U.S. patent application number 17/008432 was filed with the patent office on 2021-02-25 for disulfide bridging conjugates.
This patent application is currently assigned to Sorrento Therapeutics, Inc.. The applicant listed for this patent is Sorrento Therapeutics, Inc.. Invention is credited to Yanwen Fu, Gunnar F. Kaufmann, James T. Patterson, Tong Zhu.
Application Number | 20210052738 17/008432 |
Document ID | / |
Family ID | 1000005196824 |
Filed Date | 2021-02-25 |
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United States Patent
Application |
20210052738 |
Kind Code |
A1 |
Patterson; James T. ; et
al. |
February 25, 2021 |
Disulfide Bridging Conjugates
Abstract
Provided are bispecific conjugates having the general formula:
##STR00001## pharmaceutically acceptable salts thereof,
pharmaceutical compositions thereof, and their use in the treatment
of cancer.
Inventors: |
Patterson; James T.; (San
Diego, CA) ; Kaufmann; Gunnar F.; (San Diego, CA)
; Fu; Yanwen; (San Diego, CA) ; Zhu; Tong;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorrento Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Sorrento Therapeutics, Inc.
San Diego
CA
|
Family ID: |
1000005196824 |
Appl. No.: |
17/008432 |
Filed: |
August 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16052406 |
Aug 1, 2018 |
10799598 |
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17008432 |
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62539850 |
Aug 1, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6849 20170801;
A61K 47/55 20170801; A61K 47/6851 20170801 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 47/55 20060101 A61K047/55 |
Claims
1. An injectable formulation comprising: (i) an effective amount of
antibody Fab conjugate having a Formula I or a Formula II:
##STR00008## or a pharmaceutically acceptable salt thereof, wherein
each n is independently an integer from 2 to 20; each m is
independently an integer from 2 to 15, wherein each FAB fragment is
connected to a pyrrolidine-dione moiety or a CH.sub.2-quinoxalinyl
moiety through native cysteine residues in the Fab fragment; and
(ii) water, Ringer's solution U.S.P., isotonic sodium chloride
solution, synthetic mono- or diglycerides, or oleic acid.
2. The injectable formulation of claim 1, wherein n is an integer
from 2 to 12.
3. The injectable formulation of claim 2, wherein n is an integer
from 4 to 10.
4. The injectable formulation of claim 3, wherein n is 8.
5. The injectable formulation of claim 1, wherein m is an integer
from 2 to 10.
6. The injectable formulation of claim 5, wherein m is an integer
from 4 to 8.
7. The injectable formulation of claim 6, wherein m is 6.
8. The injectable formulation of claim 1, wherein the FAB fragment
comprises a CD3 binding region.
9. The injectable formulation of claim 1, wherein the solvent is
water.
10. The injectable formulation of claim 1, wherein the solvent is
Ringer's solution U.S.P.
11. The injectable formulation of claim 1, wherein the solvent is
an isotonic sodium chloride solution.
12. The injectable formulation of claim 1, wherein the solvent is a
synthetic monoglyceride.
13. The injectable formulation of claim 1, wherein the solvent is a
synthetic diglyceride.
14. The injectable formulation of claim 1, wherein the solvent is
an oleic acid.
15. A method of treating prostate cancer or adenocarcinoma,
comprising administering to a subject in need thereof an injectable
formulation of claim 1, or a pharmaceutically acceptable salt
thereof.
16. The method of claim 15, wherein the subject has prostate
cancer.
17. The method of claim 15, wherein the subject has adenocarcinoma.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/052,406, filed Aug. 1, 2018, which claims
priority from U.S. Provisional Application No. 62/539,850, filed
Aug. 1, 2017, the contents of each of which are incorporated herein
by reference in their entirety.
BACKGROUND
[0002] Cancer is, and still remains, the leading cause of death
worldwide. According to the American Cancer Society, the average
lifetime risk of developing cancer for American males is 40.8%, or
a little less than one in two. Prostate cancer is most predominant
cancer in males and is the second leading cause of death. While
traditional approaches for treating cancer have had success, these
approaches are often plagued by the lack of targeted treatments,
drug resistance, and specialized problems associated with
particular strains. For example, oesophageal, prostate, and
pancreatic cancer are extremely difficult to treat because they are
often slow growing, remain symptom free, and metastasize.
[0003] Bispecific antibodies or conjugates have emerged as useful
therapeutic tools to selectively target cancers. In these
instances, one portion of the conjugate is affixed a motif that is
responsible for binding a target cell and a second portion is
affixed with a synthetic agent such as a drug, protein, cytotoxin,
polymer, or nanoparticle. Clinical successes and impressive
treatment profiles for bispecific conjugates have been shown. See
e.g., MAbs. 2009 November-December; 1(6): 539-547.
[0004] Prostate-specific antigen (PSMA) is overexpressed in most
prostate cancer cells, but is present at low or undetectable levels
in normal cell. Studies have revealed that PSMA is the second most
upregulated protein in prostate cancer and that expression levels
rise depending upon the severity of the cancer. See e.g., Wang, et
al., J. Cell. Biochem. 2007, 102, 571-579). These distinctions
provide a unique opportunity for selective targeting PSMA. Taken
together with bispecific conjugates, simultaneously targeting PSMA
and presenting T-cell surface antigen (CD3) provides a new
generation of selective cancer targeting drugs.
SUMMARY
[0005] Provided herein are bispecific conjugates having the general
formula:
##STR00002##
In these conjugates, the fragment antigen binding (FAB) fragment of
an antibody is chemically bonded to a targeting moiety X through
disulfide bonds on the constant heavy (C.sub.H) and constant light
(C.sub.L) chains of the FAB fragment. Compositions comprising these
bispecific conjugates and their use for the treatment of one or
more cancers are also provided.
[0006] The bispecific conjugates and compositions described herein
can be manufactured in commercially relevant yields and quantities,
utilize digestions on off-the-shelf antibodies or cells (e.g., CHO
cells), undergo facile conjugation processes, and elicit the
exclusive formation of heterodimers (with high assembly yield).
These processes mitigate conventional requirements for extensive
protein engineering, complex genetic techniques, and laborious
biochemical processing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 illustrates an SDS-PAGE gel analysis of intermediates
involved in the synthesis of an exemplary bispecific antibody.
[0008] FIG. 2A and FIG. 2B illustrate a Hydrophobic Interaction
Chromatography (HIC) analysis of the .alpha.CD3 Fab (2A) and the
conjugate having the Formula 1 (2B).
[0009] FIG. 3A and FIG. 3B illustrate a mass spectrum analysis for
the .alpha.CD3 Fab (3A) the conjugate having the Formula 1
(3B).
[0010] FIGS. 4A and 4B show the cytotoxicity of LNCap (4A) and PC-3
cells (4B) absent PSMA treated with controls and the conjugate
having the Formula 1.
[0011] FIG. 5A and FIG. 5B show the cytotoxicity of LNCap (5A) and
PC-3 cells (5B) with PSMA treated with controls and the conjugate
having the Formula 1.
DETAILED DESCRIPTION
1. General Description of Conjugates
[0012] In one embodiment, provided herein are bispecific conjugates
having the formula:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein FAB
represents the fragment antigen binding fragment of an antibody;
V.sub.L, V.sub.H, C.sub.L, and C.sub.H represent the variable
light, variable heavy, constant light, and constant heavy portions
of the FAB fragment; the bond between C.sub.L and C.sub.H
represents a disulfide bond, and is the point to which a chemical
targeting group X is attached.
2. Definitions
[0013] The term "small molecule" means an non-peptide organic
compound having a molecular weight of less than 900 daltons and
which elicits a biological response in a subject. Small molecules
include e.g., lipids, monosacchirides, second messengers, natural
products, and metabolites.
[0014] The term "peptide" refers to compounds which are short
chains of amino acid monomers linked by amide bonds.
[0015] The term "protein" means a large biomolecule or
macromolecule comprising one or more long chains of amino acid
residues. Proteins may be made up of naturally occurring amino
acids and peptide bonds, or synthetic peptidomimetic structures.
"Amino acid" means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids. "Amino acid" also includes imino acid
residues such as proline and hydroxyproline. The side chains may be
in either the (R) or (S) configuration. Proteins include
antibodies.
[0016] The term "nucleic acid" means at least two nucleotides
covalently linked together. A nucleic acid generally contains
phosphodiester bonds, although in some cases, nucleic acid analogs
are included that may have alternate backbones, comprising, for
example, phosphoramide. Mixtures of naturally occurring nucleic
acids and analogs are contemplated. The nucleic acids may be single
stranded or double stranded, or contain portions of both double
stranded or single stranded sequence. The nucleic acid may be DNA,
both genomic and cDNA, RNA or a hybrid, where the nucleic acid
contains a combination of deoxyribo- and ribo-nucleotides. Nucleic
acids may naturally occurring nucleic acids, random nucleic acids,
or "biased" random nucleic acids. For example, digests of
prokaryotic or eukaryotic genomes may be used. Where the ultimate
expression product is a nucleic acid, at least 10, at least 12, at
least 15, or at least 21 nucleotide positions need to be
randomized.
[0017] As used herein, the term "polymer" refers to a large
molecule or marcromolecule comprised of many repeated subunits.
[0018] The term "nanoparticle" refers to a particle that is between
1 and 100 nanometers in size.
[0019] Chemical targeting groups which can be used in the present
compounds include, but are not limited to, bombesin; auristatins
such as MMAE and MMAF; maytansinoids such as DM1 and DM4;
inhibitors of MDMX, MDM2, MDMX/MDM2, STAT3, and KRAS; compounds
that bind avb3 integrin; compounds that bind a gastrin releasing
peptide receptor, compounds that bind neurokinin 1 receptor,
compounds that bind melanocortin 1 receptor, compounds that bind
neuropeptide Y receptor Erlotinib (TARCEVA.RTM., Genentech/OSI
Pharm.), Bortezomib (VELCADE.RTM., Millennium Pharm.), Fulvestrant
(FASLODEX.RTM., Astrazeneca), Sutent (SU11248, Pfizer), Letrozole
(FEMARA.RTM., Novartis), Imatinib mesylate (GLEEVECO, Novartis),
PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin.RTM., Sanofi),
5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNEO,
Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH
66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib
(IRESSA.RTM., Astrazeneca), AG1478, AG1571 (SU 5271; Sugen),
alkylating agents such as Thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gammal I and calicheamicin omegall (Angew Chem Intl.
Ed. Engl. (1994) 36 33:183-186); dynemicin, including dynemicin A;
bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, anthramycin,
azaserine, bleomycins, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINO doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanop article formulation of paclitaxel
(American Pharmaceutical Partners, Schaumberg, Ill.), and
TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; GEMZAR.RTM. gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine; NAVELBINEO vinorelbine;
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluorometlhylornithine (DMFO); retinoids such as retinoic acid;
capecitabine; tamoxifen (including NOLVADEX.RTM. tamoxifen),
raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and FARESTON toremifene; (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. megestrol acetate,
AROMASINO exemestane, formestanie, fadrozole, RIVISOR.RTM.
vorozole, FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole;
(iii) anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) aromatase
inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase
inhibitors; (vii) antisense oligonucleotides, particularly those
which inhibit expression of genes in signaling pathways implicated
in abherant cell proliferation, such as, for example, PKC-alpha,
Ralf and H-Ras; (viii) ribozymes such as a VEGF expression
inhibitor (e.g., ANGIOZYME.RTM. ribozyme) and a HER2 expression
inhibitor; (ix) vaccines such as gene therapy vaccines, for
example, ALLOVECTINO vaccine, LEUVECTINO vaccine, and VAXID.RTM.
vaccine; PROLEUKINO rIL-2; LURTOTECAN.RTM. topoisomerase 1
inhibitor; ABARELIX.RTM. rmRH; and (x) anti-angiogenic agents such
as bevacizumab (AVASTINO, Genentech).
Other targeting groups include, but are not limited to
mycophenolate mofetil such as CELLCEPT.RTM.; azathioprine
(IMURAN.RTM., AZASAN.RTM./6-mercaptopurine; bromocryptine; danazol;
dapsone; glutaraldehyde (which masks the MHC antigens, as described
in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC
antigens and MHC fragments; cyclosporin A; steroids such as
corticosteroids and glucocorticosteroids, e.g., prednisone,
prednisolone such as PEDIAPRED.RTM. (prednisolone sodium phosphate)
or ORAPRED.RTM. (prednisolone sodium phosphate oral solution),
methylprednisolone, and dexamethasone; methotrexate (oral or
subcutaneous) (RHEUMATREX.RTM., TREXALL.TM.);
hydroxycloroquine/chloroquine; sulfasalazine; leflunomide; cytokine
or cytokine receptor antagonists including anti-interferon-y, -13,
or -a antibodies, anti-tumor necrosis factor-a antibodies
(infliximab or adalimumab), anti-TNFa immunoadhesin (ENBREL.RTM.,
etanercept), anti-tumor necrosis factor-I3 antibodies,
anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-LFA-1 antibodies, including anti-CD11 a and anti-CD18
antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte
globulin; polyclonal or pan-T antibodies, or monoclonal anti-CD3 or
anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3
binding domain (WO 90/08187); streptokinase; TGF-I3;
streptodornase; RNA or DNA from the host; FK506; RS-61443;
deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S.
Pat. No. 5,114,721); T-cell receptor fragments (Ofiher et al.
Science 251: 430-432 (1991); WO 90/11294; Ianeway, Nature 341:482
(1989); and WO 91/01133); T cell receptor antibodies (EP 340,109)
such as T 1 OB9; cyclophosphamide (CYTOXAN.RTM.); and dapsone;
penicillamine.
[0020] The term "antibody" includes monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (including
bispecific antibodies), antibody fragments that can bind an antigen
(including, Fab', F(ab).sub.2, Fv, single chain antibodies,
diabodies), and recombinant peptides comprising the foregoing as
long as they exhibit the desired biological activity and antigen
binding specificity. In one embodiment, an antibody is a full
length or intact antibody. A full length antibody comprises four
polypeptide chains, two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds, as well as multimers thereof
(e.g., IgM). Each heavy chain comprises a heavy chain variable
region (abbreviated herein as HCVR or VH) and a heavy chain
constant region. The heavy chain constant region comprises three
domains, CH1, CH2 and CH3. Each light chain comprises a light chain
variable region (abbreviated herein as LCVR or VL) and a light
chain constant region. The light chain constant region comprises
one domain (CL1). The VH and VL regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs
of the antibody may be identical to the human germline sequences,
or may be naturally or artificially modified.
[0021] The disclosed conjugates may exist in various stereoisomeric
forms. Stereoisomers are compounds that differ only in their
spatial arrangement. Diastereomers are stereoisomers that contain
two or more asymmetrically substituted carbon atoms.
[0022] When the stereochemistry of a disclosed conjugate is named
or depicted by structure, it is to be understood that one of the
encompassed stereoisomers or any mixture of the encompassed
stereoisomers are included. It is to be further understood that the
stereoisomeric purity of the named or depicted stereoisomer is at
least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to
all of the other stereoisomers. The stereoisomeric purity in this
case is determined by dividing the total weight in the mixture of
the stereoisomers encompassed by the name or structure by the total
weight in the mixture of all of the stereoisomers.
[0023] When a disclosed conjugate is named or depicted by structure
without indicating the stereochemistry, it is to be understood that
the name or structure encompasses one stereoisomer free of other
stereoisomers, mixtures of stereoisomers, or mixtures of
stereoisomers in which one or more stereoisomers is enriched
relative to the other stereoisomer(s). For example, the name or
structure may encompass one stereoisomer free of other
diastereomers, mixtures of stereoisomers, or mixtures of
stereoisomers in which one or more diastereomers is enriched
relative to the other diastereomer(s).
[0024] Pharmaceutically acceptable salts of the conjugates herein
are contemplated. For use in medicines, the salts of the conjugates
described herein refer to non-toxic "pharmaceutically acceptable
salts." Pharmaceutically acceptable salt forms include
pharmaceutically acceptable acidic/anionic or basic/cationic
salts.
[0025] The term "pharmaceutically acceptable carrier" refers to a
non-toxic carrier, adjuvant, or vehicle that does not destroy the
pharmacological activity of the conjugate with which it is
formulated. Pharmaceutically acceptable carriers, adjuvants or
vehicles that may be used in the compositions of this disclosure
include, but are not limited to, organic or inorganic carriers,
excipients or diluents suitable for pharmaceutical
applications.
[0026] As used herein the terms "subject" and "patient" may be used
interchangeably, and means a mammal in need of treatment, e.g.,
companion animals (e.g., dogs, cats, and the like), farm animals
(e.g., cows, pigs, horses, sheep, goats and the like) and
laboratory animals (e.g., rats, mice, guinea pigs and the like).
Typically, the subject is a human in need of treatment.
[0027] As used herein, the terms "treatment," "treat," and
"treating" refer to reversing, alleviating, delaying the onset of,
or inhibiting the progress of a disease or disorder, or one or more
symptoms thereof, as described herein. In some embodiments,
treatment may be administered after one or more symptoms have
developed, i.e., therapeutic treatment. In other embodiments,
treatment may be administered in the absence of symptoms. For
example, treatment may be administered to a susceptible individual
prior to the onset of symptoms (e.g., in light of a history of
symptoms and/or in light of genetic or other susceptibility
factors), i.e., prophylactic treatment. Treatment may also be
continued after symptoms have resolved, for example to reduce the
likelihood of recurrence or to delay the recurrence.
[0028] The term "effective amount" as used herein, refers to an
amount of a conjugate disclosed herein, which is sufficient to
effect treatment of a disease when administered to a subject. A
therapeutically effective amount will vary depending upon the
relative activity of the conjugate and depending upon the subject
and disease condition being treated, the weight and age of the
subject, the severity of the disease condition, the manner of
administration and the like, which can readily be determined by one
of ordinary skill in the art.
3. Description of Exemplary Conjugates
[0029] In a first embodiment, provided herein are bispecific
conjugates having the formula:
##STR00004##
or a pharmaceutically acceptable salt thereof, wherein FAB
represents the fragment antigen binding fragment of an antibody;
V.sub.L, V.sub.H, C.sub.L, and C.sub.H represent the variable
light, variable heavy, constant light, and constant heavy portions
of the FAB fragment; the bond between C.sub.L and C.sub.H
represents a disulfide bond, and is the point to which a chemical
targeting group X is attached; and the targeting group X is
selected from a protein, nanoparticle, polymer, nucleic acid,
peptide, or small molecule. In one aspect, X is a small molecule or
protein. In another aspect, X is a small molecule.
[0030] In a second embodiment, the bispecific conjugates described
herein are of the Formula:
##STR00005##
or a pharmaceutically acceptable salt thereof, wherein each n is
independently an integer from 2 to 20; each m is independently an
integer from 2 to 15; and the hashed bonds indicate the points of
attachment to the FAB fragment.
[0031] In a third embodiment, n is independently an integer from 2
to 16, wherein the remaining variables are as described above in
the second embodiment. In another alternative, n is independently
an integer from 2 to 12, wherein the remaining variables are as
described above in the second embodiment. In another alternative, n
is independently an integer from 2 to 10, wherein the remaining
variables are as described above in the second embodiment. In
another alternative, n is independently an integer from 2 to 12,
wherein the remaining variables are as described above in the
second embodiment. In yet another alternative, n is independently
an integer from 4 to 8. In yet another aspect, n is 8, wherein the
remaining variables are as described above in the second
embodiment.
[0032] In a fourth embodiment, m is independently an integer from 2
to 14, wherein the remaining variables are as described above in
the second or third embodiment. In another alternative, m is
independently an integer from 2 to 12, wherein the remaining
variables are as described above in the second or third embodiment.
In another alternative, m is independently an integer from 2 to 10,
wherein the remaining variables are as described above in the
second or third embodiment. In another alternative, m is
independently an integer from 4 to 10, wherein the remaining
variables are as described above in the second or third embodiment.
In yet another alternative, m is independently an integer from 4 to
8, wherein the remaining variables are as described above in the
second or third embodiment. In yet another aspect, m is 6, wherein
the remaining variables are as described above in the second or
third embodiment.
[0033] In a fifth embodiment, the bispecific conjugates described
herein are of the Formula:
##STR00006##
or a pharmaceutically acceptable salt thereof.
4. Uses, Formulation and Administration
[0034] In certain embodiments, the present disclosure provides a
method of treating a patient (e.g., a human) with a cancer
comprising the step of administering to the patient an effective
amount of a conjugate described herein, or a pharmaceutically
acceptable salt or composition thereof.
[0035] Examples of cancers treated using the conjugates and methods
described herein include, but are not limited to, adrenal cancer,
acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma,
acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia,
acute lymphoblastic leukemia, acute megakaryoblastic leukemia,
acute monocytic leukemia, actue promyelocytic leukemia,
adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid
odontogenic tumor, adenosquamous carcinoma, adipose tissue
neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma,
aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar
rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma,
anaplastic large cell lymphoma, anaplastic thyroid cancer,
angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma,
astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic
lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell
lymphoma, basal cell carcinoma, biliary tract cancer, bladder
cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,
Burkitt's lymphoma, breast cancer, brain cancer, carcinoma,
carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma,
myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid
plexus papilloma, clear-cell sarcoma of the kidney,
craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer,
colorectal cancer, Degos disease, desmoplastic small round cell
tumor, diffuse large B-cell lymphoma, dysembryoplastic
neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine
gland neoplasm, endodermal sinus tumor, enteropathy-associated
T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma,
fibrosarcoma, follicular lymphoma, follicular thyroid cancer,
ganglioneuroma, gastrointestinal cancer, germ cell tumor,
gestational choriocarcinoma, giant cell fibroblastoma, giant cell
tumor of the bone, glial tumor, glioblastoma multiforme, glioma,
gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell
tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy
cell leukemia, hemangioblastoma, head and neck cancer,
hemangiopericytoma, hematological malignancy, hepatoblastoma,
hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, invasive lobular carcinoma, intestinal cancer, kidney
cancer, laryngeal cancer, lentigo maligna, lethal midline
carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer,
lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute
lymphocytic leukemia, acute myelogeous leukemia, chronic
lymphocytic leukemia, liver cancer, small cell lung cancer,
non-small cell lung cancer, MALT lymphoma, malignant fibrous
histiocytoma, malignant peripheral nerve sheath tumor, malignant
triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma,
mast cell leukemia, mediastinal germ cell tumor, medullary
carcinoma of the breast, medullary thyroid cancer, medulloblastoma,
melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic
urothelial carcinoma, mixed Mullerian tumor, mucinous tumor,
multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid
liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma,
neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma,
ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma,
optic nerve sheath meningioma, optic nerve tumor, oral cancer,
osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid
cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma,
pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma,
precursor T-lymphoblastic lymphoma, primary central nervous system
lymphoma, primary effusion lymphoma, preimary peritoneal cancer,
prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma
periotonei, renal cell carcinoma, renal medullary carcinoma,
retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's
transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma,
Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring
cell carcinoma, skin cancer, small blue round cell tumors, small
cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart,
spinal tumor, splenic marginal zone lymphoma, squamous cell
carcinoma, synovial sarcoma, Sezary's disease, small intestine
cancer, squamous carcinoma, stomach cancer, T-cell lymphoma,
testicular cancer, thecoma, thyroid cancer, transitional cell
carcinoma, throat cancer, urachal cancer, urogenital cancer,
urothelial carcinoma, uveal melanoma, uterine cancer, verrucous
carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer,
Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'
tumor.
[0036] In one aspect the diseases and conditions treatable by the
according to the methods using the bispecific antibody compounds
described herein are selected from non-Hodgkin lymphoma (NHL),
prostate cancer, B-cell lymphoma, acite myeloid leukemia (AML),
colon cancer, and breast cancer. In one embodiment, the bispecific
antibody compounds described herein are used as bispecific T cell
engagers, and are able to exert action on its antigen selectively
and direct the human immune system to act against a tumor cell.
[0037] In one embodiment, a subject is treated with a conjugate
described herein and a pharmaceutically acceptable carrier,
adjuvant, or vehicle, wherein said conjugate is present in an
amount to treat or ameliorate one or more of the diseases and
conditions recited above. In an alternative embodiment, the cancers
by a conjugate described herein include, any one of those described
above. In one aspect, the cancers are selected from non-Hodgkin
lymphoma (NHL), prostate cancer, B-cell lymphoma, acite myeloid
leukemia (AML), colon cancer, breast cancer, in the patient.
[0038] Compositions described herein may be administered orally,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir. The term
"parenteral" as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional and intracranial injection
or infusion techniques.
[0039] The term "pharmaceutically acceptable carrier" refers to a
non-toxic carrier, adjuvant, or vehicle that does not destroy the
pharmacological activity of the antibody with which it is
formulated. Pharmaceutically acceptable carriers, adjuvants or
vehicles that may be used in the compositions of this disclosure
include, but are not limited to, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0040] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
can be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid are used in the
preparation of injectables.
[0041] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0042] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including age, body weight, general health,
sex, diet, time of administration, rate of excretion, drug
combination, the judgment of the treating physician, and the
severity of the particular disease being treated. The amount of a
provided bispecific antibody compound in the composition will also
depend upon the particular compound in the composition.
Exemplification
[0043] As depicted in the Examples below, conjugates are prepared
according to the following general procedures. It will be
appreciated that, although the general methods depict the synthesis
of certain compounds herein, the following general methods, and
other methods known to one of ordinary skill in the art, can be
applied to all conjugates described herein.
Preparation of FAB from IgG1
[0044] Proteolytic digestion of IgG1 allowed generation of Fab.
SpeB cysteine protease, FabULOUS (Genovis), was used to digest the
hinge region of IgG1 to produce Fab and Fc fragments. A digestion
procedure was adopted using 0.1-0.15 U/.mu.g enzyme overnight
(.about.16 h) at 37.degree. C. in a buffer containing 20 mM
imidazole, 0.5 M NaCl, and 20 mM sodium phosphate (pH 7.4) with 0.1
mM dithiothreitol (DTT). Fab purification was enabled by tandem
HisTrap FF (GE) and HiTrap MabSelect SuRe (GE) purification on an
.ANG.KTA Pure FPLC system. His-tagged protease was removed by the
Ni-sepharose column, while digested Fc fragment and any undigested
IgG1 was retained in the Protein A column. Fab was collected in the
flow-through fractions and buffer exchanged into DPBS or 20 mM
sodium phosphate buffer pH 7.4. Fab purity was then assessed by
standard methods.
Bioconjugation Reaction
[0045] .alpha.CD3 Fab conjugate was prepared through reduction of
the interchain disulfide bonds followed by conjugation with
dibromo-functionalized linker containing the
2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) targeting
moiety. Fab (5 mg/mL) was typically reduced using 10 or 20
equivalents of DTT in 20 mM sodium phosphate buffer (pH 7.4) for 1
h at RT. Reducing agent was removed by Sephadex G-25 (GE) gravity
column and buffer exchange into 20 mM sodium phosphate buffer with
5 mM diethylenetriaminepentaacetic acid (DTPA) (pH 7.4). Next,
conjugation with 10 equivalents DUPA linker in buffer containing
7.5% organic co-solvent proceeded overnight at RT. Excess linker
was removed by centrifugal filtration using Amicon Ultra 10 kDa
NMWL units (Millipore). This method was used to afford conjugates
of Formula 1 and 2. See Scheme 1 below.
##STR00007##
[0046] The Hydrophobic Interaction Chromatography and Mass Spectrum
analysis for the .alpha.CD3 Fab and the product, the conjugate of
Formula 1 are shown in FIGS. 2A and 2B and FIGS. 3A and 3B,
respectively.
SDS-PAGE Analysis
[0047] Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was employed using NuPAGE Novex 4-12% Bis-Tris Protein
Gels with NuPAGE MOPS SDS Running Buffer in a XCell SureLock Mini
electrophoresis system. All samples (2 .mu.g) included NuPAGE LDS
Sample Buffer and were heated to 70.degree. C. for 5 min prior to
loading. Reduced samples also contained NuPAGE Sample Reducing
Agent. Mark12 Unstained Standard (10 .mu.L) was used for estimation
of molecular weights. After gel electrophoresis at 125 V for 1.5 h,
gels were fixed for 5 min and stained with SYPRO Ruby Protein Gel
Stain following the recommended procedures. Imaging was performed
with a Bio-Rad ChemiDoc MP System and analyzed by Image Lab
Software. Heavy chain-light chain disulfide bridging was determined
to be .about.85% efficient.
Hydrophobic Interaction Chromatography (HIC) HPLC
[0048] Analysis by HIC HPLC used a TOSOH TSKgel Butyl-NPR (4.6 mm
ID.times.10 cm, 2.5 .mu.m) column at 40.degree. C. on an Agilent
1260 Infinity system. Analytical runs were performed using 50 .mu.g
sample with a linear gradient of 0-60% B over 30 min: A=50 mM
sodium phosphate+1 M ammonium sulfate (pH 7), B=50 mM sodium
phosphate+25% isopropanol (pH 7). All data was analyzed using
OpenLAB Software. HIC HPLC of .alpha.CD3 Fab conjugation with
dibromomaleimide-PEG8-DUPA shows a major product (.about.80%)
corresponding to the addition of a single linker while only a minor
peak of doubly conjugated Fab was produced.
LCMS Analysis of Intact Protein
[0049] For intact MS analysis, 5 .mu.g .alpha.CD3 Fab or .alpha.CD3
Fab conjugate was injected onto an Agilent PLRP-S (1000 .ANG., 5
.mu.m, 2.1.times.50 mm) column at 80.degree. C. Antibody was eluted
from the column with an 8 min gradient (25-60% B, 0.5 mL/min flow
rate). Mobile phase A was 0.1% formic acid in water, and mobile
phase B was 0.1% formic acid in acetonitrile.
[0050] All experiments were performed on a Waters H class UPLC
system coupled to a Waters Xevo G2 TOF mass spectrometer. The mass
spectrometer was operated in positive ion, sensitivity mode with
detection in the range of 500-4000 m/z. Source parameters were as
follows: capillary voltage, 3.0 kV; sampling cone voltage, 40.0 V;
source temperature, 125.degree. C.; desolvation temperature,
350.degree. C.; cone gas flow, 10 L/hr; desolvation gas flow, 800
L/hr. The protein peak was deconvolved by MassLynx MaxEntl function
according to the following parameters: output resolution, 2.0
Da/channel; uniform Gaussian width at half height, 0.8 Da for
intact antibody; minimum intensity ratios 10% for left and right;
maxium number of iteration of 12.
[0051] LCMS demonstrated a rather homogeneous preparation of
.alpha.CD3 Fab from enzymatic digestion. Reduction and conjugation
of Fab with dibromomaleimide-PEG8-DUPA linker resulted in the major
conjugated product containing a single linker bridging the
inter-chain disulfide bond.
Luminescence Cytotoxicity Assay with Fab
[0052] Firefly luciferase transduced prostate cancer target cell
lines were used for cytotoxicity assays, LNCaP (ATCC.RTM.
CRL-1740.TM.), PSMA+(cultured in RPMI-1640+10% non-heat-inactivated
FBS+0.5 .mu.g/mL Puromycin) and PC-3 (ATCC.RTM. CRL-1435.TM.) PSMA
(cultured RPMI-1640+10% heat-inactivated FBS+1.0 .mu.g/mL
Puromycin). Cells were harvested with TrypLE (ThermoFisher
Scientific) then resuspended in fresh RPMI-1640+10%
heat-inactivated FBS and plated at 4,000 cells/well in 100 .mu.L.
After overnight incubation at 37.degree. C. in a humidified 5%
CO.sub.2 incubator, serial dilutions of proteins in RPMI-1640+10%
heat-inactivated medium (50 .mu.L) were added to the assay plates
at the indicated concentrations. In experiments utilizing effector
cells, freshly thawed peripheral blood mononuclear cells (PBMCs)
were washed with media and added to the assay plates at 40,000
cells in 50 .mu.L to obtain an effector:target ratio of 10:1. After
4 days incubation, 90 .mu.L was removed from assay plates and 90
.mu.L ONE Glo Luciferase Assay Reagent (Promega #E6120) was mixed
with the samples and incubated at room temperature for 10 min.
Samples were transferred to white 96-well flat bottom plates for
luminescence measurements using a PerkinElmer EnSpire multimode
plate reader. Data was analyzed using GraphPad Prism software.
[0053] Cell killing was analyzed in the absence of effector cells
(FIG. 4A and FIG. 4B) to monitor any inherent cytotoxic activities
of .alpha.CD3 Fab, DUPA small molecule, the combination of Fab and
small molecule, and .alpha.CD3-DUPA conjugate. No dose-dependent
cell killing activity was observed up to 20 nM for any of the
tested samples, independent of the presence (LNCaP) or absence
(PC-3) of PSMA antigen expression.
[0054] .alpha.PSMA and .alpha.CD3 Fabs did not show cytotoxic
activity in the presence of PMBCs (FIG. 5A and FIG. 5B). However,
cell killing of LNCaP cells but not PC-3 cells was evident with
.alpha.CD3-DUPA. The data indicate that potent PSMA-directed
cytotoxicity (190 pM) with conjugated Fab was achieved using PBMC
effector cells.
[0055] While we have described a number of embodiments, it is
apparent that our basic examples may be altered to provide other
embodiments that utilize the compounds and methods of this
invention. Therefore, it will be appreciated that the scope of this
invention is to be defined by the appended claims rather than by
the specific embodiments that have been represented by way of
example.
[0056] The contents of all references (including literature
references, issued patents, published patent applications, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated herein in their entireties by
reference. Unless otherwise defined, all technical and scientific
terms used herein are accorded the meaning commonly known to one
with ordinary skill in the art.
* * * * *