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WO2025240969A1 - Molécules de liaison à caix et à caxii et leurs méthodes d'utilisation - Google Patents

Molécules de liaison à caix et à caxii et leurs méthodes d'utilisation

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Publication number
WO2025240969A1
WO2025240969A1 PCT/US2025/030045 US2025030045W WO2025240969A1 WO 2025240969 A1 WO2025240969 A1 WO 2025240969A1 US 2025030045 W US2025030045 W US 2025030045W WO 2025240969 A1 WO2025240969 A1 WO 2025240969A1
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WO
WIPO (PCT)
Prior art keywords
seq
binding molecule
bispecific
caxii
caix
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/030045
Other languages
English (en)
Inventor
Vincent Luca
David L. Morse
Srishti Singh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
H Lee Moffitt Cancer Center and Research Institute Inc
Original Assignee
H Lee Moffitt Cancer Center and Research Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by H Lee Moffitt Cancer Center and Research Institute Inc filed Critical H Lee Moffitt Cancer Center and Research Institute Inc
Publication of WO2025240969A1 publication Critical patent/WO2025240969A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • lymph node (SLN) biopsy is the standard of care. Most often, a sentinel lymph node biopsy (SLNB) is done, during which only a few lymph nodes are removed.
  • an axillary lymph node dissection which removes more lymph nodes, might be needed. Lymph node surgery is often done as part of the main surgery to remove the breast cancer, but sometimes it might be done as a separate operation. [0005] However, these biopsies are associated with morbidities as the whole axillary lymph nodes region is removed, with complications such as lymphedema, Cording or scar tissue which makes the underarm muscles stiff , loss of upper arm movements, nerve pain, numbness and in some cases leads to death.
  • a carbonic anhydrase IX (CAIX) binding molecule (such as, for example, an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin) comprising a complimentary determining region (CDR) 1 (C1), CDR2, and CDR3, wherein CDR1 comprises GTIXXXXXM (SEQ ID NO: 1) (such as, for example, GTIXXXYXM (SEQ ID NO: 7) or GTIXXLYXM (SEQ ID NO: 8) including, but not limited to GTIFGLYDM (SEQ ID NO: 9), GTISLLYIM (SEQ ID NO:
  • the CAIX binding molecule can comprise a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 9, 14, and 3, respectively (including, but not limited to SEQ ID NO: 18 and SEQ ID NO: 19); a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 10, 15, and 4, respectively (including, but not limited to SEQ ID NO: 20 and SEQ ID NO: 21); a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 11, 16, and 5, respectively (including, but not limited to SEQ ID NO: 22 and SEQ ID NO: 23); or a CDR1, CDR2, and CDR3 as set for the SEQ ID Nos: 12, 17, and 6, respectively (including, but not limited to SEQ ID NO: 24 and SEQ ID NO: 25).
  • a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 9, 14, and 3, respectively (including, but not limited to SEQ ID NO: 18 and SEQ ID NO: 19); a
  • CAXII carbonic anhydrase XII
  • a carbonic anhydrase XII (CAXII) binding molecule such as, for example, an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin
  • CDR1 ⁇
  • CDR1 ⁇ Attorney Docket No.10110-445WO1 comprises GXXFXXXXM (SEQ ID NO: 26) (such as, for example, GSXFXXXAM (SEQ ID NO: 34) including, but not limited to GTIFVYINM (SEQ ID NO: 35), GYIFDGLYM (SEQ ID NO: 36), GSIFAPYAM (SEQ ID NO: 37), GSTFWSNAM (SEQ ID NO: 38), or GSTFGVNAM (SEQ ID NO:
  • CAXII binding molecule comprising a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 35, 40, and 29, respectively (including, but not limited to SEQ ID NO: 45 and SEQ ID NO: 46); a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 36, 41, and 30, respectively (including, but not limited to SEQ ID NO: 47 and SEQ ID NO: 48); a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 37, 42, and 31, respectively (including, but not limited to SEQ ID NO: 49 and SEQ ID NO: 50); a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 38, 43, and 32, respectively (including, but not limited to SEQ ID NO: 51 and SEQ ID NO: 52); or a CDR1, CDR2, and CDR3 as set forth in SEQ ID Nos: 39, 44, and 33, respectively (including, but not limited to SEQ ID NO: 45
  • bispecific binding molecules such as, for example a bispecific nanobody, a bispecific T cell engager (BiTE), or a diabody
  • a bispecific binding molecule comprising a CAIX binding molecules of any preceding aspect and a CAXII binding molecule of any preceding aspect.
  • the CAIX binding molecule and CAXII binding molecule of the bispecific binding molecule can be joined by a linker (such as, for example, SEQ ID NO: 55 or SEQ ID NO: 56).
  • a linker such as, for example, SEQ ID NO: 55 or SEQ ID NO: 56.
  • bispecific binding molecules of any preceding aspect comprising about 80% similarity or more to SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
  • the bispecific binding molecule further comprises a detectable signal, but not limited to fluorophore, fluorescent dye (such as, for example, near-infrared fluorescent dye), and/or radioisotope.
  • a detectable signal but not limited to fluorophore, fluorescent dye (such as, for example, near-infrared fluorescent dye), and/or radioisotope.
  • a method of detecting regional lymph node metastasis including, but not limited to axillary lymph node metastasis) in a subject, the method including: a) administering to the subject bispecific binding molecule of any preceding aspect (including, but not limited to bispecific binding molecules further comprising a detectable ⁇ Attorney Docket No.10110-445WO1 signal); and b) detecting the bispecific binding molecule (including, but not limited to detecting the detectable signal); wherein presence of the bispecific binding molecule (as, for example, indicated by an presence or increase of the detectable signal) signifies the presence of a regional lymph
  • the subject has a solid tumor (including, but not limited to a breast cancer).
  • step b) occurs from about 3 hours to about 96 hours after step a).
  • Also disclosed herein are methods of detecting regional lymph node metastasis of any preceding aspect, further comprising c) removing one or more regional lymph nodes (including, but not limited to the removal of all regional lymph nodes) from the subject when the presence of the bispecific binding molecule (including when indicated by the presence of an increase of the detectable signal) is detected in one or more regional lymph nodes, and removing no regional lymph nodes when the absence of the bispecific binding molecule (including when indicated by the absence of an decrease of the detectable signal) is detected in all regional lymph nodes.
  • the removal of lymph nodes can be augmented, supplemented, or replaced by the administration of an anticancer therapy to the subject.
  • methods of treating, preventing, ameliorating, reducing, and/or curing regional lymph node metastasis including, but not limited to axillary lymph node metastasis
  • the method including: a) administering to the subject the bispecific binding molecule of any preceding aspect (including, but not limited to bispecific binding molecules further comprising a detectable signal); b) detecting the bispecific binding molecule (including, but not limited to detecting the detectable signal); wherein presence of the bispecific binding molecule (as, for example, indicated by an presence or increase of the detectable signal) signifies the presence of a regional lymph node metastasis and absence of the bispecific binding molecule (as, for example, indicated by an absence or a decrease of the detectable signal) signifies no regional lymph node metastasis; and c) removing one or more regional lymph nodes
  • FIGURES 1A-1D depict schematic and structural models of CAIX and CAXII.
  • FIGS. 1A-1B represent CAIX
  • FIGS. 1C-1D correspond to CAXII.
  • FIGURE 2 depicts purification of nanobodies. Elution profile of the CAIX, and CAXII nanobodies following purification on a size-exclusion column, with inset SDS-PAGE gel of individual fractions.
  • FIGURE 3 depicts nanobody binding affinity for CAIX and CAXII determined using surface plasmon resonance (SPR).
  • FIGURE 4 depicts mRNA expression of CAIX and CAXII in MCF7 and MDAmb231 parental breast cancer cell lines. Cells were grown in normoxic condition prior to mRNA extraction and qRT-PCR. All data are normalized to GAPDH.
  • FIGURE 5 depicts expression of CAIX and CAXII in MDAmb231 and MCF-7 cells. Confocal images of cells incubated with CAIX or CAXII nanobodies conjugated to AlexaFluor 680 (Invitrogen) or IRDye800CW (Licor) dyes via NHS ester linkage. Cells were fixed and later incubated with 3 ⁇ g of each nanobody and images were acquired at 40X magnification.
  • FIGURE 6 depicts uptake of monovalent CAIX.2 & CAXII.3 and CAIX-CAXII Bispecific nanobody conjugates in MCF7 and MDAmb231 parental cells. Confocal images of cells incubated with bispecific nb conjugated to AlexaFluor 680 dye via maleimide linkage. Cells were fixed, incubated with nanobody conjugates, and images were acquired at 40x magnification. Panel 1(a) shows MCF7 stained with CAXII.3 nb_680nm, and panel 1(b) shows merged image including WGA (membrane marker) and DAPI (nuclear marker).
  • WGA membrane marker
  • DAPI nuclear marker
  • Panel 2(a) shows MDAmb231 stained with CAIX.2 nb_680nm.
  • Panel 3 shows MCF7 with Bispecific nb_680nm, and merged image including WGA & DAPI. This is compared with panel 4, which shows bispecific nb_680nm and blocked with 20-fold excess unlabeled nb.
  • panels 5 and 6 show MDAmb231 bispecific nb_680nm, blocked with 20-fold excess unlabeled nb. 5 ⁇ Attorney Docket No.10110-445WO1 Blocking studies demonstrates specific binding of labeled nb while controlling for non-specific interactions.
  • FIGURES 7A-7D depict hematoxylin & Eosin staining of MCF7 mammary fat pad tumor xenograft (FIG. 7A) and MDAmb231 mammary fat pad tumor xenograft (FIG. 7B).
  • FIGS.7C-7D show immunohistochemistry staining of MCF7 mammary fat pad tumor stained with CA12 rabbit antibody (FIG. 7C) and MDAmb231 mammary fat pad tumor stained with CA9 rabbit antibody (FIG.7D) (20x magnification).
  • FIGURE 8 depicts a MFP mouse model with MCF7 cells and injected with 5 ⁇ g Bispecific Nb_680nm via tail vein.
  • FIGURE 9 shows a MFP mouse model with MDAmb231 cells, injected with 5 ⁇ g Bispecific Nb_680nm via tail vein.
  • Panel 2(a) shows fluorescence image immediately after injection, compared with panel 2(b) where the mouse was imaged 48 h post injection. Uptake and clearance of nanobody was observed over the course of 96 h, with graph representing the clearance in tumour and blood circulation during the time course.
  • FIGURE 10 depicts the generation and characterization of additional non- humanized nanobodies.
  • FIGURE 11 depicts binding analysis of the individual nanobodies characterized in FIG.10.
  • FIGURE 12 depicts binding analysis of the bispecific nanobodies characterized in FIG.10.
  • FIGURE 13 depicts the generation and characterization of additional humanized nanobodies.
  • FIGURE 14 depicts binding analysis of the nanobodies characterized in FIG.13. DETAILED DESCRIPTION
  • each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater ⁇ Attorney Docket No.10110-445WO1 than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g.
  • ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub- ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein.
  • an “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material.
  • an “effective amount” of a monomer refers to an amount that is sufficient to achieve ⁇ Attorney Docket No.10110-445WO1 the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity.
  • wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.
  • therapeutically effective amount refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts.
  • the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications.
  • a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • a response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or ⁇ Attorney Docket No.10110-445WO1 medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • the amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. [0040] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.
  • the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
  • the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human).
  • Subject can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
  • the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and/or physiological effect.
  • the effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof.
  • the effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition.
  • treatment can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions.
  • treatment as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.
  • Those in need of treatment can include those already with the disorder and/or those in which the disorder is to be prevented.
  • the term "treating" can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • dose can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.
  • therapeutic can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.
  • nucleic acid and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). [0048] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins.
  • a polypeptide and/or protein is defined as a polymer of amino acids, typically of length ⁇ 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability. In this case, the functional fragment would retain the ability to perform as a telomerase.
  • exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or ⁇ Attorney Docket No.10110-445WO1 99% sequence identity to any amino acid sequence disclosed herein.
  • Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to a reference peptide, polypeptide, or protein.
  • amino acid includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), va
  • amino acid residue also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ⁇ -alanine, ⁇ -Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Me
  • the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.
  • the peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties.
  • Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group
  • glycation Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).
  • polysialylation e.g., the addition of polysialic acid
  • glypiation e.g., glycosylphosphatidylinositol (GPI) anchor formation
  • hydroxylation e.g., hydroxylation
  • iodination e.g., of thyroid hormones
  • phosphorylation e.g., the addition of a phosphat
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence.
  • a deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides.
  • a deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 ⁇ -terminal or 3 ⁇ -terminal truncation or both of a reference polynucleotide).
  • variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein.
  • a “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence.
  • a fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively.
  • a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and/or the C-terminal region of a polypeptide or the 5 ⁇ -terminal region and/or the 3 ⁇ terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.
  • variants comprising insertions or additions relative to a reference sequence are contemplated herein.
  • the words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or ⁇ Attorney Docket No.10110-445WO1 nucleotides.
  • An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.
  • Fusion proteins and fusion polynucleotides also are contemplated herein.
  • a “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof.
  • the heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini.
  • a fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein).
  • the heterologous protein(s), once part of the fusion protein may each be referred to herein as a “portion”, “region” or “signal” of the fusion protein. [0057] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein.
  • substantially isolated or purified refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • Antibodies Generally: The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. 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 or fragments thereof.
  • the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • IgA immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgG immunoglobulins
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present ⁇ Attorney Docket No.10110-445WO1 in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies.
  • disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S.
  • Patent No.5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • fragments of antibodies which maintain binding activity are included ⁇ Attorney Docket No.10110-445WO1 within the meaning of the term “antibody or fragment thereof.”
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • conjugates of antibody fragments and antigen binding proteins single chain antibodies.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody or “antibodies” can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Human antibodies can be prepared using any technique.
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
  • CDRs complementarity determining regions
  • donor non-human antibody molecule
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
  • Fc antibody constant region
  • humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent No.4,816,567 (Cabilly et al.), U.S. Patent No.5,565,332 (Hoogenboom et al.), U.S.
  • the disclosed antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • a nucleic acid preparation e.g., DNA or RNA
  • the delivery of the nucleic acid can be by any means, as disclosed herein, for example.
  • CAIX BINDING MOLECULES [0072]
  • a carbonic anhydrase IX (CAIX) binding molecule including a complimentary determining region (CDR1), CDR2, and CDR3, wherein: CDR1 includes GTIXXXXXM (SEQ ID NO: 1); CDR2 includes EXVAXIXXGXXTXY (SEQ ID NO: 2); and CDR3 includes any one of SEQ ID NOs: 3-6 wherein X can be independently selected at each occurrence from any amino acid.
  • CDR1 can include GTIXXXYXM (SEQ ID NO: 7).
  • CDR1 can include GTIXXLYXM (SEQ ID NO: 8).
  • CDR1 can include any one of SEQ ID NOs: 9-12.
  • CDR2 can include EXVAAIXXGTXTXY (SEQ ID NO: 13).
  • CDR2 can include any one of SEQ ID NOs: 14-17. TABLE 1. CDRs for CAIX binding molecules. ⁇ Attorney Docket No.10110-445WO1 [0075]
  • CDR1 can include GTIFGLYDM (SEQ ID NO: 9); CDR2 can include ELVAAIADGTSTYY (SEQ ID NO: 14); and CDR3 can include AAPYPYGSYYWGSHSY (SEQ ID NO: 3).
  • the CAIX binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 18 or SEQ ID NO: 19.
  • the CAIX binding molecule can include SEQ ID NO: 18 or SEQ ID NO: 19.
  • CAIX binding molecule can consist of SEQ ID NO: 18 or SEQ ID NO: 19.
  • CDR1 can include GTISLLYIM (SEQ ID NO: 10);
  • CDR2 can include EFVAAIDSGTNTNY (SEQ ID NO: 15); and
  • CDR3 can include AAPRFNVGTDGYYHVY (SEQ ID NO: 4).
  • the CAIX binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 20 or SEQ ID NO: 21.
  • the CAIX binding molecule can include SEQ ID NO: 20 or SEQ ID NO: 21.
  • CAIX binding molecule can consist of SEQ ID NO: 20 or SEQ ID NO: 21.
  • CDR1 can include GSIFRPPTM (SEQ ID NO: 11);
  • CDR2 can include EFVATIAEGTNTYY (SEQ ID NO: 16); and
  • CDR3 can include AVGAYDGWDYIVY (SEQ ID NO: 5).
  • the CAIX binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% ⁇ Attorney Docket No.10110-445WO1 or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 22 or SEQ ID NO: 23.
  • about 80% similarity or more e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more,
  • the CAIX binding molecule can include SEQ ID NO: 22 or SEQ ID NO: 23. In some aspects, the CAIX binding molecule can consist of SEQ ID NO: 22 or SEQ ID NO: 23.
  • CDR1 can include GTISTYYDM (SEQ ID NO: 12); CDR2 can include EFVASISAGSSTYY (SEQ ID NO: 17); and CDR3 can include AATYWYIEYSIVSHQY (SEQ ID NO: 6).
  • the CAIX binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 24 or SEQ ID NO: 25.
  • the CAIX binding molecule can include SEQ ID NO: 24 or SEQ ID NO: 25.
  • the CAIX binding molecule can consist of SEQ ID NO: 24 or SEQ ID NO: 25. TABLE 2. Non-humanized CAIX binding molecules. TABLE 3. Humanized CAIX binding molecules. ⁇ Attorney Docket No.10110-445WO1 [0079]
  • the CAIX binding molecule can be an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin.
  • Example toxins which may be used to generate immunotoxins may include, but are not limited to, Abrin, Conotoxins Diacetoxyscirpenol Bovine spongiform encephalopathy agent, Ricin, Saxitoxin, Tetrodotoxin, epsilon toxin, Botulinum neurotoxins, Shigatoxin, Staphylococcal enterotoxins, T-2 toxin, Diphtheria toxin, Tetanus toxoid, and pertussis toxin.
  • CAXII BINDING MOLECULES [0080]
  • a carbonic anhydrase XII (CAXII) binding molecule including a complimentary determining region (CDR1), CDR2, and CDR3, wherein: CDR1 can include GXXFXXXXM (SEQ ID NO: 26); CDR2 can include EXVAXIXXGXXTXY (SEQ ID NO: 27) or ELVAXIXXSGGXTYY (SEQ ID NO: 28); and CDR3 can include any one of SEQ ID NOs: 29-33; wherein X can be independently selected at each occurrence from any amino acid.
  • CDR1 can include GXXFXXXXM (SEQ ID NO: 26)
  • CDR2 can include EXVAXIXXGXXTXY (SEQ ID NO: 27) or ELVAXIXXSGGXTYY (SEQ ID NO: 28)
  • CDR3 can include any one of SEQ ID NOs
  • CDR1 can include GSXFXXXAM (SEQ ID NO: 34). In some aspects, CDR1 can include any one of SEQ ID NOs: 35-39. [0082] In some aspects, CDR2 can include any one of SEQ ID NOs: 40-44. ⁇ Attorney Docket No.10110-445WO1 TABLE 4. CDRs for CAXII binding molecules. [0083] In some aspects, CDR1 can include GTIFVYINM (SEQ ID NO: 35); CDR2 can include EFVAAIGKGGSTNY (SEQ ID NO: 40); and CDR3 can include AAASGYARAYGY (SEQ ID NO: 29).
  • the CAXII binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 45 or SEQ ID NO: 46.
  • the CAXII binding molecule can include SEQ ID NO: 45 or SEQ ID NO: 46.
  • CAXII binding molecule can consist of SEQ ID NO: 45 or SEQ ID NO: 46. ⁇ Attorney Docket No.10110-445WO1 [0084]
  • CDR1 can include GYIFDGLYM (SEQ ID NO: 36);
  • CDR2 can include ELVAGIADGTTTYY (SEQ ID NO: 41); and
  • CDR3 can include AAWSVDLYDSVAILGY (SEQ ID NO: 30).
  • the CAXII binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 47 or SEQ ID NO: 48.
  • the CAXII binding molecule can include SEQ ID NO: 47 or SEQ ID NO: 48.
  • CAXII binding molecule can consist of SEQ ID NO: 47 or SEQ ID NO: 48.
  • CDR1 can include GSIFAPYAM (SEQ ID NO: 37);
  • CDR2 can include ELVAAITGSGGS (SEQ ID NO: 42); and
  • CDR3 can include NAEPYWLPTRKYYY (SEQ ID NO: 31).
  • the CAXII binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 49 or SEQ ID NO: 50.
  • the CAXII binding molecule can include SEQ ID NO: 49 or SEQ ID NO: 50.
  • CAXII binding molecule can consist of SEQ ID NO: 49 or SEQ ID NO: 50.
  • CDR1 can include GSTFWSNAM (SEQ ID NO: 38);
  • CDR2 can include ELVAAISWSGGRTYY (SEQ ID NO: 43); and
  • CDR3 can include ATDYSD (SEQ ID NO: 32).
  • the CAXII binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 51 or SEQ ID NO: 52.
  • the CAXII binding molecule can include SEQ ID NO: 51 or SEQ ID NO: 52.
  • CAXII binding molecule can consist of SEQ ID NO: 51 or SEQ ID NO: 52. ⁇ Attorney Docket No.10110-445WO1 [0087]
  • CDR1 can include GSTFGVNAM (SEQ ID NO: 39);
  • CDR2 can include ELVATISSSGGSTYY (SEQ ID NO: 44); and
  • CDR3 can include AATHWSSRRSDAYDS (SEQ ID NO: 33).
  • the CAXII binding molecule can further include about 80% similarity or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) to SEQ ID NO: 53 or SEQ ID NO: 54.
  • the CAXII binding molecule can include SEQ ID NO: 53 or SEQ ID NO: 54.
  • the CAXII binding molecule can consist of SEQ ID NO: 53 or SEQ ID NO: 54. TABLE 5. Non-humanized CAXII binding molecules. TABLE 6. Humanized CAXII binding molecules. ⁇ Attorney Docket No.10110-445WO1 [0088]
  • the CAXII binding molecule can be an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin.
  • Example toxins which may be used to generate immunotoxins may include, but are not limited to, Abrin, Conotoxins Diacetoxyscirpenol Bovine spongiform encephalopathy agent, Ricin, Saxitoxin, Tetrodotoxin, epsilon toxin, Botulinum neurotoxins, Shigatoxin, Staphylococcal enterotoxins, T-2 toxin, Diphtheria toxin, Tetanus toxoid, and pertussis toxin.
  • BISPECIFIC BINDING MOLECULES [0089]
  • a bispecific binding molecule comprising any of the disclosed CAIX binding molecules and any of the CAXII binding molecules.
  • the CAIX binding molecule and the CAXII binding molecule can be joined by a linker.
  • the linker can include GSGSGSGSGSGS (SEQ ID NO: 55) or GGGGSGGGGSGGGGS (SEQ ID NO: 56).
  • Any combination of CAIX binding molecules, CAXII binding molecules, and optional linkers are considered herein.
  • TABLE 7 and TABLE 8 provide example ⁇ Attorney Docket No.10110-445WO1 combinations, and TABLE 9 and TABLE 10 provide example sequences of bispecific binding molecules. TABLE 7. Non-humanized bispecific binding molecules. TABLE 8. Humanized bispecific binding molecules. ⁇ Attorney Docket No.10110-445WO1 TABLE 9.
  • Example non-humanized bispecific binding molecules ⁇ Attorney Docket No.10110-445WO1 TABLE 10.
  • Example humanized bispecific binding molecules can be a bispecific nanobody, a bispecific T cell engager (BiTE), or a diabody.
  • the bispecific binding molecule can further include a detectable signal.
  • a detectable signal can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence.
  • Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorometric substrates (e.g., horseradish peroxidase).
  • fluorescent dyes also known herein as fluorochromes and fluorophores
  • enzymes that react with colorometric substrates e.g., horseradish peroxidase.
  • colorometric substrates e.g., horseradish peroxidase.
  • the use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts.
  • each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
  • Fluorophores are compounds or molecules that luminesce.
  • fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength.
  • Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6- CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Ac
  • the detectable signal can be a near-infrared fluorescent dye (e.g., IRDye 800CW, Cy7, Cy7.5, NIR CF750/770/790, DyLight 800 or Alexa Fluor 750).
  • the detectable signal can be a radioisotope (such as, for example, a gamma-emitting radioisotope or radionuclide).
  • a modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation.
  • radionuclides examples include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon- ⁇ Attorney Docket No.10110-445WO1 14, nitrogen-13, fluorine-18, gallium-67.
  • the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker.
  • radionuclides include, but are not limited to, Tc- 99m, Re-186, Ga-67, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62.
  • METHODS [0097]
  • a method of detecting regional lymph node metastasis in a subject including: a) administering to the subject any of the disclosed bispecific binding molecules; and b) detecting the bispecific binding molecule; wherein presence of the bispecific binding molecule indicates regional lymph node metastasis and absence of the bispecific binding molecule indicates no regional lymph node metastasis.
  • the method can further include c) removing one or more regional lymph nodes from the subject when the presence of the bispecific binding molecule is detected in one or more regional lymph nodes, and removing no regional lymph nodes when the absence of the bispecific binding molecule is detected in all regional lymph nodes.
  • the bispecific binding molecule can be any of the disclosed bispecific binding molecules which include a detectable signal, and step b) can include detecting the detectable signal.
  • a method of treating regional lymph node metastasis including: a) administering to the subject any of the disclosed bispecific binding molecules; b) detecting the bispecific binding molecule, wherein presence of the bispecific binding molecule indicates regional lymph node metastasis and absence of the bispecific binding molecule indicates no regional lymph node metastasis; and c) removing one or more regional lymph nodes from the subject when the presence of the bispecific binding molecule is detected in one or more regional lymph nodes.
  • the bispecific binding molecule can be any of the disclosed bispecific binding molecules which include a detectable signal, and step b) can include detecting the detectable signal.
  • the subject can have a solid tumor, and step a) can include injecting the bispecific binding molecule into said solid tumor.
  • the subject can have bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non-small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative ⁇ Attorney Docket No.10110-445WO1 breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematop
  • step b) can occur about 3 hours or more after step a) (e.g., about 4 hours or more, about 5 hours or more, about 6 hours or more, about 7 hours or more, about 8 hours or more, about 10 hours or more, about 12 hours or more, about 16 hours or more, about 20 hours or more, about 24 hours or more, about 28 hours or more, about 32 hours or more, about 36 hours or more, about 40 hours or more, about 44 hours or more, about 48 hours or more, about 52 hours or more, about 56 hours or more, about 60 hours or more, about 64 hours or more, about 68 hours or more, about 72 hours or more, about 76 hours or more, about 80 hours or more, about 84 hours or more, about 88 hours or more, about 92 hours or more, about 96 hours or more).
  • step a) e.g., about 4 hours or more, about 5 hours or more, about 6 hours or more, about 7 hours or more, about 8 hours or more, about 10 hours or more, about 12 hours or more, about 16 hours or
  • step b) can occur about 96 hours or less after step a) (e.g., about 92 hours or less, about 88 hours or less, about 84 hours or less, about 80 hours or less, about 76 hours or less, about 72 hours or less, about 68 hours or less, about 64 hours or less, about 60 hours or less, about 56 hours or less, about 52 hours or less, about 48 hours or less, about 44 hours or less, about 40 hours or less, about 36 hours or less, about 32 hours or less, about 28 hours or less, about 24 hours or less, about 20 hours or less, about 16 hours or less, about 12 hours or less, about 10 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less).
  • step a) e.g., about 92 hours or less, about 88 hours or less, about 84 hours or less, about 80 hours or less, about 76 hours or less, about 72 hours or less, about 68 hours or less
  • Step b) can occur any duration after step a) ranging from any of the minimum values described above to any of the maximum values described above.
  • step b) can occur from about 3 hours to about 96 hours after step a) (e.g., from about 4 hours to about 92 hours, from about 5 hours to about 88 hours, from about 6 hours to about 84 hours, from about 7 hours to about 80 hours, from about 8 hours to about 76 hours, from about 10 hours to about 72 hours, from about 12 hours to about 68 hours, from about 16 hours to about 64 hours, from about 20 hours to about 60 hours, from about 24 hours to about 56 hours, from about 28 hours to about 52 hours, from about 32 hours to about 48 hours, from about 36 hours to about 44 hours, from about 3 hours to about 40 hours, from about 4 hours to about 36 hours, from about 5 hours to about 32 hours, from about 6 hours to about 28 hours, from about 7 hours to about 24 hours, from about 8 hours to about 20 hours, from about 10 hours to about 16 hours, from about 40 hours to
  • step c) can include removing only regional lymph nodes which exhibit the presence of the bispecific binding molecule and not removing regional lymph nodes which exhibit the absence of the bispecific binding molecule. In other aspects, step c) can include removing all regional lymph nodes. [0103] In some aspects, steps b) and c) can occur simultaneously (e.g., such that the presence of the bispecific binding molecule guides a surgical procedure to remove one or more regional lymph nodes). [0104] In some aspects, the method can further include administering an anticancer therapy to the subject.
  • Anticancer therapies known in the art include, but are not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydro
  • the treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD- 1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, ⁇ Attorney Docket No.10110-445WO1 omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and
  • Axillary lymph node (ALN) biopsy is a surgical procedure that uses either nonspecific lymphatic mapping and biopsy, or, in patients that present metastases, the entire ALN basin is removed.
  • Axillary lymph node (ALN) biopsy is a surgical procedure that uses either nonspecific lymphatic mapping and biopsy, or, in patients that present metastases, the entire ALN basin is removed.
  • CAIX and CAXII targets are expressed in 100% of breast cancer lymph node metastases but are not expressed in normal unaffected lymph nodes or surrounding tissues.
  • Nanobody conjugates were used to determine the in vitro expression and uptake of CAIX and CAXII in the cell lines using fluorescence microscopy via live cell imaging and ICC.Female nude mice ⁇ Attorney Docket No.10110-445WO1 were injected with MCF7 and MDAmb231 cells into the mammary fat pad (MFP) and injected with the bispecific nanobody conjugate via tail vein to determine optimal concentration, uptake, and clearance. In addition, blocking studies were performed in-vivo and in-vitro to determine target specificity. [0109] MCF-7 cells have high CAXII expression and lower CAXI. MDA-mb-231 cells have high CAIX and very low CAXII.
  • FIGS. 1A-1D depict schematic and structural models of CAIX and CAXII.
  • FIG. 2 depicts results of nanobody characterization.
  • FIG. 3 depicts nanobody binding affinity for CAIX and CAXII.
  • FIG. 4 depicts in vitro mRNA expression.
  • FIG. 5 depicts in vitro protein expression using CAIX and CAXII nanobodies.
  • FIG.6 depicts in vitro protein expression using bispecific nanobody conjugates.
  • FIG.7, FIG.8, and FIG.9 depict mammary fat pad mouse models with MCF7 and MDAmb231 breast cancer parental cells. Bispecific Nb_680nm was injected via tail vein and imaged using Newton 7.0 optical imaging system. Uptake and clearance of nanobody was observed over the course of 0-120 hours.
  • FIG.10 depicts the generation of additional bispecific non-humanized nanobodies.
  • FIG.11 and FIG.12 depict the binding analysis of these individual nanobodies and a bispecific nanobody, respectively.
  • FIG. 13 depicts the humanization of these nanobodies
  • FIG. 14 depicts the binding analysis.
  • EXAMPLE ASPECTS [0114]
  • Example 1 A carbonic anhydrase IX (CAIX) binding molecule comprising a complimentary determining region (CDR1), CDR2, and CDR3, wherein: CDR1 comprises GTIXXXXXM (SEQ ID NO: 1); CDR2 comprises EXVAXIXXGXXTXY (SEQ ID NO: 2); and CDR3 comprises AAPYPYGSYYWGSHSY (SEQ ID NO: 3), AAPRFNVGTDGYYHVY (SEQ ID NO: 4), AVGAYDGWDYIVY (SEQ ID NO: 5), or AATYWYIEYSIVSHQY (SEQ ID NO: 6); wherein X is independently selected at each occurrence from any amino acid.
  • Example 2 The CAIX binding molecule of any examples herein, particularly Example 1, wherein CDR1 comprises GTIXXXYXM (SEQ ID NO: 7).
  • Example 3 The CAIX binding molecule of any examples herein, particularly Example 2, wherein CDR1 comprises GTIXXLYXM (SEQ ID NO: 8).
  • Example 4 The CAIX binding molecule of any examples herein, particularly Examples 1-3, wherein CDR1 comprises GTIFGLYDM (SEQ ID NO: 9), GTISLLYIM (SEQ ID NO: 10), GSIFRPPTM (SEQ ID NO: 11), or GTISTYYDM (SEQ ID NO: 12).
  • Example 5 The CAIX binding molecule of any examples herein, particularly Examples 1-4, wherein CDR2 comprises EXVAAIXXGTXTXY (SEQ ID NO: 13).
  • Example 6 The CAIX binding molecule of any examples herein, particularly Examples 1-5, wherein CDR2 comprises ELVAAIADGTSTYY (SEQ ID NO: 14), EFVAAIDSGTNTNY (SEQ ID NO: 15), EFVATIAEGTNTYY (SEQ ID NO: 16), or EFVASISAGSSTYY (SEQ ID NO: 17).
  • Example 7 The CAIX binding molecule of any examples herein, particularly Examples 1-6, wherein: CDR1 comprises GTIFGLYDM (SEQ ID NO: 9); CDR2 comprises ELVAAIADGTSTYY (SEQ ID NO: 14); and CDR3 comprises AAPYPYGSYYWGSHSY (SEQ ID NO: 3).
  • Example 8 The CAIX binding molecule of any examples herein, particularly Example 7, further comprising about 80% similarity or more to SEQ ID NO: 18 or SEQ ID NO: 19.
  • Example 9 The CAIX binding molecule of any examples herein, particularly Examples 1-6, wherein: CDR1 comprises GTISLLYIM (SEQ ID NO: 10); CDR2 comprises EFVAAIDSGTNTNY (SEQ ID NO: 15); and CDR3 comprises AAPRFNVGTDGYYHVY (SEQ ID NO: 4). [0123] Example 10: The CAIX binding molecule of any examples herein, particularly Example 9, further comprising about 80% similarity or more to SEQ ID NO: 20 or SEQ ID NO: 21.
  • Example 11 The CAIX binding molecule of any examples herein, particularly Examples 1-6, wherein: CDR1 comprises GSIFRPPTM (SEQ ID NO: 11); CDR2 comprises EFVATIAEGTNTYY (SEQ ID NO: 16); and CDR3 comprises AVGAYDGWDYIVY (SEQ ID NO: 5).
  • Example 12 The CAIX binding molecule of any examples herein, particularly Example 11, further comprising about 80% similarity or more to SEQ ID NO: 22 or SEQ ID NO: 23.
  • Example 13 The CAIX binding molecule of any examples herein, particularly Examples 1-6, wherein: CDR1 comprises GTISTYYDM (SEQ ID NO: 12); CDR2 comprises EFVASISAGSSTYY (SEQ ID NO: 17); and CDR3 comprises AATYWYIEYSIVSHQY (SEQ ID NO: 6). [0127]
  • Example 14 The CAIX binding molecule of any examples herein, particularly Example 13, further comprising about 80% similarity or more to SEQ ID NO: 24 or SEQ ID NO: 25.
  • Example 15 The CAIX binding molecule of any examples herein, particularly Examples 1-14, wherein the binding molecule is an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin.
  • the binding molecule is an antibody, a nanobody, a bispecific nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin.
  • Example 16 A carbonic anhydrase XII (CAXII) binding molecule comprising a complimentary determining region (CDR1), CDR2, and CDR3, wherein: CDR1 comprises GXXFXXXXM (SEQ ID NO: 26); CDR2 comprises EXVAXIXXGXXTXY (SEQ ID NO: 27) or ELVAXIXXSGGXTYY (SEQ ID NO: 28); and CDR3 comprises AAASGYARAYGY (SEQ ID NO: 29), AAWSVDLYDSVAILGY (SEQ ID NO: 30), NAEPYWLPTRKYYY (SEQ ID NO: 31), ATDYSD (SEQ ID NO: 32), or AATHWSSRRSDAYDS (SEQ ID NO: 33); wherein X is independently selected at each occurrence from any amino acid.
  • CDR1 comprises GXXFXXXXM (SEQ ID NO: 26)
  • CDR2 comprises EXVAXIXXGXTXY (
  • Example 17 The CAXII binding molecule of any examples herein, particularly Example 16, wherein CDR1 comprises GSXFXXXAM (SEQ ID NO: 34).
  • Example 18 The CAXII binding molecule of any examples herein, particularly Examples 16-17, wherein CDR1 comprises GTIFVYINM (SEQ ID NO: 35), GYIFDGLYM (SEQ ID NO: 36), GSIFAPYAM (SEQ ID NO: 37), GSTFWSNAM (SEQ ID NO: 38), or GSTFGVNAM (SEQ ID NO: 39).
  • Example 19 The CAXII binding molecule of any examples herein, particularly Examples 16-18, wherein CDR2 comprises EFVAAIGKGGSTNY (SEQ ID NO: 40), ELVAGIADGTTTYY (SEQ ID NO: 41), ELVAAITGSGGS (SEQ ID NO: 42), ELVAAISWSGGRTYY (SEQ ID NO: 43), or ELVATISSSGGSTYY (SEQ ID NO: 44).
  • CDR2 comprises EFVAAIGKGGSTNY (SEQ ID NO: 40), ELVAGIADGTTTYY (SEQ ID NO: 41), ELVAAITGSGGS (SEQ ID NO: 42), ELVAAISWSGGRTYY (SEQ ID NO: 43), or ELVATISSSGGSTYY (SEQ ID NO: 44).
  • Example 20 The CAXII binding molecule of any examples herein, particularly Examples 16-19, wherein: CDR1 comprises GTIFVYINM (SEQ ID NO: 35); CDR2 comprises EFVAAIGKGGSTNY (SEQ ID NO: 40); and CDR3 comprises AAASGYARAYGY (SEQ ID NO: 29). ⁇ Attorney Docket No.10110-445WO1 [0134]
  • Example 21 The CAXII binding molecule of any examples herein, particularly Example 20, further comprising about 80% similarity or more to SEQ ID NO: 45 or SEQ ID NO: 46.
  • Example 22 The CAXII binding molecule of any examples herein, particularly Examples 16-19, wherein: CDR1 comprises GYIFDGLYM (SEQ ID NO: 36); CDR2 comprises ELVAGIADGTTTYY (SEQ ID NO: 41); and CDR3 comprises AAWSVDLYDSVAILGY (SEQ ID NO: 30).
  • Example 23 The CAXII binding molecule of any examples herein, particularly Example 22, further comprising about 80% similarity or more to SEQ ID NO: 47 or SEQ ID NO: 48.
  • Example 24 The CAXII binding molecule of any examples herein, particularly Examples 16-19, wherein: CDR1 comprises GSIFAPYAM (SEQ ID NO: 37); CDR2 comprises ELVAAITGSGGS (SEQ ID NO: 42); and CDR3 comprises NAEPYWLPTRKYYY (SEQ ID NO: 31).
  • Example 25 The CAXII binding molecule of any examples herein, particularly Example 24, further comprising about 80% similarity or more to SEQ ID NO: 49 or SEQ ID NO: 50.
  • Example 26 The CAXII binding molecule of any examples herein, particularly Examples 16-19, wherein: CDR1 comprises GSTFWSNAM (SEQ ID NO: 38); CDR2 comprises ELVAAISWSGGRTYY (SEQ ID NO: 43); and CDR3 comprises ATDYSD (SEQ ID NO: 32). [0140]
  • Example 27 The CAXII binding molecule of any examples herein, particularly Example 26, further comprising about 80% similarity or more to SEQ ID NO: 51 or SEQ ID NO: 52.
  • Example 28 The CAXII binding molecule of any examples herein, particularly Examples 16-19, wherein: CDR1 comprises GSTFGVNAM (SEQ ID NO: 39); CDR2 comprises ELVATISSSGGSTYY (SEQ ID NO: 44); and CDR3 comprises AATHWSSRRSDAYDS (SEQ ID NO: 33).
  • Example 29 The CAXII binding molecule of any examples herein, particularly Example 28, further comprising about 80% similarity or more to SEQ ID NO: 53 or SEQ ID NO: 54.
  • Example 30 The CAXII binding molecule of any examples herein, particularly Examples 16-29, wherein the binding molecule is an antibody, a nanobody, a bispecific ⁇ Attorney Docket No.10110-445WO1 nanobody, an scFv, a Fab, a chimeric antigen receptor (CAR), a diabody, a bispecific T cell engager (BiTE), or an immunotoxin.
  • Example 31 A bispecific binding molecule comprising the CAIX binding molecule of any examples herein, particularly Examples 1-15 and the CAXII binding molecule of any examples herein, particularly Examples 16-30.
  • Example 32 The bispecific binding molecule of any examples herein, particularly Example 31, wherein the CAIX binding molecule and the CAXII binding molecule are joined by a linker.
  • Example 33 The bispecific binding molecule of any examples herein, particularly Example 32, wherein the linker comprises GSGSGSGSGS (SEQ ID NO: 55) or GGGGSGGGGSGGGGS (SEQ ID NO: 56).
  • Example 34 The bispecific binding molecule of any examples herein, particularly Examples 31-33, further comprising about 80% similarity or more to SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
  • Example 35 The bispecific binding molecule of any examples herein, particularly Examples 31-34, wherein the binding molecule is a bispecific nanobody, a bispecific T cell engager (BiTE), or a diabody.
  • Example 36 The bispecific binding molecule of any examples herein, particularly Examples 31-35, further comprising a detectable signal.
  • Example 37 The bispecific binding molecule of any examples herein, particularly Example 36, wherein the detectable signal is a fluorophore or fluorescent dye.
  • Example 38 The bispecific binding molecule of any examples herein, particularly Example 37, wherein the fluorophore or fluorescent dye is a near-infrared fluorescent dye.
  • Example 39 The bispecific binding molecule of any examples herein, particularly Example 36, wherein the detectable signal is a radioisotope.
  • Example 40 The bispecific binding molecule of any examples herein, particularly Example 39, wherein the detectable signal is Ga-67.
  • Example 41 A method of detecting regional lymph node metastasis in a subject, the method comprising: a) administering to the subject the bispecific binding molecule of any examples herein, particularly Examples 31-40; and b) detecting the bispecific binding molecule; wherein presence of the bispecific binding molecule indicates regional lymph node metastasis and absence of the bispecific binding molecule indicates no regional lymph node metastasis.
  • Example 42 The method of any examples herein, particularly Example 41, wherein the subject has a solid tumor, and wherein step a) comprises injecting the bispecific binding molecule into said solid tumor.
  • Example 43 The method of any examples herein, particularly Examples 41-42, wherein the subject has breast cancer; and wherein the method is used to detect axillary lymph node metastasis.
  • Example 44 The method of any examples herein, particularly Examples 41-43, wherein step b) occurs from about 3 hours to about 96 hours after step a).
  • Example 45 The method of any examples herein, particularly Examples 41-44, further comprising: c) removing one or more regional lymph nodes from the subject when the presence of the bispecific binding molecule is detected in one or more regional lymph nodes, and removing no regional lymph nodes when the absence of the bispecific binding molecule is detected in all regional lymph nodes.
  • Example 46 The method of any examples herein, particularly Example 45, wherein step c) comprises removing only regional lymph nodes which exhibit the presence of the bispecific binding molecule and not removing regional lymph nodes which exhibit the absence of the bispecific binding molecule.
  • Example 47 The method of any examples herein, particularly Example 45, wherein step c) comprises removing all regional lymph nodes.
  • Example 48 The method of any examples herein, particularly Examples 45-47, wherein steps b) and c) occur simultaneously.
  • Example 49 The method of any examples herein, particularly Examples 41-48, further comprising administering an anticancer therapy to the subject.
  • Example 50 A method of treating regional lymph node metastasis, the method comprising: a) administering to the subject the bispecific binding molecule of any examples herein, particularly Examples 31-40; b) detecting the bispecific binding molecule, wherein presence of the bispecific binding molecule indicates regional lymph node metastasis and absence of the bispecific binding molecule indicates no regional lymph node metastasis; and c) removing one or more regional lymph nodes from the subject when the presence of the bispecific binding molecule is detected in one or more regional lymph nodes.
  • Example 51 The method of any examples herein, particularly Example 50, wherein the subject has a solid tumor, and wherein step a) comprises injecting the bispecific binding molecule into said solid tumor.
  • Example 52 The method of any examples herein, particularly Examples 50-51, wherein the subject has breast cancer; and wherein the method is used to treat axillary lymph node metastasis.
  • Example 53 The method of any examples herein, particularly Examples 50-52, wherein step b) occurs from about 3 hours to about 96 hours after step a).
  • Example 54 The method of any examples herein, particularly Examples 50-53, wherein step c) comprises removing only regional lymph nodes which exhibit the presence of the bispecific binding molecule and not removing regional lymph nodes which exhibit the absence of the bispecific binding molecule.
  • Example 55 The method of any examples herein, particularly Examples 50-54, wherein step c) comprises removing all regional lymph nodes.
  • Example 56 The method of any examples herein, particularly Examples 50-55, wherein steps b) and c) occur simultaneously.
  • Example 57 The method of any examples herein, particularly Examples 50-56, further comprising administering an anticancer therapy to the subject.

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Abstract

L'invention concerne des molécules de liaison à l'anhydrase carbonique IX (CAIX) et à l'anhydrase carbonique XII (CAXII), des molécules de liaison bispécifiques formées à partir de celles-ci et des méthodes de détection et/ou de traitement de métastases des ganglions lymphatiques régionaux, utilisant lesdites molécules de liaison bispécifiques.
PCT/US2025/030045 2024-05-17 2025-05-19 Molécules de liaison à caix et à caxii et leurs méthodes d'utilisation Pending WO2025240969A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20200291482A1 (en) * 2017-06-21 2020-09-17 Exosomics S.p.A. Methods and kits relating to the capture of ca-ix positive exosomes
US20210054095A1 (en) * 2017-12-19 2021-02-25 Philogen S.P.A. Antibodies To Tumour Antigens
WO2022040506A2 (fr) * 2020-08-21 2022-02-24 Yale University Compositions de nanocorps et leurs procédés d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200291482A1 (en) * 2017-06-21 2020-09-17 Exosomics S.p.A. Methods and kits relating to the capture of ca-ix positive exosomes
US20210054095A1 (en) * 2017-12-19 2021-02-25 Philogen S.P.A. Antibodies To Tumour Antigens
WO2022040506A2 (fr) * 2020-08-21 2022-02-24 Yale University Compositions de nanocorps et leurs procédés d'utilisation

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