in HCAbs contains only one single-variable domain. This domain is termed VHH and is also known as a single-domain antibody (sdAb) or nanobody (Nb). Uniquely, the monomeric state of these single domain antibodies provides the ability to recognize and bind antigens independently. With a smaller biomolecule in the size range of 12-15 kDa and a higher affinity and stability, single domain antibodies are contemplated for use herein.

[0067] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human).

[0068] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. As used herein, the term “monoclonal antibody” refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, an antibody group wherein the antibodies constituting the group are homogeneous except for naturally occurring mutants that may exist in a small amount. Monoclonal antibodies are highly specific and interact with a single antigenic site. Furthermore, each monoclonal antibody targets a single antigenic determinant (epitope) on an antigen, as compared to common polyclonal antibody preparations that typically contain various antibodies against diverse antigenic determinants. In addition to their specificity, monoclonal antibodies are advantageous in that they are typically produced from hybridoma cultures not contaminated with other immunoglobulins.

[0069] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal X, Clustal W, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0070] The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein.

[0071] The term “variant” polypeptide refers to a polypeptide derived from the native protein but has substitution of one or more amino acids at one or more sites in the native protein, or deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C -terminal end of the native protein; or deletion or addition of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0072] Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.

[0073] Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). As described herein, by changing the amino acids, e.g., using directed mutations, the properties of the resulting molecules can be altered, including to decrease the binding affinity of an antibody.

[0074] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991); Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0075] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g, 81%-84%, at least 85%, e.g, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0076] “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0077] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, culture medium may represent less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0078] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0079] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0080] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0081] "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0082] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0083] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0084] The invention will now be illustrated by the following non-limiting Examples.

[0085] Example 1.

[0086] Making the monomers: Lower affinity monomers that form the basis of multimers may be isolated, e.g., from a specifically generated antigen library, either from immunization in a camelid or modified mouse, by selection from a naive cell library, or by selection from a synthetic sdAb library. They may also be created from known high-affinity sequences by selective amino acid mutation to alter the chemistry to decrease the binding affinity.

[0087] Making the multimers: Multimers can be designed with the addition of flexible protein linkers, generally from 3-30 amino acids in length, between monomers. These linkers form the chain that connects individual monomers together. DNA sequences can be created to encode the entire sequence of the multimer, including all monomers and all linkers, and a single multimer protein can be produced as a single unit from one DNA sequence. Multimers can be composed of repeats of a single monomer subunit, or a combination of monomers against multiple different antigen targets to create a multi-specific multimer. (also, see, e.g., Sargentini-Maier et al., (2019). Expert Review of Clinical Pharmacology, 12(6), 537-545; Beirnaert et al., Front Immunol. 2017 Jul 31;8:867; Muyldermans, S. (2021), A guide to: generation and design of nanobodies. FEBS J, 288: 2084-2102; Feng et al., J Nucl Med. 2023 Jan;64(l): 124-130; and Dekempeneer et al., Mol Pharm. 2019 Aug 5;16(8):3524-3533.

[0088] Conjugating emitter to sdAb: Emitters can be conjugated to the multimer in different ways, e.g., by a chemical reaction with amino groups in the monomer, but can also be sitespecific. Example 2.

[0089] Multimers are evaluated in terms of kinetic binding parameters by surface plasmon resonance and / or biolayer inferometry. Their relative binding will be assessed through binding of target-expressing cells in vitro, and their relative enrichment in tumor tissue will be assessed through quantification of the conjugated radioemitter in various tissues. They may also be assessed for their ability to be labeled with a halogen radioemitter and rate of internalization.

[0090] In certain embodiments, high and low affinity ranges for monomers are as follows:

[0091] High affinity Kd <=10 nM; Low affinity Kd > 10 nM. Example s

[0092] Examples of cancers that can be targeted using the monomers and multimers described herein include metastatic cancers, solid tumors, lung cancer, breast cancer, head and neck cancer, GI cancer, ovarian cancer, prostate cancer, bladder cancer. Glioblastoma.

[0093] Antigen targets for the monomers and multimers described herein include HER2, EGFR, HER3, TROP2, c-MET, PD-L1, PDGFRA, VEGFR2, PSMA, GPC3, EPCAM, FAP, B7H3, and Nectin-4.

Claims

07805.001W01CLAIMSWhat is claimed is:

1. An antibody multimer, wherein the antibody multimer comprises at least 2 single domain antibody monomers, wherein at least one of the single domain antibody monomers is conjugated to21'Astatine.

2. The antibody multimer of claim 1, wherein the antibody multimer comprises 2, 3, or 4 single domain antibodies.

3. The antibody multimer of claim 1 or 2, where each single domain antibody monomer of the multimer is conjugated to21 1Astatine.

4. The antibody multimer of any one of claims 1-3, wherein at least one of the single domain antibody monomers is a low affinity single domain antibody.

5. The antibody multimer of any one of claims 1-4, wherein each of the single domain antibody monomers is a low affinity single domain antibody.

6. The antibody multimer of any one of claims 1-5, wherein at least one of the monomers is targeted to HER2, EGFR, HER3, TROP2, c-MET, PD-L1, PDGFRA, VEGFR2, PSMA, GPC3, EPCAM, FAP, B7H3, or Nectin-4.

7. The antibody multimer of any one of claims 1-6, wherein the monomers are with connected flexible protein linkers between the monomers.

8. The antibody multimer of claim 7, wherein each linker is 3-30 amino acids in length.

9. The antibody multimer of claim 7 or 8, wherein the linkers form the chain that connects individual monomers together.

10. A DNA sequence encoding the antibody multimer described in any one of claims 1-9.07805.001W0111. A pharmaceutical composition comprising the antibody multimer of any one of the preceding claims and a pharmaceutically acceptable carrier.

12. An antibody multimer of any one of the preceding claims for the prophylactic or therapeutic treatment of cancer in a subject.

13. The use of an antibody multimer of any one of the preceding claims to prepare a medicament for the treatment of cancer in a subject.

14. The antibody multimer or use of claim 12 or 13, wherein the cancer is a metastatic cancer, a solid tumor, a lung cancer, a breast cancer, a head and neck cancer, a GI cancer, an ovarian cancer, a prostate cancer, a bladder cancer or a glioblastoma.

15. An antibody multimer of any one of the preceding claims for use in medical therapy.

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