U.S. patent application number 17/497157 was filed with the patent office on 2022-08-25 for antibody molecule-drug conjugates and uses thereof.
The applicant listed for this patent is VISTERRA, INC.. Invention is credited to James C. Delaney, Obadiah Joseph Plante, Boopathy Ramakrishnan, Zachary Holmes Shriver, Karthik Viswanathan, Andrew M. Wollacott.
Application Number | 20220267418 17/497157 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220267418 |
Kind Code |
A1 |
Plante; Obadiah Joseph ; et
al. |
August 25, 2022 |
ANTIBODY MOLECULE-DRUG CONJUGATES AND USES THEREOF
Abstract
Antibody molecule-drug conjugates (ADCs) that specifically bind
to lipopolysaccharides (LPS) are disclosed. The antibody
molecule-drug conjugates can be used to treat, prevent, and/or
diagnose bacterial infections and related disorders.
Inventors: |
Plante; Obadiah Joseph;
(Danvers, MA) ; Delaney; James C.; (Cambridge,
MA) ; Viswanathan; Karthik; (Acton, MA) ;
Ramakrishnan; Boopathy; (Braintree, MA) ; Shriver;
Zachary Holmes; (Winchester, MA) ; Wollacott; Andrew
M.; (Milton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VISTERRA, INC. |
Waltham |
MA |
US |
|
|
Appl. No.: |
17/497157 |
Filed: |
October 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15348436 |
Nov 10, 2016 |
11168131 |
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17497157 |
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62375800 |
Aug 16, 2016 |
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62253345 |
Nov 10, 2015 |
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International
Class: |
C07K 16/12 20060101
C07K016/12; C07K 7/08 20060101 C07K007/08; C07K 14/00 20060101
C07K014/00; C07K 7/06 20060101 C07K007/06; A61K 47/68 20060101
A61K047/68; A61K 38/10 20060101 A61K038/10; A61K 38/16 20060101
A61K038/16; A61K 39/40 20060101 A61K039/40; A61K 45/06 20060101
A61K045/06; C07K 16/44 20060101 C07K016/44 |
Claims
1. An antibody molecule-drug conjugate (ADC) comprising: a) an
antibody molecule that binds to lipopolysaccharide (LPS); and b) an
antimicrobial peptide.
2. The ADC of claim 1, wherein: (i) the ADC or antibody molecule
comprises a heavy chain variable region (VH), wherein the VH
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), and wherein the VH comprises: (a) an
HCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an
HCDR2 comprising the amino acid sequence of any of SEQ ID NOS: 109,
145, or 146; and an HCDR3 comprising the amino acid sequence of SEQ
ID NO: 107, or (b) an HCDR1 comprising the amino acid sequence of
SEQ ID NO: 105; an HCDR2 comprising the amino acid sequence of SEQ
ID NO: 106; and an HCDR3 comprising the amino acid sequence of SEQ
ID NO: 107; or (ii) the ADC or antibody molecule comprises a light
chain variable region (VL), wherein VL comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), and
wherein the VL comprises: an LCDR1 comprising the amino acid
sequence of any of SEQ ID NOS: 110, 138, 140, or 144; an LCDR2
comprising the amino acid sequence of any of SEQ ID NOS: 111, 139,
141, 142, or 143; and an LCDR3 comprising the amino acid sequence
of any of SEQ ID NO: 112.
3. (canceled)
4. The ADC of claim 1, wherein the ADC or antibody molecule
comprises a VH and a VL, wherein the VH comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the VL comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), and wherein the ADC or antibody
molecule comprises: (a) an HCDR1 comprising the amino acid sequence
of SEQ ID NO: 108; an HCDR2 comprising the amino acid sequence of
any of SEQ ID NOS: 109, 145, or 146; an HCDR3 comprising the amino
acid sequence of SEQ ID NO: 107; an LCDR1 comprising the amino acid
sequence of any of SEQ ID NOS: 110, 138, 140, or 144; an LCDR2
comprising the amino acid sequence of any of SEQ ID NOS: 111, 139,
141, 142, or 143; and an LCDR3 comprising the amino acid sequence
of any of SEQ ID NO: 112, or (b) an HCDR1 comprising the amino acid
sequence of SEQ ID NO: 105; an HCDR2 comprising the amino acid
sequence of SEQ ID NO: 106; an HCDR3 comprising the amino acid
sequence of SEQ ID NO: 107; an LCDR1 comprising the amino acid
sequence of any of SEQ ID NOS: 110, 138, 140, or 144; an LCDR2
comprising the amino acid sequence of any of SEQ ID NOS: 111, 139,
141, 142, or 143; and an LCDR3 comprising the amino acid sequence
of any of SEQ ID NO: 112.
5. The ADC of claim 1, wherein the ADC or antibody molecule
comprises a VH and a VL, wherein the VH comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the VL comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the VH comprises: (a)(i)
an HCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an
HCDR2 comprising the amino acid sequence of SEQ ID NO: 109; and an
HCDR3 comprising the amino acid sequence of SEQ ID NO: 107, (a)(ii)
an HCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an
HCDR2 comprising the amino acid sequence of SEQ ID NO: 145; and an
HCDR3 comprising the amino acid sequence of SEQ ID NO: 107,
(a)(iii) an HCDR1 comprising the amino acid sequence of SEQ ID NO:
108; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 146;
and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 107,
or (b) an HCDR1 comprising the amino acid sequence of SEQ ID NO:
105; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 106;
and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 107,
and/or wherein the VL comprises: (i) an LCDR1 comprising the amino
acid sequence of SEQ ID NO: 110; an LCDR2 comprising the amino acid
sequence of SEQ ID NO: 111; and an LCDR3 comprising the amino acid
sequence of any of SEQ ID NO: 112, (ii) an LCDR1 comprising the
amino acid sequence of SEQ ID NO: 138; an LCDR2 comprising the
amino acid sequence of SEQ ID NO: 139; and an LCDR3 comprising the
amino acid sequence of any of SEQ ID NO: 112, (iii) an LCDR1
comprising the amino acid sequence of SEQ ID NO: 138; an LCDR2
comprising the amino acid sequence of SEQ ID NO: 111; and an LCDR3
comprising the amino acid sequence of any of SEQ ID NO: 112, (iv)
an LCDR1 comprising the amino acid sequence of SEQ ID NO: 140; an
LCDR2 comprising the amino acid sequence of SEQ ID NO: 111; and an
LCDR3 comprising the amino acid sequence of any of SEQ ID NO: 112,
(v) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 110;
an LCDR2 comprising the amino acid sequence of SEQ ID NO: 141; and
an LCDR3 comprising the amino acid sequence of any of SEQ ID NO:
112, (vi) an LCDR1 comprising the amino acid sequence of SEQ ID NO:
110; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 142;
and an LCDR3 comprising the amino acid sequence of any of SEQ ID
NO: 112, (vii) an LCDR1 comprising the amino acid sequence of SEQ
ID NO: 138; an LCDR2 comprising the amino acid sequence of SEQ ID
NO: 143; and an LCDR3 comprising the amino acid sequence of any of
SEQ ID NO: 112, (viii) an LCDR1 comprising the amino acid sequence
of SEQ ID NO: 138; an LCDR2 comprising the amino acid sequence of
SEQ ID NO: 139; and an LCDR3 comprising the amino acid sequence of
any of SEQ ID NO: 112, (ix) an LCDR1 comprising the amino acid
sequence of SEQ ID NO: 144; an LCDR2 comprising the amino acid
sequence of SEQ ID NO: 142; and an LCDR3 comprising the amino acid
sequence of any of SEQ ID NO: 112, or (x) an LCDR1 comprising the
amino acid sequence of SEQ ID NO: 138; an LCDR2 comprising the
amino acid sequence of SEQ ID NO: 142; and an LCDR3 comprising the
amino acid sequence of any of SEQ ID NO: 112.
6. The ADC of claim 1, wherein the ADC or antibody molecule
comprises a VH and a VL, wherein the VH comprises the amino acid
sequence of any of SEQ ID NOS: 103 or 115-118, or comprises an
amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from, or has
at least 85, 90, 95, 96, 97, 98, 99, or 100% homology with, the
amino acid sequence of any of SEQ ID NOS: 103 or 115-118, and/or
wherein the VL comprises the amino acid sequence of any of SEQ ID
NOS: 104 or 119-137, or comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of any
of SEQ ID NOS: 104 or 119-137.
7. The ADC of claim 1, wherein: (i) the antibody molecule is a
monoclonal antibody molecule, a humanized antibody molecule, an
isolated antibody molecule, or a synthetic antibody molecule; (ii)
the ADC or antibody molecule comprises two VHs and two VLs, or
comprises a Fab, a F(ab')2, an Fv, or a single chain Fv fragment
(scFv); (iii) the ADC or antibody molecule further comprises a
heavy chain constant region of an IgG1, IgG2, IgG3, or IgG4, and/or
a light chain constant region of a kappa or lambda chain; or (iv)
the ADC or antibody molecule binds to a core pentasaccharide region
of the LPS, wherein the core pentasaccharide region comprises one
or more Kdo residues and one or more Hep residues.
8-10. (canceled)
11. The ADC of claim 1, wherein the ADC or antibody molecule binds
to: (a) one or more Gram-negative bacteria, or one or more
antibiotic-resistant or multidrug-resistant (MDR) bacteria. (b) a
species of Enterobacteriaceae chosen from a species of Klebsiella,
Enterobacter, Shigella, Escherichia, Salmonella, Yersinia, or
Citrobacter, a species of Pseudomonas, a species of Acinetobacter,
or any combination thereof; or (c) Klebsiella pneumonia,
Enterobacter cancerogenous, Enterobacter cloacae, Enterobacter
hormaechei, Enterobacter asburiae, Shigella boydii, Shigella
dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli,
Escherichia fergusonii, Salmonella choleraesuis, Salmonella
enteritidis, Salmonella virchow, Salmonella paratyphi B, Salmonella
typhimurium, Salmonella paratyphi A, Salmonella typhi, Salmonella
bongori, Citrobacter sedlakii, Citrobacter braakii, Citrobacter
werkmanii, Citrobacter freundii, Citrobacter youngae, Citrobacter
amalonaticus, Yersinia enterocolitica, Yersinia frederiksenii,
Yersinia pestis, Yersinia pseudotuberculosis, Pseudomonas
aeruginosa, Acinetobacter baumannii, or any combination
thereof.
12. The ADC of claim 1, wherein: (i) the ADC or antibody molecule
binds to LPS with a K.sub.D that is less than about 10 nM as
measured by an ELISA method, and/or has an opsonophagocytic
activity against a bacterium; (ii) the antibody molecule is fused
to the antimicrobial peptide; (iii) the antibody molecule
comprises: (a) a VH fused to the antimicrobial peptide, wherein the
VH is N-terminal or C-terminal to the antimicrobial peptide; (b) a
VL fused to the antimicrobial peptide, wherein the VL is N-terminal
or C-terminal to the antimicrobial peptide; or (c) both (a) and
(b); optionally wherein the C-terminus of the VH or VL is fused to
the N-terminus of the antimicrobial peptide indirectly via a
constant region, a linker, or both; (iv) the antibody molecule is
fused to the antimicrobial peptide by a sortase; (v) the ADC or
antimicrobial peptide comprises the amino acid sequence of any of
SEQ ID NOS: 67-80, 94-102, 147-156, 158-159, or 163-164, or
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, or 5 amino acid residues from, or has at least 80, 85, 90,
95, 96, 97, 98, 99, or 100% homology with, the amino acid sequence
of any of SEQ ID NOS: 67-80, 94-102, 147-156, 158-159, or 163-164;
(vi) the antimicrobial peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, or more D-amino acids, or at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, of
the amino acid residues in the antimicrobial peptide are D-amino
acids; (vii) the antimicrobial peptide comprises a first cysteine
residue and a second cysteine residue, and wherein the first
cysteine residue is cross-linked to the second cysteine residue;
(viii) the ADC comprises at least two, three, or four antimicrobial
peptides; or comprising at least two, three, or four identical, or
substantially identical, antimicrobial peptides, each of which is
fused to a VH or VL indirectly via a constant region, a linker, or
both; (ix) the antimicrobial peptide has an antimicrobial activity
against 2, 3, or all of the following: (a) at least one species of
Enterobacteriaceae chosen from one or more species of Klebsiella,
Enterobacter, Shigella, Escherichia, Salmonella, Yersinia, or
Citrobacter, (b) at least one species of Pseudomonas; or (c) at
least one species of Acinetobacter; or (x) the antimicrobial
peptide has a PLC to MIC ratio for a Gram-negative bacteria which
is greater than 4:1, wherein the PLC is determined by a red blood
cell hemolysis assay.
13-20. (canceled)
21. The ADC of claim 1, wherein (ii) the ADC has (a) a minimum
inhibitory concentration (MIC) for a Gram-negative bacterium that
is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,
90, or 100 fold lower on a molar basis than the MIC of the
antimicrobial peptide alone; (b) a minimum bactericidal
concentration (MBC) for a Gram-negative bacterium that is at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100
fold lower on a molar basis than the MBC of the antimicrobial
peptide alone; or (c) both (a) and (b); (ii) the ADC has (d) a MIC
for a Gram-negative bacterium that is at least 2, 5, 10, 20, 50,
100, 200, 500, or 1000 fold lower on a molar basis than a MIC for a
Gram-positive bacterium; (e) an MBC for a Gram-negative bacterium
that is at least 2, 5, 10, 20, 50, 100, 200, 500, or 1000 fold
lower on a molar basis than an MBC for a Gram-positive bacterium;
or (f) both (d) and (e); or (iii) the antimicrobial peptide has:
(a) a MIC of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or
5 .mu.g/ml, for Escherichia coli, Pseudomonas aeruginosa, or both;
(b) an MBC of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or
5 .mu.g/ml, for Escherichia coli, Pseudomonas aeruginosa, or both;
or (c) both (a) and (b).
22-25. (canceled)
26. A pharmaceutical composition comprising the ADC of claim 1 and
a pharmaceutically acceptable carrier.
27. A method of treating or preventing a bacterial infection,
comprising administering to a subject in need thereof the ADC of
claim 1, in an amount effective to treat or prevent the bacterial
infection.
28. The method of claim 27, wherein: (i) the bacterial infection is
associated with a Gram-negative bacterium, or is a nosocomial
infection or a hospital-acquired infection; (ii) the ADC is
administered at a dose of 1-10 mg/kg, and/or is administered
intravenously, subcutaneously, or intranasally or by inhalation;
(iii) the ADC is administered prior to or after onset of a symptom
associated with the bacterial infection; (iv) the method further
comprises administering to the subject a second antimicrobial agent
or therapy.
29-30. (canceled)
31. The method of claim 27, wherein the subject: (a) has one or
more of: pneumonia, a urinary tract infection (UTI), septicemia,
meningitis, diarrhea, a soft tissue infection, a skin infection,
bacteremia, a respiratory system infection, endocarditis, an
intra-abdominal infection, septic arthritis, osteomyelitis, a CNS
infection, an ophthalmic infection, cholecystitis, cholangitis,
meningitis, typhoid fever, food poisoning, gastroenteritis, enteric
fever, shigellosis, a blood stream infection, intra-abdominal
sepsis, a brain abscess, meningitis, sepsis, a joint infection, a
bone infection, a gastrointestinal infection, or a wound infection.
(b) is a human or an animal; (c) is an immunocompromised patient or
a health professional; (d) has, or is at risk of having, an HIV
infection or AIDS, a cancer, a solid organ transplantation, a stem
cell transplantation, a sickle cell disease or asplenia, a
congenital immune deficiency, a chronic inflammatory condition, a
cochlear implant, malnutrition, or a cerebrospinal fluid leak; or
(e) is 18 years old or younger, 15 years old or younger, 12 years
old or younger, 9 years old or younger, or 6 years old or younger,
or is at least 60 years old, at least 65 years old, at least 70
years old, at least 75 years old, or at least 80 years old.
32. (canceled)
33. The method of claim 28, wherein the second antimicrobial agent
or therapy comprises an antibiotic or a phage therapy, wherein the
antibiotic is chosen from: (a) an aminoglycoside, an ansamycin, a
carbacephem, a carbapenem, a cephalosporin, a glycopeptide, a
lincosamide, a lipopeptide, a macrolide, a monobactam, a
nitrofuran, an oxazolidinone, a penicillin, a penicillin
combination, a polypeptide, a quinolone or fluoroquinolone, a
sulfonamide, a tetracycline, or a drug against mycobacteria; or (b)
amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin,
paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin,
or rifaximin, loracarbef, ertapenem, doripenem,
imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin,
cefalothin, cephalexin, cefaclor, cefamandole, cefoxitin,
cefprozil, cefuroxime, cefixime, cefdinir, cefditoren,
cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,
ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil,
ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin,
oritavancin, clindamycin, lincomycin, daptomycin, azithromycin,
clarithromycin, dirithromycin, erythromycin, roxithromycin,
troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone,
nitrofurantoin, linezolid, posizolid, radezolid, torezolid,
amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,
dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin,
oxacillin, penicillin g, penicillin v, piperacillin, penicillin g,
temocillin, ticarcillin, amoxicillin/clavulanate,
ampicillin/sulbactam, piperacillin/tazobactam,
ticarcillin/clavulanate, bacitracin, colistin, polymyxin b,
ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin,
lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin,
trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide,
sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine,
sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine,
sulfisoxazole, trimethoprim-sulfamethoxazole (co-trimoxazole),
sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline,
oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin,
cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,
rifampin, rifabutin, rifapentine, streptomycin, arsphenamine,
chloramphenicol, fosfomycin, fusidic acid, metronidazole,
mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol,
tigecycline, tinidazole, or trimethoprim; and/or wherein the second
antimicrobial agent or therapy is administered before the ADC is
administered, concurrently with the administration of the ADC, or
after the ADC is administered.
34. (canceled)
35. A method of inhibiting or reducing a bacterial infection,
comprising contacting a cell the ADC of claim 1 in an amount
effective to inhibit or reduce the bacterial infection.
36. A kit comprising: the ADC of claim 1 and instructions for use
of the ADC.
37. A container comprising the ADC of claim 1.
38. A nucleic acid molecule encoding the ADC of claim 1.
39. A vector comprising the nucleic acid molecule of claim 38.
40. A cell comprising the nucleic acid molecule of 38.
41. A method of producing an ADC, the method comprising culturing
the cell of claim 40 under conditions that allow production of an
ADC, thereby producing the ADC.
42. A method of producing an ADC, the method comprising contacting
an antibody molecule that binds to LPS with a peptide comprising an
antimicrobial peptide, and optionally, a sortase donor sequence, in
the presence of a sortase, under conditions that allow a
sortase-mediated reaction to occur, thereby producing the ADC.
43. An antibody molecule that binds to LPS, wherein: (i) the
antibody molecule comprises a VH and a VL, wherein the VH comprises
three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the VL comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), and
wherein the ADC or antibody molecule comprises: (a) an HCDR1
comprising the amino acid sequence of SEQ ID NO: 108; an HCDR2
comprising the amino acid sequence of any of SEQ ID NOS: 109, 145,
or 146; an HCDR3 comprising the amino acid sequence of SEQ ID NO:
107; an LCDR1 comprising the amino acid sequence of any of SEQ ID
NOS: 110, 138, 140, or 144; an LCDR2 comprising the amino acid
sequence of any of SEQ ID NOS: 111, 139, 141, 142, or 143; and an
LCDR3 comprising the amino acid sequence of SEQ ID NO: 112, or (b)
an HCDR1 comprising the amino acid sequence of SEQ ID NO: 105; an
HCDR2 comprising the amino acid sequence of SEQ ID NO: 106; an
HCDR3 comprising the amino acid sequence of SEQ ID NO: 107; an
LCDR1 comprising the amino acid sequence of any of SEQ ID NOS: 110,
138, 140, or 144; an LCDR2 comprising the amino acid sequence of
any of SEQ ID NOS: 111, 139, 141, 142, or 143; and an LCDR3
comprising the amino acid sequence of SEQ ID NO: 112; or (ii) the
VH comprises an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid
residues from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100%
homology with, the amino acid sequence of any of SEQ ID NOS: 103 or
115-118, and wherein the VL comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of any
of SEQ ID NOS: 104 or 119-137.
44. (canceled)
45. An antimicrobial peptide that comprises an amino acid sequence
that differs by no more than 1, 2, 3, 4, or 5 amino acid residues
from, or has at least 80, 85, 90, 95, 96, 97, 98, 99, or 100%
homology with, the amino acid sequence of any of SEQ ID NOS: 67-80,
94-102, 147-156, 158-159, or 163-164.
46. A nucleic acid molecule encoding the antibody molecule of claim
43.
47. A nucleic acid molecule encoding the antimicrobial peptide of
claim 45.
48. An antibody molecule that: (i) binds to the same epitope, or
substantially the same epitope, or (ii) competes for binding with
the antibody molecule of claim 43.
49. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/348,436, filed Nov. 10, 2016, which claims
the benefit of U.S. Provisional Application No. 62/253,345, filed
Nov. 10, 2015, and U.S. Provisional Application No. 62/375,800,
filed Aug. 16, 2016. The contents of the aforementioned
applications are hereby incorporated by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 10, 2016, is named P2029-7003WO_SL.txt and is 85,211 bytes
in size.
BACKGROUND
[0003] A wide range of bacteria can cause infections that lead to
mild to serious illnesses. Bacterial infections are often treated
by antibiotics. However, the emergence of antibiotic-resistant
bacterial strains has complicated the treatment of infections.
Antibiotic-resistant infections often result in greater disability
and death compared with infections that are easily treatable with
antibiotics. According to the Centers for Disease Control and
Prevention (CDC), each year in the United States, at least 2
million people acquire serious infections with bacteria that are
resistant to one or more of the antibiotics designed to treat those
infections. At least 23,000 people die each year as a direct result
of these antibiotic-resistant infections. These estimates were
based on conservative assumptions and are likely minimum estimates.
More patients may die from other conditions that were complicated
by a bacterial infection. When first-line and then second-line
antibiotic treatment options are limited by resistance or are
unavailable, healthcare providers are forced to use antibiotics
that may be more toxic to the patient and frequently more expensive
and less effective. In many cases, antibiotic-resistant infections
require prolonged or costlier treatments, extend hospital stays,
and necessitate additional doctor visits and healthcare use.
[0004] The use of antibiotics is one of the most important factors
leading to antibiotic resistance around the world. Antibiotics are
among the most commonly prescribed drugs used in human medicine.
However, according to CDC, up to 50% of all the antibiotics
prescribed for people are not needed or are not optimally effective
as prescribed. Antibiotics are also commonly used in food animals
to prevent, control, and treat disease, and to promote the growth
of food-producing animals. The resistant strains of bacteria may
spread from person to person, or from the non-human sources in the
environment, including food.
[0005] There is a need for developing new approaches for treating,
preventing and diagnosing bacterial infections.
SUMMARY
[0006] This disclosure provides, at least in part, antibody
molecules or antibody molecule-drug conjugates (ADC) that bind to
bacteria, e.g., Gram-negative bacteria, e.g., lipopolysaccharides
(LPS) on the outer membrane of Gram-negative bacteria, and that
comprise functional and structural properties disclosed herein. In
an embodiment, the antibody molecule or ADC binds to a core
pentasaccharide region of the LPS. In an embodiment, the antibody
molecule or ADC binds to, inhibits, and/or reduces the viability
of, one or more bacteria, e.g., Gram-negative bacteria, of
different genera, species, and/or subspecies. In an embodiment, the
antibody molecule is selected from Table 1 or 8. In an embodiment,
the ADC comprises an antibody molecule that is selected from Table
1 or 8. In an embodiment, the antibody molecule or ADC comprises
one or more heavy chain variable regions and/or one or more light
chain variable regions described in Table 1 or 8. In an embodiment,
the antibody molecule or ADC comprises one or more heavy chain CDRs
and/or one or more light chain CDRs described in Table 1 or 8. In
an embodiment, the ADC comprises an antimicrobial peptide, e.g., an
antimicrobial peptide described herein, e.g., in Tables 3 or 6A-6B,
or in FIG. 4 or 15A-15B. While not wishing to be bound by theory,
it is believed that in an embodiment, the conjugation of an
antibody molecule with an antimicrobial peptide may improve one or
more properties of the antibody molecule and/or antimicrobial
peptide, e.g., improve the ability of an antimicrobial peptide to
inhibit, or reduce the viability, of one or more bacteria, e.g.,
one or more Gram-negative bacteria, of different genera, species,
and/or subspecies. Nucleic acid molecules encoding the antibody
molecules, ADCs, or antimicrobial peptides, expression vectors,
host cells, compositions (e.g., pharmaceutical compositions), kits,
and methods for making the antibody molecules, ADCs, or
antimicrobial peptides are also provided. The antibody molecules,
ADCs, and antimicrobial peptides disclosed herein can be used
(alone or in combination with other agents or therapeutic
modalities) to treat, prevent and/or diagnose bacterial infections
or related disorders, e.g., caused by or associated with
Gram-negative bacteria.
[0007] Accordingly, in certain aspects, this disclosure provides an
antibody molecule-drug conjugate (ADC), e.g., an ADC comprising an
antibody molecule described herein and an antimicrobial peptide
(e.g., an antimicrobial peptide described herein), having one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or all) of the
following properties: [0008] a) Binds to one or more (e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10, or more) Gram-negative bacteria of different
genera, species, and/or subspecies (e.g., one or more bacteria from
Enterobacteriaceae (e.g., Klebsiella, Enterobacter, Shigella,
Escherichia, Salmonella, or Citrobacter, e.g., pan-resistant
Enterobacteriaceae), one or more bacteria from Pseudomonas, one or
more bacteria from Acinetobacter, or any combination thereof) with
high affinity, e.g., with a dissociation constant (K.sub.D) of less
than about 100 nM, typically about 10 nM, and more typically, about
10-0.01 nM, about 5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or
stronger, e.g., less than about 80, 70, 60, 50, 40, 30, 20, 10, 8,
6, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM, [0009] b) Binds to
lipopolysaccharide (LPS) with high affinity, e.g., with a
dissociation constant (K.sub.D) of less than about 100 nM,
typically about 10 nM, and more typically, about 10-0.01 nM, about
5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger, e.g., less
than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5,
0.2, 0.1, 0.05, or 0.01 nM, [0010] c) Inhibits one or more (e.g.,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram-negative bacteria of
different genera, species, and/or subspecies (e.g., one or more
Gram-negative bacteria described herein), e.g., as determined by
measuring the minimum inhibitory concentration (MIC) of the ADC,
e.g., by a method described herein, [0011] d) Inhibits one or more
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram-negative bacteria
of different genera, species, and/or subspecies (e.g., one or more
Gram-negative bacteria described herein) with a lower MIC compared
to the antimicrobial peptide alone, e.g., at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500,
1,000-fold lowered MIC, e.g., on a molar basis, e.g., as measured
by a method described herein, [0012] e) Reduces the viability of
one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
Gram-negative bacteria of different genera, species, and/or
subspecies (e.g., one or more Gram-negative bacteria described
herein), e.g., as determined by measuring the minimum bactericidal
concentration (MBC), e.g., by a method described herein, [0013] f)
Reduces the viability of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) Gram-negative bacteria of different genera, species,
and/or subspecies (e.g., one or more Gram-negative bacteria
described herein) with a lower MBC compared to the antimicrobial
peptide alone, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 500, 1,000-fold lowered MBC,
e.g., on a molar basis, e.g., as measured by a method described
herein, [0014] g) Displays an opsonophagocytic activity (OPA),
e.g., determined by an OPA assay, e.g., as described herein, [0015]
h) Binds specifically to an epitope on LPS, e.g., the same or
similar epitope as the epitope recognized by an antibody molecule
described in Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0016] i) Shows the
same or similar binding affinity or specificity, or both, as an
antibody molecule described in Table 1 or 8, e.g., mAb001 (e.g., a
humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
[0017] j) Shows the same or similar binding affinity or
specificity, or both, as an ADC comprising an antibody molecule
described in Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0018] k) Shows the
same or similar binding affinity or specificity, or both, as an
antibody molecule comprising a heavy chain variable region and/or
light chain variable region described in Table 1 or 8, e.g., a
heavy chain variable region and/or light chain variable region of
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0019] l) Shows the same or similar binding affinity
or specificity, or both, as an ADC comprising a heavy chain
variable region and/or light chain variable region described in
Table 1 or 8, e.g., a heavy chain variable region and/or light
chain variable region of mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0020] m) Shows the
same or similar binding affinity or specificity, or both, as an
antibody molecule comprising one or more (e.g., two or three) heavy
chain CDRs and/or one or more (e.g., two or three) light chain CDRs
described in Table 1 or 8, e.g., one or more (e.g., two or three)
heavy chain CDRs and/or one or more (two or three) light chain CDRs
of mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7,
3G1, 2C7, or 3D6, [0021] n) Shows the same or similar binding
affinity or specificity, or both, as an ADC comprising one or more
(e.g., two or three) heavy chain CDRs and/or one or more (e.g., two
or three) light chain CDRs described in Table 1 or 8, e.g., one or
more (e.g., two or three) heavy chain CDRs and/or one or more (two
or three) light chain CDRs of mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0022] o) Shows the
same or similar binding affinity or specificity, or both, as an
antibody molecule comprising an amino acid sequence shown in Table
1 or 8, [0023] p) Shows the same or similar binding affinity or
specificity, or both, as an ADC comprising an amino acid sequence
shown in Table 1 or 8, [0024] q) Inhibits, e.g., competitively
inhibits, the binding of a second antibody molecule to a
Gram-negative bacterium, LPS, or both, wherein the second antibody
molecule is an antibody molecule chosen from Table 1 or 8, e.g.,
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0025] r) Inhibits, e.g., competitively inhibits, the
binding of a second ADC comprising a second antibody molecule to a
Gram-negative bacterium, LPS, or both, wherein the second antibody
molecule is an antibody molecule chosen from Table 1 or 8, e.g.,
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0026] s) Binds the same or an overlapping epitope as
a second antibody molecule, wherein the second antibody molecule is
an antibody molecule chosen from Table 1 or 8, e.g., mAb001 (e.g.,
a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
[0027] t) Binds the same or an overlapping epitope with a second
ADC comprising a second antibody molecule, wherein the second
antibody molecule is an antibody molecule chosen from Table 1 or 8,
e.g., mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1,
3E7, 3G1, 2C7, or 3D6, [0028] u) Competes for binding with a second
antibody molecule to a Gram-negative bacterium, LPS, or both,
wherein the second antibody molecule is an antibody molecule chosen
from Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0029] v) Competes
for binding with a second ADC comprising a second ADC to a
Gram-negative bacterium, LPS, or both, wherein the second antibody
molecule is an antibody molecule chosen from Table 1 or 8, e.g.,
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0030] w) Has one or more biological properties of an
antibody molecule chosen from Table 1 or 8, e.g., mAb001 (e.g., a
humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
[0031] x) Has one or more biological properties of an ADC
comprising an antibody molecule chosen from Table 1 or 8, e.g.,
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0032] y) Has one or more pharmacokinetic properties
of ADC comprising an antibody molecule chosen from Table 1 or 8,
e.g., mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1,
3E7, 3G1, 2C7, or 3D6, [0033] z) Reduces the viability of
Gram-negative bacteria from a first genus, species, or subspecies
(e.g., Pseudomonas) with high selectivity, compared to the
reduction of viability of Gram-negative bacteria from a second
genus, species, or subspecies (e.g., E. coli, Klebsiella spp., or
both), e.g., at least at least 2, 5, 10, 20, 50, 100, 200, 500, or
1000 fold more in % killing, e.g., as determined by a mixed
microbial killing assay described herein, [0034] aa) Binds to one
or more P. aeruginosa strains (e.g., one or more P. aeruginosa
strains described in Table 7) with high affinity, e.g., with an
avidity EC.sub.50 of about 200 pM or less, e.g., less than about
150 pM or less, about 120 pM or less, about 100 pM or less, about
80 pM or less, about 60 pM or less, or about 40 pM or less, e.g.,
between about 40 pM and about 120 pM, between about 50 pM and 110
pM, between about 60 pM and 100 pM, between about 40 pM and 80 pM,
or between 80 pM and 120 pM, or [0035] bb) Inhibits one or more
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram-negative bacteria
of different genera, species, and/or subspecies (e.g., one or more
Gram-negative bacteria described herein, e.g., P. aeruginosa) in
vivo, e.g., at least 2, 5, 10, 20, 50, 100, 200, 500, 1000, or more
fold reduction in bacterial burden, e.g., as determined using an
animal model, e.g., a murine acute pneumonia model described
herein.
[0036] Accordingly, in certain aspects, this disclosure provides an
antibody molecule, e.g., an antibody molecule described herein,
having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, or all) of the following properties: [0037] a) Binds to
one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
Gram-negative bacteria of different genera, species, and/or
subspecies (e.g., one or more bacteria from Enterobacteriaceae
(e.g., Klebsiella, Enterobacter, Shigella, Escherichia, Salmonella,
or Citrobacter, e.g., pan-resistant Enterobacteriaceae), one or
more bacteria from Pseudomonas, one or more bacteria from
Acinetobacter, or any combination thereof) with high affinity,
e.g., with a dissociation constant (K.sub.D) of less than about 100
nM, typically about 10 nM, and more typically, about 10-0.01 nM,
about 5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger,
e.g., less than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3,
2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM, [0038] b) Binds to
lipopolysaccharide (LPS) with high affinity, e.g., with a
dissociation constant (K.sub.D) of less than about 100 nM,
typically about 10 nM, and more typically, about 10-0.01 nM, about
5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger, e.g., less
than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5,
0.2, 0.1, 0.05, or 0.01 nM, [0039] c) Displays an
opsonophagocytosis activity (OPA), e.g., determined by an OPA
assay, e.g., as described herein, [0040] d) Binds specifically to
an epitope on LPS, e.g., the same or similar epitope as the epitope
recognized by an antibody molecule described in Table 1 or 8, e.g.,
mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, [0041] e) Shows the same or similar binding affinity
or specificity, or both, as an antibody molecule described in Table
1 or 8, e.g., mAb001 (e.g., a humanized mAb001), A001-25, hWN01,
hWNv1, 3E7, 3G1, 2C7, or 3D6, [0042] f) Shows the same or similar
binding affinity or specificity, or both, as an ADC comprising an
antibody molecule described in Table 1 or 8, e.g., mAb001 (e.g., a
humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
[0043] g) Shows the same or similar binding affinity or
specificity, or both, as an antibody molecule comprising a heavy
chain variable region and/or light chain variable region described
in Table 1 or 8, e.g., a heavy chain variable region and/or light
chain variable region of mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0044] h) Shows the
same or similar binding affinity or specificity, or both, as an
antibody molecule comprising one or more (e.g., two or three) heavy
chain CDRs and/or one or more (e.g., two or three) light chain CDRs
described in Table 1 or 8, e.g., one or more (e.g., two or three)
heavy chain CDRs and/or one or more (two or three) light chain CDRs
of mAb001 (e.g., a humanized mAb001), A001-25, hWN01, hWNv1, 3E7,
3G1, 2C7, or 3D6, [0045] i) Shows the same or similar binding
affinity or specificity, or both, as an antibody molecule
comprising an amino acid sequence shown in Table 1 or 8, [0046] j)
Inhibits, e.g., competitively inhibits, the binding of a second
antibody molecule to a Gram-negative bacterium, LPS, or both,
wherein the second antibody molecule is an antibody molecule chosen
from Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0047] k) Binds the
same or an overlapping epitope as a second antibody molecule,
wherein the second antibody molecule is an antibody molecule chosen
from Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0048] l) Competes
for binding with a second antibody molecule to a Gram-negative
bacterium, LPS, or both, wherein the second antibody molecule is an
antibody molecule chosen from Table 1 or 8, e.g., mAb001 (e.g., a
humanized mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
[0049] m) Has one or more biological properties of an antibody
molecule chosen from Table 1 or 8, e.g., mAb001 (e.g., a humanized
mAb001), A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0050] n)
Has one or more biological properties of an antibody molecule
chosen from Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001),
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, [0051] o) Has one or
more pharmacokinetic properties of an antibody molecule chosen from
Table 1 or 8, e.g., mAb001 (e.g., a humanized mAb001), A001-25,
hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or [0052] p) Binds to one or
more P. aeruginosa strains (e.g., one or more P. aeruginosa strains
described in Table 7) with high affinity, e.g., with an avidity
EC.sub.50 of about 200 pM or less, e.g., less than about 150 pM or
less, about 120 pM or less, about 100 pM or less, about 80 pM or
less, about 60 pM or less, or about 40 pM or less, e.g., between
about 40 pM and about 120 pM, between about 50 pM and 110 pM,
between about 60 pM and 100 pM, between about 40 pM and 80 pM, or
between 80 pM and 120 pM.
[0053] In an aspect, the disclosure features an antibody
molecule-drug conjugate (ADC) comprising a) an antibody molecule
that binds to lipopolysaccharide (LPS) and b) an antimicrobial
peptide.
[0054] In an embodiment, the ADC or antibody molecule binds to a
core pentasaccharide region of the LPS. In an embodiment, the core
pentasaccharide region comprises one or more (e.g., two) Kdo
residues and one or more (e.g., two or three) Hep residues. In an
embodiment, the ADC or antibody molecule binds to one or more
(e.g., two) Kdo residues, or one or more (e.g., two or three) Hep
residues, or any combination thereof.
[0055] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable
region comprises one, two, or all of the following: an HCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 108); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS:
109, 145, or 146); or an HCDR3 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR3 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 107).
[0056] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 108); an HCDR2 comprising the amino acid sequence
of the HCDR2 of antibody mAb001 or humanized mAb001 (e.g., any of
SEQ ID NOS: 109, 145, or 146); or an HCDR3 comprising the amino
acid sequence of the HCDR3 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 107).
[0057] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 108); an
HCDR2 comprising the amino acid sequence of the HCDR2 of antibody
mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 109, 145, or
146); and an HCDR3 comprising the amino acid sequence of the HCDR3
of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 107).
[0058] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 110,
138, 140, or 144); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody mAb001 or humanized mAb001 (e.g.,
any of SEQ ID NOS: 111, 139, 141, 142, or 143); or an LCDR3
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR3 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 112).
[0059] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody mAb001
or humanized mAb001 (e.g., any of SEQ ID NOS: 111, 139, 141, 142,
or 143); or an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO:
112).
[0060] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 110,
138, 140, or 144); an LCDR2 comprising the amino acid sequence of
the LCDR2 of antibody mAb001 or humanized mAb001 (e.g., any of SEQ
ID NOS: 111, 139, 141, 142, or 143); and an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody mAb001 or humanized
mAb001 (e.g., SEQ ID NO: 112).
[0061] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3),
[0062] wherein the heavy chain variable region comprises one, two,
or all of the following: an HCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 108); an HCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR2 of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 109, 145, or 146); or an HCDR3 comprising
an amino acid sequence that differs by no more than 1, 2, or 3
amino acid residues from, or has at least 85, 90, 95, 99 or 100%
homology with, the amino acid sequence of the HCDR3 of antibody
mAb001 or humanized mAb001 (e.g., SEQ ID NO: 107), and
[0063] wherein the light chain variable region comprises one, two,
or all of the following: an LCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR1 of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR2 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 111, 139,
141, 142, or 143); or an LCDR3 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR3 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 112).
[0064] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 108); an HCDR2 comprising the amino acid sequence
of the HCDR2 of antibody mAb001 or humanized mAb001 (e.g., any of
SEQ ID NOS: 109, 145, or 146); or an HCDR3 comprising the amino
acid sequence of the HCDR3 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 107), and the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising
the amino acid sequence of the LCDR1 of antibody mAb001 or
humanized mAb001 (e.g., any of SEQ ID NOS: 110, 138, 140, or 144);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 111,
139, 141, 142, or 143); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody mAb001 or humanized mAb001 (e.g.,
SEQ ID NO: 112).
[0065] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 108); an
HCDR2 comprising the amino acid sequence of the HCDR2 of antibody
mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 109, 145, or
146); and an HCDR3 comprising the amino acid sequence of the HCDR3
of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 107), and
the light chain variable region comprises an LCDR1 comprising the
amino acid sequence of the LCDR1 of antibody mAb001 or humanized
mAb001 (e.g., any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody mAb001
or humanized mAb001 (e.g., any of SEQ ID NOS: 111, 139, 141, 142,
or 143); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO:
112).
[0066] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable
region comprises one, two, or all of the following: an HCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 105); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 106); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 107).
[0067] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 105); an HCDR2 comprising the amino acid sequence
of the HCDR2 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID
NO: 106); or an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO:
107).
[0068] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 105); an
HCDR2 comprising the amino acid sequence of the HCDR2 of antibody
mAb001 or humanized mAb001 (e.g., SEQ ID NO: 106); and an HCDR3
comprising the amino acid sequence of the HCDR3 of antibody mAb001
or humanized mAb001 (e.g., SEQ ID NO: 107).
[0069] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 110,
138, 140, or 144); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody mAb001 or humanized mAb001 (e.g.,
any of SEQ ID NOS: 111, 139, 141, 142, or 143); or an LCDR3
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR3 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 112).
[0070] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody mAb001
or humanized mAb001 (e.g., any of SEQ ID NOS: 111, 139, 141, 142,
or 143); or an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO:
112).
[0071] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 110,
138, 140, or 144); an LCDR2 comprising the amino acid sequence of
the LCDR1 of antibody mAb001 or humanized mAb001 (e.g., any of SEQ
ID NOS: 111, 139, 141, 142, or 143); and an LCDR3 comprising the
amino acid sequence of the LCDR1 of antibody mAb001 or humanized
mAb001 (e.g., SEQ ID NO: 112).
[0072] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3),
[0073] wherein the heavy chain variable region comprises one, two,
or all of the following: an HCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 105); an HCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR2 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 106); or an HCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody mAb001 or
humanized mAb001 (e.g., SEQ ID NO: 107), and
[0074] wherein the light chain variable region comprises one, two,
or all of the following: an LCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR1 of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR2 of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 111,
139, 141, 142, or 143); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody mAb001 or
humanized mAb001 (e.g., SEQ ID NO: 112).
[0075] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 or humanized mAb001
(e.g., SEQ ID NO: 105); an HCDR2 comprising the amino acid sequence
of the HCDR2 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID
NO: 106); or an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO:
107), and
[0076] the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody mAb001 or humanized mAb001 (e.g., any of SEQ
ID NOS: 110, 138, 140, or 144); an LCDR2 comprising the amino acid
sequence of the LCDR2 of antibody mAb001 or humanized mAb001 (e.g.,
any of SEQ ID NOS: 111, 139, 141, 142, or 143); or an LCDR3
comprising the amino acid sequence of the LCDR3 of antibody mAb001
or humanized mAb001 (e.g., SEQ ID NO: 112).
[0077] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 105); an
HCDR2 comprising the amino acid sequence of the HCDR2 of antibody
mAb001 or humanized mAb001 (e.g., SEQ ID NO: 106); and an HCDR3
comprising the amino acid sequence of the HCDR3 of antibody mAb001
or humanized mAb001 (e.g., SEQ ID NO: 107), and the light chain
variable region comprises an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody mAb001 or humanized mAb001 (e.g.,
any of SEQ ID NOS: 110, 138, 140, or 144); an LCDR2 comprising the
amino acid sequence of the LCDR2 of antibody mAb001 or humanized
mAb001 (e.g., any of SEQ ID NOS: 111, 139, 141, 142, or 143); and
an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody mAb001 or humanized mAb001 (e.g., SEQ ID NO: 112).
[0078] In an embodiment, the ADC or antibody molecule further
comprises one or more human or human derived heavy or light chain
variable region frameworks.
[0079] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises an amino acid sequence that differs by no more
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino
acid residues from, or has at least 85, 90, 95, 96, 97, 98, 99, or
100% homology with, the amino acid sequence of the VH of antibody
mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 103 or
115-118). In an embodiment, the heavy chain variable region
comprises the amino acid sequence of the VH of antibody mAb001 or
humanized mAb001 (e.g., any of SEQ ID NOs: 103 or 115-118).
[0080] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises an amino acid sequence that differs by no more
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino
acid residues from, or has at least 85, 90, 95, 96, 97, 98, 99, or
100% homology with, the amino acid sequence of the VL of antibody
mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 104 or
119-137). In an embodiment, the light chain variable region
comprises the amino acid sequence of the VL of antibody mAb001 or
humanized mAb001 (e.g., any of SEQ ID NOS: 104 or 119-137).
[0081] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises an amino
acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 amino acid residues from, or has at
least 85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino
acid sequence of the VH of antibody mAb001 or humanized mAb001
(e.g., any of SEQ ID NOS: 103 or 115-118), and wherein the light
chain variable region comprises an amino acid sequence that differs
by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15 amino acid residues from, or has at least 85, 90, 95, 96, 97,
98, 99, or 100% homology with, the amino acid sequence of the VL of
antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS: 104
or 119-137).
[0082] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of antibody mAb001 or humanized
mAb001 (e.g., any of SEQ ID NOS: 103 or 115-118) and the light
chain variable region comprises the amino acid sequence of the VL
of antibody mAb001 or humanized mAb001 (e.g., any of SEQ ID NOS:
104 or 119-137).
[0083] In an embodiment, the ADC or antibody molecule comprises or
consists of two heavy chain variable regions and two light chain
variable regions. In an embodiment, the ADC or antibody molecule
comprises a Fab, a F(ab')2, an Fv, or a single chain Fv fragment
(scFv).
[0084] In an embodiment, the ADC or antibody molecule further
comprises a heavy chain constant region, a light chain constant
region, or both. In an embodiment, the ADC or antibody molecule is
an IgG antibody molecule, e.g., IgG1, IgG2, IgG3, or IgG4 antibody
molecule. In an embodiment, the antibody molecule is not an IgM
antibody molecule. In an embodiment, the ADC or antibody molecule
comprises a light chain constant region from a kappa or lambda
light chain.
[0085] In an embodiment, the antibody molecule is a monoclonal
antibody molecule. In an embodiment, the antibody molecule is a
humanized antibody molecule. In an embodiment, the antibody
molecule is an isolated antibody molecule. In an embodiment, the
antibody molecule is a synthetic antibody molecule.
[0086] In an embodiment, the ADC or antibody molecule binds to one
or more bacteria, e.g., one or more Gram-negative bacteria, e.g.,
of different genera, species, subspecies, and/or strains.
[0087] In an embodiment, the one or more Gram-negative bacteria are
selected from a species of Enterobacteriaceae (e.g., a species in
Klebsiella, Enterobacter, Shigella, Escherichia, Salmonella,
Yersinia, or Citrobacter, e.g., pan-resistant Enterobacteriaceae),
a species of Pseudomonas, a species of Acinetobacter, or any
combination thereof.
[0088] In an embodiment, the ADC or antibody molecule binds to one
or more of: Klebsiella pneumonia (e.g., Klebsiella pneumoniae
subsp. ozaenae, Klebsiella pneumoniae subsp. pneumoniae, or
Klebsiella pneumoniae subsp. rhinoscleromatis), Enterobacter
cancerogenous, Enterobacter cloacae, Enterobacter hormaechei,
Enterobacter asburiae, Shigella boydii, Shigella dysenteriae,
Shigella flexneri, Shigella sonnei, Escherichia coli (e.g.,
Escherichia coli ATCC 11775, Escherichia coli ATCC 25922,
Escherichia coli ATCC 35401, or Escherichia coli ATCC 43895),
Escherichia fergusonii, Salmonella choleraesuis, Salmonella
choleraesuis subsp. indica, Salmonella enteritidis, Salmonella
virchow, Salmonella paratyphi B, Salmonella typhimurium, Salmonella
paratyphi A, Salmonella typhi, Salmonella choleraesuis subsp.
arizonae, Salmonella choleraesuis subsp. diarizonae, Salmonella
choleraesuis subsp. houtenae, Salmonella bongori, Citrobacter
sedlakii, Citrobacter braakii, Citrobacter werkmanii, Citrobacter
freundii, Citrobacter youngae, Citrobacter amalonaticus, Yersinia
enterocolitica, Yersinia frederiksenii, Yersinia pestis, Yersinia
pseudotuberculosis, Pseudomonas aeruginosa, Acinetobacter
baumannii, or any combination thereof. In an embodiment, the ADC or
antibody molecule binds to Pseudomonas aeruginosa.
[0089] In an embodiment, the one or more bacteria are one or more
antibiotic-resistant bacteria, e.g., one or more
multidrug-resistant Gram-negative bacteria.
[0090] In an embodiment, the one or more antibiotic-resistant
bacteria are selected from Pseudomonas (e.g., P. aeruginosa),
Enterobacteriaceae (e.g., Klebsiella pneumonia or E. coli), or
Acinetobacter (e.g., A. baumannii).
[0091] In an embodiment, the ADC or antibody molecule binds to one
or more of: Enterococcus faecium (e.g., vancomycin-resistant (VRE)
Enterococcus faecium), Staphylococcus aureus (e.g.,
methicillin-resistant (MRSA) Staphylococcus aureus), Clostridium
difficile, Acinetobacter baumannii (e.g., multidrug resistant (MDR)
Acinetobacter), Pseudomonas aeruginosa (e.g., multidrug resistant
(MDR) P. aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis).
[0092] In an embodiment, the ADC or antibody molecule binds to one
or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) P. aeruginosa
strains in Table 7. In another embodiment, the ADC or antibody
molecule binds to one or more (e.g., 2, 3, 4, 5, 6, or all)
multidrug-resistant P. aeruginosa strains in Table 7.
[0093] In an embodiment, the ADC or antibody molecule binds to LPS
with high affinity, e.g., with a K.sub.D that is less than about 10
nM, e.g., measured by an ELISA method.
[0094] In an embodiment, the ADC or antibody molecule binds to LPS
with a K.sub.off slower than 1.times.10.sup.-4, 5.times.10.sup.-5,
or 1.times.10.sup.-5 s.sup.-1. In an embodiment, the ADC or
antibody molecule binds to LPS with a K.sub.on faster than
1.times.10.sup.4, 5.times.10.sup.4, 1.times.10.sup.5, or
5.times.10.sup.5 M.sup.-1 s.sup.-1.
[0095] In an embodiment, the antibody molecule has opsonophagocytic
activity, e.g., as determined by an OPA assay, e.g., as described
herein.
[0096] In an embodiment, the ADC or antibody molecule binds to an
epitope comprising one or more (e.g., two) Kdo residues and/or one
or more (e.g., two or three) Hep residues in LPS.
[0097] In an embodiment, a) the antibody molecule that binds to
lipopolysaccharide (LPS) is coupled (e.g., fused) to b) the
antimicrobial peptide.
[0098] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
is coupled (e.g., fused) to the antimicrobial peptide. In an
embodiment, the heavy chain variable region is N-terminal to the
antimicrobial peptide. In another embodiment, the heavy chain
variable region is C-terminal to the antimicrobial peptide. In an
embodiment, the VH is fused to the antimicrobial peptide to form a
fusion polypeptide, e.g., encoded by an open reading frame.
[0099] In an embodiment, the heavy chain variable region is coupled
(e.g., fused) to the antimicrobial peptide indirectly, e.g.,
wherein the C-terminus of the heavy chain variable region is
coupled (e.g., fused) to the N-terminus of the antimicrobial
peptide via a constant region, a linker, or both.
[0100] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
is coupled (e.g., fused) to the antimicrobial peptide. In an
embodiment, the light chain variable region is N-terminal to the
antimicrobial peptide. In another embodiment, the heavy chain
variable region is C-terminal to the antimicrobial peptide. In an
embodiment, the VL is fused to the antimicrobial peptide to form a
fusion polypeptide, e.g., encoded by an open reading frame.
[0101] In an embodiment, the light chain variable region is coupled
(e.g., fused) to the antimicrobial peptide indirectly, e.g.,
wherein the C-terminus of the light chain variable region is
coupled (e.g., fused) to the N-terminus of the antimicrobial
peptide via a constant region, a linker, or both.
[0102] In an embodiment, the antibody molecule comprises a sortase
acceptor sequence comprising a sortase recognition sequence. For
example, the sortase recognition sequence can have the amino acid
sequence of LPXTG (e.g., for Staphylococcus aureus sortase A) (SEQ
ID NO: 160) or LPXTA (e.g., for Streptococcus pyogenes sortase A)
(SEQ ID NO: 161), wherein X can be any amino acid residue. In an
embodiment, the sortase recognition sequence is LPETG (SEQ ID NO:
162). The sortase acceptor sequence may contain additional
sequence(s) other than the sortase recognition sequence. In an
embodiment, the sortase acceptor sequence further comprises a
linker sequence, e.g., a tandem repeat of glycine-serine peptide
linker sequences (e.g., (GS).sub.15 (SEQ ID NO: 157)). In an
embodiment, a heavy chain of the antibody molecule comprises a
sortase acceptor sequence, e.g., at the C-terminus. In an
embodiment, a light chain of the antibody molecule comprises a
sortase acceptor sequence, e.g., at the C-terminus.
[0103] In an embodiment, a heavy chain of the antibody molecule
comprises a first sortase acceptor sequence and a light chain of
the antibody molecule comprises a second sortase acceptor sequence.
In an embodiment, the sortase acceptor sequence, e.g., the first
sortase recognition sequence, comprises the amino acid sequence of
(GS).sub.6LPETGGG (SEQ ID NO: 24). In another embodiment, the
sortase acceptor sequence, e.g., the second sortase acceptor
sequence, comprises the amino acid sequence of
P(G.sub.4S).sub.2LPETGGSG (SEQ ID NO: 26).
[0104] In an embodiment, the ADC comprises two or more (e.g.,
three, four, five, six, seven, eight, or more) antimicrobial
peptides. In an embodiment, at least two of the antimicrobial
peptides are identical or substantially identical. In an
embodiment, at least two of the antimicrobial peptides are
different. For example, a plurality of antimicrobial peptides can
be coupled (e.g., fused) to the antibody molecule (e.g., a heavy
chain (or a portion thereof), a light chain (or a portion thereof),
or both).
[0105] In an embodiment, the ADC comprises two or more (e.g., three
or four) identical, or substantially identical, antimicrobial
peptides, each is coupled (e.g., fused) to a heavy chain variable
region, e.g., indirectly, e.g., via a constant region, linker, or
both. In an embodiment, the ADC comprises two or more (e.g., three
or four) identical, or substantially identical, antimicrobial
peptides, each is coupled (e.g., fused) to a light chain variable
region, e.g., indirectly, e.g., via a constant region, linker, or
both. In an embodiment, the ADC comprises two identical, or
substantially identical, antimicrobial peptides, each is coupled
(e.g., fused) to a heavy chain variable region, e.g., indirectly,
e.g., via a constant region, linker, or both. In an embodiment, the
ADC comprises two identical, or substantially identical,
antimicrobial peptides, each is coupled (e.g., fused) to a light
chain variable region, e.g., indirectly, e.g., via a constant
region, linker, or both. In an embodiment, the ADC comprises at
least four identical, or substantially identical, antimicrobial
peptides. In an embodiment, the ADC or antibody molecule comprises
two heavy chain variable regions and two light chain variable
regions, and each of the heavy and light chain variable regions is
coupled (e.g., fused) with at least one antimicrobial peptide,
e.g., indirectly, e.g., via a constant region, linker, or both.
[0106] In an embodiment, the antimicrobial peptide is coupled to
the antibody molecule by enzymatic conjugation (e.g., a sortase
reaction). In an embodiment the antimicrobial peptide is coupled to
the antibody molecule by chemical conjugation.
[0107] In an embodiment, the ADC is more effective in inhibiting,
e.g., inhibiting the growth, virulence, or infectivity of, a
Gram-negative bacterium (e.g., a Gram-negative bacterium described
herein) than the antimicrobial peptide or antibody molecule alone,
e.g., having a minimum inhibitory concentration (MIC) that is
lower, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100 fold lower, than the MIC of the
antimicrobial peptide alone.
[0108] In an embodiment, the ADC is more effective in reducing the
viability of, e.g., killing, a Gram-negative bacterium (e.g., a
Gram-negative bacterium described herein) than the antimicrobial
peptide or antibody molecule alone, e.g., having a minimum
bactericidal concentration (MBC) that is lower, e.g., at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100
fold lower, than the MIC of the antimicrobial peptide alone.
[0109] In an embodiment, the ADC has opsonophagocytosis activity
(e.g., is phagocytized when bound to the Fc receptor (FcR) of a
neutrophil), e.g., as determined by an OPA assay, e.g., as
described herein.
[0110] In an embodiment, the ADC does not inhibit, e.g., does not
inhibit the growth, virulence, or infectivity of, a Gram-positive
bacterium (e.g., a Gram-positive bacterium described herein), e.g.,
having a minimum inhibitory concentration (MIC) for a Gram-negative
bacterium (e.g., a Gram-negative bacterium) that is lower, e.g., at
least 2, 5, 10, 20, 50, 100, 200, 500, or 1000 fold lower, than a
MIC for a Gram-positive bacterium (e.g., a Gram-positive
bacterium).
[0111] In an embodiment, the ADC does not reduce the viability of,
e.g., does not kill, a Gram-positive bacterium (e.g., a
Gram-positive bacterium described herein), e.g., having a minimum
bactericidal concentration (MBC) for a Gram-negative bacterium
(e.g., a Gram-negative bacterium) that is lower, e.g., at least 2,
5, 10, 20, 50, 100, 200, 500, or 1000 fold lower fold lower, than a
MBC for a Gram-positive bacterium (e.g., a Gram-positive
bacterium). In an embodiment, the Gram-positive bacterium is
Staphylococcus aureus.
[0112] In an embodiment, the ADC does not alter, or does not
significantly alter microbiome (e.g., is microbiome sparing).
[0113] In an embodiment, the antimicrobial peptide comprises or
consists of an alpha-helical antimicrobial peptide, e.g., a peptide
comprising turns where residues i and i+4 are on the same face.
[0114] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, or 5 amino acid residues from, or has at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with, an amino acid
sequence described herein, e.g., any of SEQ ID NOS: 67-80, 94-102,
147-156, 158-159, or 163-164. In an embodiment, the antimicrobial
peptide comprises or consists of an amino acid sequence described
herein e.g., any of SEQ ID NOS: 67-80, 94-102, 147-156, 158-159, or
163-164. In an embodiment, the antimicrobial peptide comprises a
carboxamide group (e.g., a C-terminal carboxamide functional
group).
[0115] In an embodiment, the antimicrobial peptide comprises 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more D-amino acids. In another
embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
or 100% of the amino acid residues of the antimicrobial peptide are
D-amino acids.
[0116] In an embodiment, the antimicrobial peptide comprises a
first cysteine residue and a second cysteine residue, and wherein
the first cysteine residue is cross-linked to the second cysteine
residue.
[0117] In an embodiment, the antimicrobial peptide is a broad
spectrum antimicrobial peptide, e.g., having antimicrobial activity
against 2, 3, or all of the following: at least one species of
Enterobacteriaceae (e.g., one or more species of Klebsiella,
Enterobacter, Shigella, Escherichia, Salmonella, Yersinia, or
Citrobacter, e.g., pan-resistant Enterobacteriaceae), at least one
species of Pseudomonas, or at least one species of
Acinetobacter.
[0118] In an embodiment, the antimicrobial peptide is a broad
spectrum antimicrobial peptide, e.g., having antimicrobial activity
against 2, 3, 4, 5, 6, or all of the following: at least one
species of Klebsiella, at least one species of Enterobacter, at
least one species of Shigella, at least one species of Escherichia,
at least one species of Salmonella, at least one species of
Yersinia, or at least one species of Citrobacter.
[0119] In an embodiment, the antimicrobial peptide has a minimum
inhibitory concentration (MIC) of less than 100 .mu.g/ml, e.g.,
less than 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 .mu.g/ml, for a
bacterial strain described herein, e.g., Escherichia coli (e.g.,
Escherichia coli ATCC 25922), Pseudomonas aeruginosa (e.g.,
Pseudomonas aeruginosa ATCC27853), or both.
[0120] In an embodiment, the antimicrobial peptide has a minimum
bactericidal concentration (MBC) of less than 100 .mu.g/ml, e.g.,
less than 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 .mu.g/ml, for a
bacterial strain described herein, e.g., Escherichia coli (e.g.,
Escherichia coli ATCC 25922), Pseudomonas aeruginosa (Pseudomonas
aeruginosa ATCC27853), or both.
[0121] In an embodiment, the antimicrobial peptide has low
hemolytic activity, e.g., has a partial lytic concentration (PLC)
to MIC ratio for a Gram-negative bacterium (e.g., a Gram-negative
bacterium described herein) which is greater than 4:1 (e.g.,
greater than 8:1, 16:1, 24:1, or 32:1), e.g., as determined by a
red blood cell hemolysis assay and an MIC assay, respectively. In
an embodiment, the PLC is the concentration (e.g., minimum
concentration) that results in lysis of 50% of the red blood
cells.
[0122] In an embodiment, the antimicrobial peptide has low
hemolytic activity, e.g., has a partial lytic concentration (MLC)
to MIC ratio for a Gram-negative bacterium (e.g., a Gram-negative
bacterium described herein) which is greater than 4:1 (e.g.,
greater than 8:1, 16:1, 24:1, or 32:1), e.g., as determined by a
red blood cell hemolysis assay and an MIC assay, respectively. In
an embodiment, the MLC is the concentration (e.g., minimum
concentration) that results in lysis of 100% of the red blood
cells.
[0123] In an aspect, the disclosure features a composition, e.g., a
pharmaceutical composition, comprising an ADC described herein and
a pharmaceutically acceptable carrier.
[0124] In an aspect, the disclosure features a method of treating
or preventing a bacterial infection or a related disorder,
comprising administering to a subject in need thereof an ADC
described herein, or a pharmaceutical composition described herein,
in an amount effective to treat or prevent the bacterial infection
or related disorder.
[0125] In an embodiment, the bacterial infection is a Gram-negative
bacterial infection. In an embodiment, the disorder is caused by,
or associated with, a Gram-negative bacterial infection.
[0126] In an embodiment, the ADC is administered at a dose of
0.1-100 mg/kg, e.g., 0.1-50 mg/kg or 1-10 mg/kg, e.g., 1-5 or 5-10
mg/kg. In an embodiment, the ADC is administered intravenously,
subcutaneously, or intranasally or by inhalation.
[0127] In an embodiment, the ADC is administered prior to onset of
a symptom associated with the bacterial infection or related
disorder. In an embodiment, the ADC is administered at or after
onset of a symptom associated with the bacterial infection or
related disorder.
[0128] In an embodiment, the subject has one or more of pneumonia
(e.g., community-acquired pneumonia and hospital-acquired
pneumonia), a urinary tract infection (UTI), septicemia,
meningitis, diarrhea (e.g., traveler's diarrhea), a soft tissue
infection, a skin infection, bacteremia, a respiratory system
infection (e.g., a lower respiratory tract infection),
endocarditis, an intra-abdominal infection, septic arthritis,
osteomyelitis, a CNS infection, an ophthalmic infection,
cholecystitis, cholangitis, meningitis (e.g., neonatal meningitis),
typhoid fever, food poisoning, gastroenteritis, enteric fever,
shigellosis, a blood stream infection, intra-abdominal sepsis, a
brain abscess, meningitis, sepsis (e.g., neonatal sepsis), a joint
infection, a bone infection, a gastrointestinal infection, or a
wound infection.
[0129] In an embodiment, the bacterial infection is a nosocomial
infection or a hospital-acquired infection. In an embodiment, the
disorder related to bacterial infection is associated with a
nosocomial infection or a hospital-acquired infection.
[0130] In an embodiment, the subject is a human or an animal. In an
embodiment, the subject is an immunocompromised patient, e.g., a
subject having an HIV infection or AIDS, cancer, solid organ
transplantation, stem cell transplantation, sickle cell disease or
asplenia, a congenital immune deficiency, a chronic inflammatory
condition, a cochlear implant, malnutrition, or a cerebrospinal
fluid leak. In an embodiment, the subject is a health
professional.
[0131] In an embodiment, the subject is 18 years old or younger,
e.g., 15 years old or younger, 12 years old or younger, 9 years old
or younger, 6 years old or younger, or 3 years old or younger. In
an embodiment, the subject is at least 60 years old, e.g., at least
65 years old, at least 70 years old, at least 75 years old, or at
least 80 years old.
[0132] In an embodiment, the method further comprises administering
to the subject a second antimicrobial agent or therapy, e.g., an
antibiotic or phage therapy.
[0133] In an embodiment, the antibiotic is selected from the group
consisting of an aminoglycoside (e.g., amikacin, gentamicin,
kanamycin, neomycin, netilmicin, tobramycin, paromomycin,
streptomycin, or spectinomycin), an ansamycin (e.g., geldanamycin,
herbimycin, or rifaximin), a carbacephem (e.g., loracarbef), a
carbapenem (e.g., ertapenem, doripenem, imipenem/cilastatin, or
meropenem), a cephalosporin (cefadroxil, cefazolin, cefalotin,
cefalothin, cephalexin, cefaclor, cefamandole, cefoxitin,
cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone
(e.g., in combination with sulbactam), cefotaxime, cefpodoxime,
ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,
ceftaroline fosamil, or ceftobiprole), a glycopeptide (e.g.,
teicoplanin, vancomycin, telavancin, dalbavancin, or oritavancin),
a lincosamide (e.g., clindamycin or lincomycin), lipopeptide (e.g.,
daptomycin), a macrolide (e.g., azithromycin, clarithromycin,
dirithromycin, erythromycin, roxithromycin, troleandomycin,
telithromycin, or spiramycin), a monobactam (e.g., aztreonam), a
nitrofuran (e.g., furazolidone or nitrofurantoin), an oxazolidinone
(e.g., linezolid, posizolid, radezolid, torezolid), a penicillin
(e.g., amoxicillin, ampicillin, azlocillin, carbenicillin,
cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,
methicillin, nafcillin, oxacillin, penicillin g, penicillin v,
piperacillin, penicillin g, temocillin, or ticarcillin), a
penicillin combination (e.g., amoxicillin/clavulanate,
ampicillin/sulbactam, piperacillin/tazobactam, or
ticarcillin/clavulanate), a polypeptide (e.g., bacitracin,
colistin, or polymyxin b), a quinolone/fluoroquinolone (e.g.,
ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin,
lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin,
trovafloxacin, grepafloxacin, sparfloxacin, or temafloxacin), a
sulfonamide (e.g., mafenide, sulfacetamide, sulfadiazine, silver
sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole,
sulfanilimide, sulfasalazine, sulfisoxazole,
trimethoprim-sulfamethoxazole (co-trimoxazole), or
sulfonamidochrysoidine), a tetracycline (e.g., demeclocycline,
doxycycline, minocycline, oxytetracycline, or tetracycline), a drug
against mycobacteria (e.g., clofazimine, dapsone, capreomycin,
cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,
rifampin, rifabutin, rifapentine, or streptomycin), or others
(e.g., arsphenamine, chloramphenicol, fosfomycin, fusidic acid,
metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin,
thiamphenicol, tigecycline, tinidazole, or trimethoprim).
[0134] In an embodiment, the antibiotic is selected from
levofloxacin, ciprofloxacin, gentamicin, ceftriaxone, ofloxacin,
amikacin, tobramycin, aztreonam, or imipenem/cilastatin.
[0135] In an embodiment, the second antimicrobial agent or therapy
is administered before the ADC is administered, concurrently with
the administration of the ADC, or after the ADC is
administered.
[0136] In an aspect, the disclosure features a method of inhibiting
or reducing a bacterial infection (e.g., Gram-negative bacterial
infection), comprising contacting a cell (e.g., in a sample) or a
subject in need thereof an ADC described herein, or a
pharmaceutical composition described herein, in an amount effective
to inhibit or reduce the bacterial infection.
[0137] In an embodiment, the ADC is contacted with the cell or
subject in vitro, ex vivo, or in vivo.
[0138] In an aspect, the disclosure features an ADC described
herein for use in treating or preventing a bacterial infection
(e.g., a Gram-negative bacterial infection) or related disorder
described herein.
[0139] In another aspect, the disclosure features use of an ADC
described herein in the manufacture of a medicament for treating or
preventing a bacterial infection (e.g., a Gram-negative bacterial
infection) or related disorder described herein.
[0140] In an aspect, the disclosure features a kit comprising an
ADC described herein or a pharmaceutical composition described
herein.
[0141] In an embodiment, the kit further comprises instructions for
use of the ADC or pharmaceutical composition.
[0142] In an aspect, the disclosure features a container comprising
an ADC described herein or a pharmaceutical composition described
herein.
[0143] In an aspect, the disclosure features a nucleic acid
molecule (e.g., an isolated nucleic acid molecule) that encodes an
ADC described herein, e.g., a VH, a VL; or both, or a heavy chain,
a light chain, or both, of an antibody molecule described herein,
coupled (e.g., fused) to an antimicrobial peptide, as described
herein.
[0144] In an embodiment, the nucleic acid molecule comprises a
nucleotide sequence described in Table 2, e.g., any of SEQ ID NOS:
81-93 or 113-114.
[0145] In an aspect, the disclosure features a vector comprising a
nucleic acid molecule described herein.
[0146] In an aspect, the disclosure features a cell (e.g., an
isolated cell) comprising a nucleic acid molecule described herein
or a vector described herein.
[0147] In an aspect, the disclosure features a method of producing
an ADC described herein, the method comprising culturing a cell
described herein under conditions that allow production of an ADC,
thereby producing the ADC described herein.
[0148] In another aspect, the disclosure features a method of
producing an ADC described herein, the method comprises contacting
an antibody molecule (e.g., an antibody molecule described herein)
with a peptide (e.g., a peptide comprising an antimicrobial peptide
described herein, and optionally, a sortase donor sequence), in the
presence of a sortase, under conditions that allow a
sortase-mediated reaction to occur, thereby producing the ADC.
[0149] In an embodiment, the antibody molecule comprises a sortase
acceptor sequence, e.g., a sortase acceptor sequence described
herein. In an embodiment, a heavy chain of the antibody molecule
comprises a sortase acceptor sequence, e.g., at the C-terminus. In
an embodiment, a light chain of the antibody molecule comprises a
sortase acceptor sequence, e.g., at the C-terminus. In an
embodiment, the sortase acceptor sequence further comprises a
linker sequence, e.g., a tandem repeat of glycine-serine peptide
linker sequences.
[0150] In an embodiment, a heavy chain of the antibody molecule
comprises a first sortase acceptor sequence and a light chain of
the antibody molecule comprises a second sortase acceptor sequence.
In an embodiment, the sortase acceptor sequence, e.g., the first
sortase recognition sequence, comprises the amino acid sequence of
(GS).sub.6LPETGGG (SEQ ID NO: 24). In another embodiment, the
sortase acceptor sequence, e.g., the second sortase acceptor
sequence, comprises the amino acid sequence of
P(G.sub.4S).sub.2LPETGGSG (SEQ ID NO: 26).
[0151] In an embodiment, the peptide comprises or consists of an
amino acid sequence that differs by no more than 1, 2, 3, 4, or 5
amino acid residues from, or has at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% homology with, an amino acid sequence
described herein, e.g., any of SEQ ID NOS: 67-80, 94-102, 147-156,
158-159, or 163-164. In an embodiment, the antimicrobial peptide
comprises or consists of an amino acid sequence described herein
e.g., any of SEQ ID NOS: 67-80, 94-102, 147-156, 158-159, or
163-164. In an embodiment, the antimicrobial peptide comprises a
carboxamide group (e.g., a C-terminal carboxamide functional
group).
[0152] In an embodiment, 1.5 mg/mL antibody molecule is contacted
with 20 mol equivalents of sortase donor peptide per sortase
acceptor sequence, and 1 mol equivalent of sortase per 75 mol
equivalents of sortase acceptor sequence. In an embodiment, the
contacting is performed in the presence of 150 mM NaCl, 50 mM Tris
(pH 7.5), and 10 mM CaCl.sub.2. In an embodiment, the contacting is
performed at 18.degree. C. to 37.degree. C., e.g., at 25.degree.
C., e.g., for 2 to 48 hours, e.g., 18 to 24 hours, e.g., 20 hours.
In an embodiment, the sortase is a sortase A pentamutant.
[0153] In an embodiment, the further comprises detecting the
sortase-mediated reaction, e.g., by Q-TOF mass spectrometry. In an
embodiment, the further comprises purifying the ADC, e.g., by gel
electrophoresis.
[0154] In an aspect, the disclosure features a reaction mixture
comprising:
[0155] (i) a sortase, e.g., a sortase described herein, and
[0156] (ii) an antibody molecule described herein, an antimicrobial
peptide described herein, or both.
[0157] In an aspect, the disclosure features an antibody molecule
described herein.
[0158] In an aspect, the disclosure features a nucleic acid
molecule (e.g., an isolated nucleic acid molecule) that encodes an
antibody molecule described herein, e.g., a VH, a VL, or both; or a
heavy chain, a light chain, or both, of the antibody molecule
described herein.
[0159] In an aspect, the disclosure features a vector comprising a
nucleic acid molecule encoding an antibody molecule described
herein.
[0160] In an aspect, the disclosure features a cell (e.g., an
isolated cell) comprising a nucleic acid molecule encoding an
antibody molecule described herein or a vector described
herein.
[0161] In an aspect, the disclosure features a method of producing
an antibody molecule described herein, the method comprising
culturing a cell described herein under conditions that allow
production of an antibody molecule, thereby producing the antibody
molecule described herein.
[0162] In an aspect, the disclosure features an antimicrobial
peptide (e.g., an isolated or synthetic antimicrobial peptide)
described herein.
[0163] In an aspect, the disclosure features a nucleic acid
molecule (e.g., an isolated nucleic acid molecule) that encodes an
antimicrobial peptide described herein.
[0164] In an aspect, the disclosure features a vector comprising a
nucleic acid molecule encoding an antimicrobial peptide described
herein.
[0165] In an aspect, the disclosure features a cell (e.g., an
isolated cell) comprising a nucleic acid molecule encoding an
antimicrobial peptide described herein or a vector of described
herein.
[0166] In an aspect, the disclosure features a method of producing
an antimicrobial peptide described herein, the method comprising
culturing a cell of described herein under conditions that allow
production of an antimicrobial peptide, thereby producing the
antimicrobial peptide described herein.
[0167] In an aspect, the disclosure features an antibody molecule
that binds to the same epitope, or substantially the same epitope,
as an antibody molecule described herein.
[0168] In an aspect, the disclosure features an ADC comprising: a)
an antibody molecule that binds to the same epitope, or
substantially the same epitope, as an antibody molecule described
herein; and b) an antimicrobial peptide, e.g., an antimicrobial
peptide described herein.
[0169] In an aspect, the disclosure features an antibody molecule
that competes for binding with an antibody molecule described
herein.
[0170] In an aspect, the disclosure features an ADC comprising: a)
an antibody molecule that competes for binding with an antibody
molecule described herein; and b) an antimicrobial peptide, e.g.,
an antimicrobial peptide described herein.
[0171] The disclosure contemplates all combinations of any one or
more of the foregoing aspects and/or embodiments, as well as
combinations with any one or more of the embodiments set forth in
the detailed description and examples.
[0172] Other features, objects, and advantages of the compositions
and methods herein will be apparent from the description and
drawings, and from the claims.
[0173] Figures and Tables are provided herewith.
BRIEF DESCRIPTION OF THE DRAWINGS
[0174] FIG. 1 depicts the composition and structure of core
pentasaccharides of several exemplary Gram-negative bacteria.
[0175] FIG. 2 depicts the binding of antibody 3E7 to E. coli (Eco)
and K. pneumoniae (Kpn) as determined by ELISA.
[0176] FIG. 3 depicts the binding of antibody A001-25 to E. coli
(Eco) and K. pneumoniae (Kpn) as determined by ELISA.
[0177] FIG. 4 depicts the process of crosslinking an exemplary
alpha-helical peptide (SEQ ID NO: 158).
[0178] FIG. 5 depicts the readout of an exemplary MIC assay.
[0179] FIG. 6 depicts the mass spectrometry characterization of
Sortase-ligation products using QTof. All samples were reduced with
DTT prior to analysis. Top: The Sortase-tagged heavy chain at
52,201 corresponds with the theoretical molecular weight based on
the peptide sequence; Bottom: The Sortase-ligated reaction product
shows a strong signal at 55002 corresponding to the theoretical
molecular weight for the ligated construct.
[0180] FIG. 7 depicts the binding of antibody 2C7 to representative
E. coli (Eco), K. pneumoniae (Kpn) and S. typhimurium (Sty)
strains.
[0181] FIG. 8 depicts the binding of antibody 3D6 to representative
E. coli (Eco), K. pneumoniae (Kpn) and S. typhimurium (Sty)
strains.
[0182] FIG. 9 depicts the binding of antibody 3E7 to representative
E. coli (Eco), K. pneumoniae (Kpn) and S. typhimurium (Sty)
strains.
[0183] FIG. 10 depicts the binding of antibody 3G1 to
representative E. coli (Eco), K. pneumoniae (Kpn) and S.
typhimurium (Sty) strains.
[0184] FIG. 11A depicts the % killing of Pseudomonas, E. coli, and
Klebsiella spp. in a mixed microbial killing assay using an
exemplary ADC at 8 .mu.g/ml.
[0185] FIG. 11B depicts the % killing of Pseudomonas, E. coli, and
Klebsiella spp. in a mixed microbial killing assay using an
exemplary antibody alone at 125 .mu.g/ml.
[0186] FIG. 11C depicts the % killing of Pseudomonas, E. coli, and
Klebsiella spp. in a mixed microbial killing assay using an
exemplary peptide alone at 0.3 .mu.g/ml.
[0187] FIG. 11D depicts the % killing of Pseudomonas, E. coli, and
Klebsiella spp. in a mixed microbial killing assay using a
combination of antibody at 125 .mu.g/ml and peptide at 0.3
.mu.g/ml.
[0188] FIG. 12 depicts the binding of an exemplary ADC to P.
aeruginosa strains.
[0189] FIG. 13 depicts the binding of an exemplary ADC to bacterial
surface.
[0190] FIG. 14 depicts the reduction of bacterial burden in lung in
a murine acute pneumonia model.
[0191] FIG. 15A depicts the serum stability of an exemplary AMP
without stapling. FIG. 15A discloses SEQ ID NO: 101.
[0192] FIG. 15B depicts the serum stability of an exemplary AMP
with stapling. FIG. 15B discloses SEQ ID NO: 159.
[0193] FIG. 15C depicts the difference in serum stability between
an unoptimized payload and an optimized payload.
[0194] FIG. 16 depicts the effect of antibody mAb001 on endotoxin
(Pseudomonas LPS) signal measured by a cell-based colorimetric
assay.
[0195] FIG. 17 depicts the selective killing activity of an
exemplary ADC against Pseudomonas.
[0196] FIG. 18A depicts the reduction of bacterial burden in lung
by an exemplary ADC in a murine acute pneumonia model
(co-administration study arm).
[0197] FIG. 18B depicts the reduction of bacterial burden in lung
by an exemplary ADC in a murine acute pneumonia model (intranasal
study arm).
[0198] FIG. 19 depicts the bioavailability of an exemplary ADC.
[0199] FIG. 20 depicts the stability of an exemplary L-amino
acid-containing antimicrobial peptide in human serum.
[0200] FIG. 21 depicts the stability of an exemplary D-amino
acid-containing antimicrobial peptide in human serum.
BRIEF DESCRIPTION OF THE TABLES
[0201] Table 1 depicts the amino acid sequences of the heavy chain
variable regions (VHs), light chain variable regions (VLs), heavy
chain CDRs (HCDRs), and light chain CDRs (LCDRs) of the exemplary
antibody molecules. Heavy and light chain CDRs defined according to
Chothia system and Kabat system are shown.
[0202] Table 2 depicts the nucleotide sequences of the heavy chain
variable regions (VHs) and light chain variable regions (VLs) of
the exemplary antibody molecules.
[0203] Table 3 depicts the amino acid sequences of the exemplary
antimicrobial peptides.
[0204] Table 4 depicts exemplary MIC control compound values.
[0205] Table 5 depicts targeted in vitro activity of exemplary ADC,
antibody molecule and peptide.
[0206] Table 6A depicts the inhibitory and hemolytic activities of
exemplary antimicrobial peptides.
[0207] Table 6B depicts the structure-activity relationships for
exemplary AMPs.
[0208] Table 7 depicts the binding avidity of an exemplary ADC to
P. aeruginosa (EC.sub.50).
[0209] Table 8 depicts the amino acid sequences of heavy chain
variable regions (VHs), light chain variable regions (VLs), heavy
chain CDRs (HCDRs), and light chain CDRs (LCDRs) of exemplary
humanized antibody molecules. Heavy and light chain CDRs defined
according to Chothia system and Kabat system are shown.
[0210] Table 9 depicts the microbial killing activity of exemplary
ADCs against P. aeruginosa stains.
[0211] Table 10 depicts the microbial killing activity of exemplary
ADCs against MDR strains.
DETAILED DESCRIPTION
[0212] Disclosed herein are antibody molecules and antibody
molecule-drug conjugates (ADCs) that bind to bacteria, e.g.,
Gram-negative bacteria, e.g., lipopolysaccharides on the outer
membrane of Gram-negative bacteria, with high affinity and
specificity. The ADCs disclosed herein can include an antibody
molecule and an antimicrobial peptide (AMP). Advantageously,
compared to antimicrobial peptides alone, several of the ADCs
describe herein have improved ability to inhibit or reduce the
viability of one or more bacteria (e.g., Gram-negative bacteria) of
different genera, species, and/or subspecies. Nucleic acid
molecules encoding the antibody molecules, ADCs, and antimicrobial
peptides, expression vectors, host cells, compositions (e.g.,
pharmaceutical compositions), kits, and methods for making the
antibody molecules, ADCs, and antimicrobial peptides, are also
provided. The antibody molecules, ADCs, antimicrobial peptides, and
pharmaceutical compositions disclosed herein can be used (alone or
in combination with other agents or therapeutic modalities) to
treat, prevent and/or diagnose bacterial infections or related
disorders and conditions, e.g., caused by Gram-negative
bacteria.
[0213] In an embodiment, the ADCs described herein can have one or
more of the following properties: (i) is capable of treating or
preventing a bacterial disease in a patient with unmet medical
need; (ii) is capable of treating a genus and species of bacteria
causing a bacterial disease; acts via a new mechanism of action;
(iii) has an added inhibitor that reduces or neutralizes a
mechanism of resistance; or (iv) has an alteration in the structure
of the molecule that reduces or neutralizes a mechanism of
resistance.
[0214] In an embodiment, the ADC has an opsonophagocytosis activity
(e.g., via an antibody molecule), a bactericidal activity (e.g.,
via a payload, e.g., an antimicrobial peptide), or both. Without
wishing to be bound by theory, it is believed that in an
embodiment, the use of an antimicrobial peptide in ADC can achieve
one or more of the following goals: a new target with low
resistance, synergistic efficacy with lower spontaneous resistance
probability, improved directed treatment, or rapid bactericidal
activity. In an embodiment, the ADC targets a core LPS region,
e.g., to reduce the probability of resistance caused by potential
target site alteration. In another embodiment, peptide stapling is
used to increase activity and/or stability, e.g., to reduce or
prevent potential antibiotic inactivation.
Definitions
[0215] As used herein, the articles "a" and "an" refer to one or to
more than one (e.g., to at least one) of the grammatical object of
the article.
[0216] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or", unless context clearly
indicates otherwise.
[0217] "About" and "approximately" shall generally mean an
acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Exemplary degrees of error
are within 20 percent (%), typically, within 10%, and more
typically, within 5% of a given value or range of values.
[0218] The compositions and methods disclosed herein encompass
polypeptides and nucleic acids having the sequences specified, or
sequences substantially identical or similar thereto, e.g.,
sequences at least 85%, 90%, 95% identical or higher to the
sequence specified.
[0219] In the context of an amino acid sequence, the term
"substantially identical" is used herein to refer to a first amino
acid that contains a sufficient or minimum number of amino acid
residues that are i) identical to, or ii) conservative
substitutions of aligned amino acid residues in a second amino acid
sequence such that the first and second amino acid sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to a reference sequence, e.g., a
sequence provided herein.
[0220] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic acid sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to a reference sequence, e.g., a
sequence provided herein.
[0221] The term "functional variant" refers polypeptides that have
a substantially identical amino acid sequence to the
naturally-occurring sequence, or are encoded by a substantially
identical nucleotide sequence, and are capable of having one or
more activities of the naturally-occurring sequence.
[0222] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0223] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a typical
embodiment, the length of a reference sequence aligned for
comparison purposes is at least 30%, e.g., at least 40%, 50%, 60%,
e.g., at least 70%, 80%, 90%, 100% of the length of the reference
sequence. The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that
position.
[0224] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0225] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In some embodiments, the percent identity
between two amino acid sequences is determined using the Needleman
and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has
been incorporated into the GAP program in the GCG software package
(available at www.gcg.com), using either a Blossum 62 matrix or a
PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a
length weight of 1, 2, 3, 4, 5, or 6. In certain embodiments, the
percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (available at
www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40,
50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One
suitable set of parameters (and the one that should be used unless
otherwise specified) are a Blossum 62 scoring matrix with a gap
penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5.
[0226] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller ((1989) CABIOS, 4:11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0227] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to a nucleic acid as described herein. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
protein molecules described herein. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When
utilizing BLAST and gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be used.
See www.ncbi.nlm.nih.gov.
[0228] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous
and nonaqueous methods are described in that reference and either
can be used. Specific hybridization conditions referred to herein
are as follows: 1) low stringency hybridization conditions in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at
50.degree. C. (the temperature of the washes can be increased to
55.degree. C. for low stringency conditions); 2) medium stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C.; 3) high stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and preferably 4) very
high stringency hybridization conditions are 0.5M sodium phosphate,
7% SDS at 65.degree. C., followed by one or more washes at
0.2.times.SSC, 1% SDS at 65.degree. C. Very high stringency
conditions 4) are suitable conditions and the ones that should be
used unless otherwise specified.
[0229] It is understood that the molecules described herein may
have additional conservative or non-essential amino acid
substitutions, which do not have a substantial effect on their
functions.
[0230] The term "amino acid" is intended to embrace all molecules,
whether natural or synthetic, which include both an amino
functionality and an acid functionality and capable of being
included in a polymer of naturally-occurring amino acids. Exemplary
amino acids include naturally-occurring amino acids; analogs,
derivatives and congeners thereof; amino acid analogs having
variant side chains; and all stereoisomers of any of any of the
foregoing. As used herein the term "amino acid" includes both the
D- or L-optical isomers and peptidomimetics.
[0231] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0232] The terms "polypeptide," "peptide" and "protein" (if single
chain) are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer may be linear or branched,
it may comprise modified amino acids, and it may be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified; for example, disulfide bond formation,
glycosylation, lipidation, acetylation, phosphorylation, or any
other manipulation, such as conjugation with a labeling component.
The polypeptide can be isolated from natural sources, can be a
produced by recombinant techniques from a eukaryotic or prokaryotic
host, or can be a product of synthetic procedures.
[0233] The terms "nucleic acid," "nucleic acid sequence,"
"nucleotide sequence," or "polynucleotide sequence," and
"polynucleotide" are used interchangeably. They refer to a
polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof. The
polynucleotide may be either single-stranded or double-stranded,
and if single-stranded may be the coding strand or non-coding
(antisense) strand. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
The sequence of nucleotides may be interrupted by non-nucleotide
components. A polynucleotide may be further modified after
polymerization, such as by conjugation with a labeling component.
The nucleic acid may be a recombinant polynucleotide, or a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin
which either does not occur in nature or is linked to another
polynucleotide in a non-natural arrangement.
[0234] The term "isolated," as used herein, refers to material that
is removed from its original or native environment (e.g., the
natural environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide, separated by human intervention from some or all of
the co-existing materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a composition, and
still be isolated in that such vector or composition is not part of
the environment in which it is found in nature.
[0235] As used herein, the term "treat," e.g., a bacterial
infection or related disorder, means that a subject (e.g., a human)
who has a bacterial infection or related disorder, and/or
experiences a symptom of a bacterial infection or related disorder,
will, in an embodiment, suffer less a severe symptom and/or recover
faster when an antibody molecule, ADC, or antimicrobial peptide is
administered than if the antibody molecule, ADC, or antimicrobial
peptide were never administered. In an embodiment, when an
infection or related disorder is treated, an assay to detect
bacteria in the subject will detect fewer bacteria after effective
treatment for the infection or disorder. For example, a diagnostic
assay using an antibody molecule or ADC, such as an antibody
molecule or ADC described herein, will detect fewer or no bacteria
in a biological sample of a subject after administration of an
antibody molecule, ADC, or antimicrobial peptide for the effective
treatment of the infection or disorder. Other assays, such as PCR
(e.g., qPCR) can also be used to monitor treatment in a patient, to
detect the presence, e.g., decreased presence (or absence) after
treatment of bacterial infection or disorder in the subject.
Treatment can, e.g., partially or completely alleviate, ameliorate,
relieve, inhibit, or reduce the severity of, and/or reduce
incidence and optionally, delay onset of, one or more
manifestations of the effects or symptoms, features, and/or causes
of a particular infection, disease, disorder, and/or condition
(e.g., a bacterial infection). In an embodiment, treatment is of a
subject who does not exhibit certain signs of the relevant
infection, disease, disorder and/or condition and/or of a subject
who exhibits only early signs of the infection, disease, disorder,
and/or condition. In an embodiment, treatment is of a subject who
exhibits one or more established signs of the relevant infection,
disease, disorder and/or condition. In an embodiment, treatment is
of a subject diagnosed as suffering from a bacterial infection or
related disorder.
[0236] As used herein, the term "prevent," e.g., a bacterial
infection, means that a subject (e.g., a human) is less likely to
have a bacterial infection if the subject receives the antibody
molecule, ADC, or antimicrobial peptide prior to (e.g., 1 day, 2
days, 1 week, 2 weeks, 3 weeks, or 1 month of more) being exposed
to the bacteria that cause the infection.
[0237] As used herein, the term "minimum inhibitory concentration"
or "MIC" refers to the lowest concentration of an antimicrobial
agent, e.g., an antibody molecule, ADC, or antimicrobial peptide,
that will inhibit the growth (e.g., visible growth) of a bacterium,
e.g., after incubation (e.g., overnight incubation). Methods for
determining minimum inhibitory concentration or MIC are described,
e.g., in Andrews, J. Antimicrob. Chemother. 2001; 48 Suppl 1:5-16
(Erratum in J. Antimicrob. Chemother. 2002; 49(6):1049). For
example, MIC can be determined by using the following procedure:
preparation of antibiotic stock solution, preparation of antibiotic
dilution range, preparation of agar dilution plates, preparation of
inoculum, inoculation, incubation, and reading and interpreting
results. MICs can also be determined by agar dilution or broth
microdilution, usually following the guidelines of a reference body
such as the Clinical & Laboratory Standards Institute (CLSI),
British Society for Antimicrobial Chemotherapy (BSAC), or European
Committee on Antimicrobial Susceptibility Testing (EUCAST). In an
embodiment, the MIC is the lowest concentration of an antimicrobial
agent, e.g., an antibody molecule, ADC, or antimicrobial peptide,
that inhibits growth of a bacterium, by at least 60%, 65%, 70%,
75%, 80%, 85%, 90%, or 95%. In an embodiment, the MIC is the lowest
concentration of an antimicrobial agent, e.g., an antibody
molecule, ADC, or antimicrobial peptide, that inhibits growth of a
bacterium, by at least 80%. Exemplary method for determining MIC is
also described in Example 2.
[0238] As used herein, the term "minimum bactericidal
concentration" or "MBC" refers to the lowest concentration of an
antimicrobial agent, e.g., an antibody molecule, ADC, or
antimicrobial peptide, required to kill a particular bacterium. In
an embodiment, minimum bactericidal concentration or MBC can be
determined from broth dilution minimum inhibitory concentration
(MIC) tests by subculturing to agar plates that do not contain the
test agent. In an embodiment, the MBC is identified by determining
the lowest concentration of antimicrobial agent that reduces the
viability of the initial bacterial inoculum by >99.9%. In an
embodiment, antimicrobial agents are usually regarded as
bactericidal if the MBC is no more than four times the MIC (French,
J Antimicrob Chemother. 2006; 58(6):1107-1117).
[0239] Various aspects of the compositions and methods herein are
described in further detail below. Additional definitions are set
out throughout the specification.
Lipopolysaccharides
[0240] Disclosed herein are antibody molecules and antibody
molecule-drug conjugates (ADCs) that can bind to
lipopolysaccharides (LPS), e.g., on the outer membrane of
Gram-negative bacteria. In an embodiment, the antibody molecule or
ADC binds to a core pentasaccharide region of the LPS. Without
wishing to be bound by theory, it is believed that in an
embodiment, a core LPS region is targeted, at least in part,
because it has one or more of the following properties: high
density, conserved within species, accessible, or essential for
adhesion (Raetz and Whitfield Annu. Rev. Biochem. 2002; 71:
635-700; de Kievit and Lam J Bacteriol. 1994; 176(23):7129-39;
Schmengler et al. Eur J Cell Biol. 2010; 89(1):25-33; Pier et al.
Am J Respir Crit Care Med. 1996; 154(4 Pt 2):S175-82).
[0241] LPS, also known as lipoglycans or endotoxin, are large
molecules including a lipid and a polysaccharide composed of
O-antigen, outer core and inner core joined by a covalent bond. LPS
is found, e.g., in the outer membrane of Gram-negative bacteria,
which may elicit strong immune responses in animals. LPS
contributes to the structural integrity of the bacteria and
protects the membrane from certain kinds of chemical attack. It
also increases the negative charge of the cell membrane and helps
stabilize the overall membrane structure. LPS may induce a strong
immune response in animals. It has also been implicated in
non-pathogenic aspects of bacterial ecology, including surface
adhesion, bacteriophage sensitivity, and interactions with
predators such as amoebae. LPS is required for the proper
conformation of omptin activity; however, smooth LPS will
sterically hinder omptins. As LPS is a major component of the outer
membrane of Gram-negative bacteria, mutation or removal of LPS can
result in death of Gram-negative bacteria.
[0242] LPS comprises three parts: O antigen (or O polysaccharide),
Core oligosaccharide, and Lipid A.
[0243] O antigen, also known as O polysaccharide or O side-chain,
is a repetitive glycan polymer contained within an LPS of the
bacteria. The O antigen is attached to the core oligosaccharide,
and comprises the outermost domain of the LPS molecule. The
composition of the O chain varies from strain to strain. For
example, there are over 160 different O antigen structures produced
by different E. coli strains (Raetz and Whitfield Annu. Rev.
Biochem. 2002; 71: 635-700). The presence or absence of O chains
determines whether the LPS is considered rough or smooth.
Full-length O-chains would render the LPS smooth, whereas the
absence or reduction of O-chains would make the LPS rough (Rittig
et al. J. Leukoc. Biol. 2003; 74 (6): 1045-55). Bacteria with rough
LPS usually have more penetrable cell membranes to hydrophobic
antibiotics, since a rough LPS is more hydrophobic (Tsujimoto et
al. J. Infect. Chemother. 1999, 5 (4): 196-200. O antigen is
exposed on the very outer surface of the bacterial cell, and, can
be targeted for recognition by host antibodies.
[0244] The Core oligosaccharide or core domain contains an
oligosaccharide component that attaches directly to lipid A and
commonly contains sugars such as heptose (Hep) and
3-deoxy-D-mannooctulosonic acid (also known as Kdo or
keto-deoxyoctulosonate) (Hershberger and Binkley, J. Biol. Chem.
1968; 243 (7): 1578-1584). A typical core pentasaccharide or core
pentasaccharide region includes, e.g., two Kdo residues and one,
two or three Hep residues. The composition and structure of core
pentasaccharides from exemplary bacteria are shown in FIG. 1. The
LPS Cores of many bacteria also contain non-carbohydrate
components, such as phosphate, amino acids, and ethanolamine
substituents.
[0245] A LPS core can include an inner core and an outer core.
[0246] The "base" of the inner core is 1-3 Kdo residues. The last
Kdo is often modified with a phosphate or ethanolamine group. From
the Kdo residues, there are attached 2-3 heptose residues (e.g.,
L-glycero-D-mannoheptulose) that are usually phosphorylated. These
Kdo and heptose residues form the "inner core." The ketosidic bond
between Kdo and lipid A (a2.fwdarw.6) is especially susceptible to
acid cleavage. The lipid and polysaccharide portions of LPS can be
separated by a weak acid treatment. An LPS molecule that includes
only a lipid A and an inner core (or less) is referred to as
"deep-rough LPS."
[0247] The outer core is made of hexose residues that are attached
to the last heptose residue in the inner core. Hexoses often found
in the outer core include, e.g., D-glucose, D-mannose, or
D-galactose. There is usually at least three hexoses bound
.beta.1.fwdarw.3, with the O antigen being ligated to the third
hexose. Other hexoses are often found attached to the outer core,
branching from the main oligomer. LPS that include lipid A and a
complete core oligosaccharide (inner and outer) is referred to as
"rough LPS."
[0248] Lipid A is, in some circumstances, a phosphorylated
glucosamine disaccharide decorated with multiple fatty acids. These
hydrophobic fatty acid chains anchor the LPS into the bacterial
membrane, and the rest of the LPS projects from the cell surface.
The lipid A domain is responsible for much of the toxicity of
Gram-negative bacteria. When bacterial cells are lysed by the
immune system, fragments of membrane containing lipid A are
released into the circulation, causing fever, diarrhea, and
possible fatal endotoxic shock (also called septic shock).
[0249] In an embodiment, a core glycan of P. aeruginosa is
targeted. Without wishing to be bound by theory, it is believed
that in an embodiment, core glycans of P. aeruginosa are conserved
across strains, are accessible in vivo, and/or have limited phase
variation/resistance elements.
[0250] In an embodiment, the antibody molecule or ADC binds to
phosphorylated LPS, e.g., a phosphorylated core pentasaccharide
region of the LPS. In an embodiment, the antibody molecule or ADC
described herein binds to one or more (e.g., two or three)
phosphate groups in the core LPS region of P. aeruginosa. In an
embodiment, the phosphate group is bound by an Arginine residue in
the antibody molecule or ADC. In an embodiment, the phosphate group
is inhibited or neutralized by an Arginine residue in the antibody
molecule or ADC. In an embodiment, the antibody molecule or ADC
binds to at least three phosphate groups (e.g., Hep2,4,6-PO.sub.4)
in the core LPS region of P. aeruginosa, each of which is bound,
inhibited, or neutralized by an Arginine residue (e.g., R100, R101,
or R103) in the antibody molecule or ADC. For example, the H3 and
L3 cavity of the antibody molecule can accommodate a
monosaccharide.
Antibody Molecules
[0251] Disclosed herein are antibody molecules that bind to
bacteria (e.g., Gram-negative bacteria) and/or lipopolysaccharides
(LPS). The antibody molecule-drug conjugates (ADCs) disclosed
herein can include an antibody molecule disclosed herein.
[0252] As used herein, the term "antibody molecule" refers to a
protein, e.g., an immunoglobulin chain or a fragment thereof,
comprising at least one immunoglobulin variable domain sequence.
The term "antibody molecule" includes, for example, full-length,
mature antibodies and antigen-binding fragments of an antibody. For
example, an antibody molecule can include a heavy (H) chain
variable domain sequence (abbreviated herein as VH), and a light
(L) chain variable domain sequence (abbreviated herein as VL). In
another example, an antibody molecule includes two heavy (H) chain
variable domain sequences and two light (L) chain variable domain
sequence, thereby forming two antigen binding sites, such as Fab,
Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv for
example), single variable domain antibodies, diabodies (Dab)
(bivalent and bispecific), and chimeric (e.g., humanized)
antibodies, which may be produced by the modification of whole
antibodies or those synthesized de novo using recombinant DNA
technologies. These functional antibody fragments retain the
ability to selectively bind with their respective antigen or
receptor. Antibodies and antibody fragments can be from any class
of antibodies including, but not limited to, IgG, IgA, IgM, IgD,
and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4)
of antibodies. The antibody molecules can be monoclonal or
polyclonal. The antibody molecule can also be a human, humanized,
CDR-grafted, or in vitro generated antibody. The antibody molecule
can have a heavy chain constant region chosen from, e.g., IgG1,
IgG2, IgG3, or IgG4. The antibody molecule can also have a light
chain chosen from, e.g., kappa or lambda. The term "immunoglobulin"
(Ig) is used interchangeably with the term "antibody" herein.
[0253] Examples of antigen-binding fragments include: (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a diabody (dAb) fragment, which
consists of a VH domain; (vi) a camelid or camelized variable
domain; (vii) a single chain Fv (scFv), see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody.
These antibody fragments may be obtained using any suitable method,
including several conventional techniques known to those with skill
in the art, and the fragments can be screened for utility in the
same manner as are intact antibodies.
[0254] The term "antibody" includes intact molecules as well as
functional fragments thereof. Constant regions of the antibodies
can be altered, e.g., mutated, to modify the properties of the
antibody (e.g., to increase or decrease one or more of: Fc receptor
binding, antibody glycosylation, the number of cysteine residues,
effector cell function, or complement function).
[0255] The antibody molecule can be a single chain antibody. A
single-chain antibody (scFv) may be engineered (see, for example,
Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter,
Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can
be dimerized or multimerized to generate multivalent antibodies
having specificities for different epitopes of the same target
protein.
[0256] The antibody molecules disclosed herein can also be single
domain antibodies. Single domain antibodies can include antibodies
whose complementary determining regions are part of a single domain
polypeptide. Examples include, but are not limited to, heavy chain
antibodies, antibodies naturally devoid of light chains, single
domain antibodies derived from conventional 4-chain antibodies,
engineered antibodies and single domain scaffolds other than those
derived from antibodies. Single domain antibodies may be any of the
art, or any future single domain antibodies. Single domain
antibodies may be derived from any species including, but not
limited to mouse, human, camel, llama, fish, shark, goat, rabbit,
and bovine. According to some aspects, a single domain antibody is
a naturally occurring single domain antibody known as heavy chain
antibody devoid of light chains. Such single domain antibodies are
disclosed in International Publication No. WO 94/04678, for
example. For clarity reasons, this variable domain derived from a
heavy chain antibody naturally devoid of light chain is known
herein as a VHH or nanobody to distinguish it from the conventional
VH of four chain immunoglobulins. Such a VHH molecule can be
derived from antibodies raised in Camelidae species, for example in
camel, llama, dromedary, alpaca and guanaco. Other species besides
Camelidae may produce heavy chain antibodies naturally devoid of
light chain; such VHHs are also contemplated.
[0257] The VH and VL regions can be subdivided into regions of
hypervariability, termed "complementarity determining regions"
(CDR), interspersed with regions that are more conserved, termed
"framework regions" (FR or FW). The terms "complementarity
determining region," and "CDR," as used herein refer to the
sequences of amino acids within antibody variable regions which
confer antigen specificity and binding affinity. As used herein,
the terms "framework," "FW" and "FR" are used interchangeably.
[0258] The extent of the framework region and CDRs has been
precisely defined by a number of methods (see, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol.
196:901-917; and the AbM definition used by Oxford Molecular's AbM
antibody modeling software. See, generally, e.g., Protein Sequence
and Structure Analysis of Antibody Variable Domains. In: Antibody
Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R.,
Springer-Verlag, Heidelberg). In an embodiment, the following
definitions are used: AbM definition of CDR1 of the heavy chain
variable domain and Kabat definitions for the other CDRs. In an
embodiment, Kabat definitions are used for all CDRs. In addition,
embodiments described with respect to Kabat or AbM CDRs may also be
implemented using Chothia hypervariable loops. Each VH and VL
typically includes three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0259] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence which can form the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
or may not include one, two, or more N- or C-terminal amino acids,
or may include other alterations that are compatible with formation
of the protein structure.
[0260] The term "antigen-binding region" refers to the part of an
antibody molecule that comprises determinants that form an
interface that binds to an antigen, e.g., lipopolysaccharide (LPS),
or an epitope thereof. With respect to proteins (or protein
mimetics), the antigen-binding region typically includes one or
more loops (of at least, e.g., four amino acids or amino acid
mimics) that form an interface that binds to the antigen, e.g.,
LPS. Typically, the antigen-binding region of an antibody molecule
includes at least one or two CDRs and/or hypervariable loops, or
more typically at least three, four, five or six CDRs and/or
hypervariable loops.
[0261] The terms "compete" or "cross-compete" are used
interchangeably herein to refer to the ability of an antibody
molecule to interfere with binding of an anti-LPS antibody
molecule, e.g., an anti-LPS antibody molecule provided herein, to a
target, e.g., LPS on a Gram-negative bacterium. The interference
with binding can be direct or indirect (e.g., through an allosteric
modulation of the antibody molecule or the target). The extent to
which an antibody molecule is able to interfere with the binding of
another antibody molecule to the target, and therefore whether it
can be said to compete, can be determined using a competition
binding assay, for example, a FACS assay, an ELISA, an SPR assay,
or an OCTET.RTM. assay (ForteBio). In an embodiment, a competition
binding assay is a quantitative competition assay. In an
embodiment, a first anti-LPS antibody molecule is said to compete
for binding to the target with a second anti-LPS antibody molecule
when the binding of the first antibody molecule to the target is
reduced by 10% or more, e.g., 20% or more, 30% or more, 40% or
more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or
more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 98% or more, 99% or more in a competition binding assay
(e.g., a competition assay described herein).
[0262] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. A monoclonal antibody can be made by
hybridoma technology or by methods that do not use hybridoma
technology (e.g., recombinant methods).
[0263] An "effectively human" protein is a protein that does not
evoke a neutralizing antibody response, e.g., the human anti-murine
antibody (HAMA) response. HAMA can be problematic in a number of
circumstances, e.g., if the antibody molecule is administered
repeatedly, e.g., in treatment of a chronic or recurrent disease
condition. A HAMA response can make repeated antibody
administration potentially ineffective because of an increased
antibody clearance from the serum (see, e.g., Saleh et al., Cancer
Immunol. Immunother., 32:180-190 (1990)) and also because of
potential allergic reactions (see, e.g., LoBuglio et al.,
Hybridoma, 5:5117-5123 (1986)).
[0264] The antibody molecule can be a polyclonal or a monoclonal
antibody. In some embodiments, the antibody can be recombinantly
produced, e.g., produced by any suitable phage display or
combinatorial methods.
[0265] Various phage display and combinatorial methods for
generating antibodies are known in the art (as described in, e.g.,
Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International
Publication No. WO 92/18619; Dower et al. International Publication
No. WO 91/17271; Winter et al. International Publication WO
92/20791; Markland et al. International Publication No. WO
92/15679; Breitling et al. International Publication WO 93/01288;
McCafferty et al. International Publication No. WO 92/01047;
Garrard et al. International Publication No. WO 92/09690; Ladner et
al. International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the
contents of all of which are incorporated by reference herein).
[0266] In an embodiment, the antibody molecule is a fully human
antibody (e.g., an antibody made in a mouse which has been
genetically engineered to produce an antibody from a human
immunoglobulin sequence), or a non-human antibody, e.g., a rodent
(mouse or rat), goat, primate (e.g., monkey), camel antibody. In an
embodiment, the non-human antibody is a rodent (mouse or rat
antibody). Methods of producing rodent antibodies are known in the
art.
[0267] Human monoclonal antibodies can be generated using
transgenic mice carrying the human immunoglobulin genes rather than
the mouse system. Splenocytes from these transgenic mice immunized
with the antigen of interest are used to produce hybridomas that
secrete human mAbs with specific affinities for epitopes from a
human protein (see, e.g., Wood et al. International Application WO
91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg
et al. International Application WO 92/03918; Kay et al.
International Application 92/03917; Lonberg, N. et al. 1994 Nature
368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21;
Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA
81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon
et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol
21:1323-1326).
[0268] An antibody can be one in which the variable region, or a
portion thereof, e.g., the CDRs, are generated in a non-human
organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and
humanized antibodies are within the invention. Antibodies generated
in a non-human organism, e.g., a rat or mouse, and then modified,
e.g., in the variable framework or constant region, to decrease
antigenicity in a human are within the invention.
[0269] Chimeric antibodies can be produced by any suitable
recombinant DNA technique. Several are known in the art (see
Robinson et al., International Patent Publication PCT/US86/02269;
Akira, et al., European Patent Application 184,187; Taniguchi, M.,
European Patent Application 171,496; Morrison et al., European
Patent Application 173,494; Neuberger et al., International
Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;
Cabilly et al., European Patent Application 125,023; Better et al.
(1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443;
Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS
84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et
al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl
Cancer Inst. 80:1553-1559).
[0270] A humanized or CDR-grafted antibody will have at least one
or two but generally all three recipient CDRs (of heavy and or
light immunoglobulin chains) replaced with a donor CDR. The
antibody may be replaced with at least a portion of a non-human CDR
or only some of the CDRs may be replaced with non-human CDRs. It is
only necessary to replace the number of CDRs required for binding
of the humanized antibody to lipopolysaccharide. In an embodiment,
the donor will be a rodent antibody, e.g., a rat or mouse antibody,
and the recipient will be a human framework or a human consensus
framework. Typically, the immunoglobulin providing the CDRs is
called the "donor" and the immunoglobulin providing the framework
is called the "acceptor." In some embodiments, the donor
immunoglobulin is a non-human (e.g., rodent). The acceptor
framework is typically a naturally-occurring (e.g., a human)
framework or a consensus framework, or a sequence about 85% or
higher, e.g., 90%, 95%, 99% or higher identical thereto.
[0271] As used herein, the term "consensus sequence" refers to the
sequence formed from the most frequently occurring amino acids (or
nucleotides) in a family of related sequences (See e.g., Winnaker,
From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987).
In a family of proteins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence. A
"consensus framework" refers to the framework region in the
consensus immunoglobulin sequence.
[0272] An antibody can be humanized by any suitable method, and
several such methods known in the art (see e.g., Morrison, S. L.,
1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques
4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and
5,693,762, the contents of all of which are hereby incorporated by
reference).
[0273] Humanized or CDR-grafted antibodies can be produced by
CDR-grafting or CDR substitution, wherein one, two, or all CDRs of
an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No.
5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al.
1988 Science 239:1534; Beidler et al. 1988 J. Immunol.
141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all
of which are hereby expressly incorporated by reference. Winter
describes a CDR-grafting method which may be used to prepare
humanized antibodies (UK Patent Application GB 2188638A, filed on
Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of
which is expressly incorporated by reference.
[0274] Also provided are humanized antibodies in which specific
amino acids have been substituted, deleted or added. Criteria for
selecting amino acids from the donor are described in, e.g., U.S.
Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089,
the contents of which are hereby incorporated by reference. Other
techniques for humanizing antibodies are described in Padlan et al.
EP 519596 A1, published on Dec. 23, 1992.
[0275] In an embodiment, the antibody molecule has a heavy chain
constant region chosen from, e.g., the heavy chain constant regions
of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;
particularly, chosen from, e.g., the heavy chain constant regions
(e.g., the human heavy chain constant regions) of IgG1, IgG2, IgG3,
and IgG4. In another embodiment, the antibody molecule has a light
chain constant region chosen from, e.g., the light chain constant
regions (e.g., the human light chain constant regions) of kappa or
lambda. The constant region can be altered, e.g., mutated, to
modify the properties of the antibody molecule (e.g., to increase
or decrease one or more of: Fc receptor binding, antibody
glycosylation, the number of cysteine residues, effector cell
function, and/or complement function). In an embodiment, the
antibody molecule has effector function and can fix complement. In
another embodiment, the antibody molecule does not recruit effector
cells or fix complement. In certain embodiments, the antibody
molecule has reduced or no ability to bind an Fc receptor. For
example, it may be an isotype or subtype, fragment or other mutant,
which does not support binding to an Fc receptor, e.g., it has a
mutagenized or deleted Fc receptor binding region. In an
embodiment, the antibody molecule comprises an Fc region that is
altered to increase an ADCC activity. In an embodiment, the ADCC
activity is increased by 10-fold or more, e.g., 25-fold or more,
50-fold or more, 100-fold or more, 200-fold or more, 400-fold or
more, 600-fold or more, 800-fold or more, or 1000-fold or more,
e.g., between 100-fold and 1000-fold or between 250-fold and
750-fold. In an embodiment, the antibody molecule comprises an Fc
region that is altered to modulate engagement with an Fc.gamma.
receptor or an opsonophagocytosis activity. In an embodiment, the
antibody molecule comprises an Fc region that is altered to
modulate engagement with an FcRn receptor.
[0276] In an embodiment, a constant region of the antibody molecule
is altered. Methods for altering an antibody constant region are
known in the art. Antibody molecules with altered function, e.g.
altered affinity for an effector ligand, such as FcR on a cell, or
the Cl component of complement can be produced by replacing at
least one amino acid residue in the constant portion of the
antibody with a different residue (see e.g., EP 388,151 A1, U.S.
Pat. Nos. 5,624,821 and 5,648,260, the contents of all of which are
hereby incorporated by reference) Amino acid mutations which
stabilize antibody structure, such as S228P (EU nomenclature, S241P
in Kabat nomenclature) in human IgG4 are also contemplated. Similar
type of alterations could be described which if applied to the
murine, or other species immunoglobulin would reduce or eliminate
these functions.
[0277] In an embodiment, the only amino acids in the antibody
molecule are canonical amino acids. In an embodiment, the antibody
molecule comprises naturally-occurring amino acids; analogs,
derivatives and congeners thereof; amino acid analogs having
variant side chains; and/or all stereoisomers of any of any of the
foregoing. The antibody molecule may comprise the D- or L-optical
isomers of amino acids and peptidomimetics.
[0278] A polypeptide of an antibody molecule described herein may
be linear or branched, it may comprise modified amino acids, and it
may be interrupted by non-amino acids. The antibody molecule may
also be modified; for example, by disulfide bond formation,
glycosylation, lipidation, acetylation, phosphorylation, or any
other manipulation, such as conjugation with a labeling component.
The polypeptide can be isolated from natural sources, can be a
produced by recombinant techniques from a eukaryotic or prokaryotic
host, or can be a product of synthetic procedures.
[0279] The antibody molecule described herein can be used alone in
unconjugated form, or can be bound to a substance, e.g., a toxin or
moiety (e.g., a therapeutic drug (e.g., an antibiotic); a compound
emitting radiation; molecules of plant, fungal, or bacterial
origin; or a biological protein (e.g., a protein toxin) or particle
(e.g., a recombinant viral particle, e.g., via a viral coat
protein). For example, the anti-LPS antibody can be coupled to a
radioactive isotope such as an .alpha.-, .beta.-, or
.gamma.-emitter, or a .beta.- and .gamma.-emitter.
[0280] An antibody molecule can be derivatized or linked to another
functional molecule (e.g., another peptide or protein). As used
herein, a "derivatized" antibody molecule is one that has been
modified. Methods of derivatization include but are not limited to
the addition of a fluorescent moiety, a radionucleotide, a toxin,
an enzyme or an affinity ligand such as biotin. Accordingly, the
antibody molecules are intended to include derivatized and
otherwise modified forms of the antibodies described herein,
including immunoadhesion molecules. For example, an antibody
molecule can be functionally linked (by chemical coupling, genetic
fusion, noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a toxin, a
pharmaceutical agent, and/or a protein or peptide that can mediate
association of the antibody or antibody portion with another
molecule (such as a streptavidin core region or a polyhistidine
tag).
[0281] Some types of derivatized antibody molecule are produced by
crosslinking two or more antibodies (of the same type or of
different types, e.g., to create bispecific antibodies). Suitable
crosslinkers include those that are heterobifunctional, having two
distinctly reactive groups separated by an appropriate spacer
(e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or
homobifunctional (e.g., disuccinimidyl suberate). Such linkers are
available from Pierce Chemical Company, Rockford, Ill.
[0282] Useful detectable agents with which an anti-LPS antibody
molecule may be derivatized (or labeled) to include fluorescent
compounds, various enzymes, prosthetic groups, luminescent
materials, bioluminescent materials, fluorescent emitting metal
atoms, e.g., europium (Eu), and other anthanides, and radioactive
materials (described below). Exemplary fluorescent detectable
agents include fluorescein, fluorescein isothiocyanate, rhodamine,
5dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the
like. An antibody may also be derivatized with detectable enzymes,
such as alkaline phosphatase, horseradish peroxidase,
.beta.-galactosidase, acetylcholinesterase, glucose oxidase and the
like. When an antibody is derivatized with a detectable enzyme, it
is detected by adding additional reagents that the enzyme uses to
produce a detectable reaction product. For example, when the
detectable agent horseradish peroxidase is present, the addition of
hydrogen peroxide and diaminobenzidine leads to a colored reaction
product, which is detectable. An antibody molecule may also be
derivatized with a prosthetic group (e.g., streptavidin/biotin and
avidin/biotin). For example, an antibody may be derivatized with
biotin, and detected through indirect measurement of avidin or
streptavidin binding. Examples of suitable fluorescent materials
include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of bioluminescent materials include
luciferase, luciferin, and aequorin.
[0283] Labeled antibody molecule can be used, for example,
diagnostically and/or experimentally in a number of contexts,
including (i) to isolate a predetermined antigen by standard
techniques, such as affinity chromatography or immunoprecipitation;
(ii) to detect a predetermined antigen (e.g., in a cellular lysate
or cell supernatant) in order to evaluate the abundance and pattern
of expression of the protein; (iii) to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to determine
the efficacy of a given treatment regimen.
[0284] An antibody molecule may be conjugated to another molecular
entity, typically a label or a therapeutic (e.g., antimicrobial
(e.g., antibacterial or bactericidal), immunomodulatory,
immunostimularoty, cytotoxic, or cytostatic) agent or moiety.
Radioactive isotopes can be used in diagnostic or therapeutic
applications. Radioactive isotopes that can be coupled to the
antibody molecules include, but are not limited to .alpha.-,
.beta.-, or .gamma.-emitters, or .beta.- and .gamma.-emitters. Such
radioactive isotopes include, but are not limited to iodine
(.sup.131I or .sup.125I), yttrium (.sup.90Y), lutetium
(.sup.177Lu), actinium (.sup.225Ac), praseodymium, astatine
(.sup.211At), rhenium (.sup.186Re), bismuth (.sup.212Bi or
.sup.213Bi), indium (.sup.111In), technetium (.sup.99mTc),
phosphorus (.sup.32P), rhodium (.sup.188Rh), sulfur (.sup.35S),
carbon (.sup.14C), tritium (.sup.3H), chromium (.sup.51Cr),
chlorine (.sup.36Cl), cobalt (.sup.57Co or .sup.58Co), iron
(.sup.59Fe), selenium (.sup.75Se), or gallium (.sup.67Ga).
Radioisotopes useful as therapeutic agents include yttrium
(.sup.90Y), lutetium (.sup.177Lu), actinium (.sup.225Ac),
praseodymium, astatine (.sup.211At), rhenium (.sup.186Re), bismuth
(.sup.212Bi or .sup.213Bi), and rhodium (.sup.188Rh). Radioisotopes
useful as labels, e.g., for use in diagnostics, include iodine
(.sup.131I or .sup.125I), indium (.sup.111In), technetium
(.sup.99mTc), phosphorus (.sup.32P), carbon (.sup.14C), and tritium
(.sup.3H), or one or more of the therapeutic isotopes listed
above.
[0285] The present disclosure provides radiolabeled antibody
molecules and methods of labeling the same. In an embodiment, a
method of labeling an antibody molecule is disclosed. The method
includes contacting an antibody molecule, with a chelating agent,
to thereby produce a conjugated antibody. The conjugated antibody
is radiolabeled with a radioisotope, e.g., .sup.111Indium,
.sup.90Yttrium and .sup.177Lutetium, to thereby produce a labeled
antibody molecule.
[0286] As is discussed above, the antibody molecule can be
conjugated to a therapeutic agent. Therapeutically active
radioisotopes have already been mentioned. Examples of other
therapeutic agents include antimicrobial (e.g., anti-bacterial)
agents, e.g., antimicrobial peptides. In an embodiment, an
antimicrobial peptide can be coupled (e.g., fused) to the antibody
molecule, e.g., a heavy chain or light chain of the antibody
molecule. In an embodiment, the antimicrobial peptide is coupled
(e.g., fused) to the N-terminus of the heavy chain or light chain
or a functional fragment thereof. In an embodiment, the
antimicrobial peptide is coupled (e.g., fused) to the C-terminus of
the heavy chain or light chain or a functional fragment thereof. In
an embodiment, the antimicrobial peptide is coupled (e.g., fused)
to a constant region or a portion thereof. In an embodiment, the
heavy chain or light chain, or a portion thereof, and the
antimicrobial peptide, forms a fusion polypeptide, e.g., encoded by
an open reading frame (ORF). One or more antimicrobial peptides can
be coupled (e.g., fused) to the antibody molecule. In an
embodiment, at least two of the antimicrobial peptides are
identical or substantially identical. In another embodiment, at
least two of the antimicrobial peptides are different. In an
embodiment, all of the antimicrobial peptides are identical or
substantially identical.
[0287] In some aspects, this disclosure provides a method of making
an antibody molecule disclosed herein. The method includes:
providing an antigen, e.g., a bacteria (e.g., a Gram-negative
bacteria) or LPS; obtaining an antibody molecule that specifically
binds to the antigen; evaluating efficacy of the antibody molecule
in modulating activity of the antigen and/or organism expressing
the antigen, e.g., a bacterium (e.g., a Gram-negative bacterium).
The method can further include administering the antibody molecule,
including a derivative thereof (e.g., a humanized antibody
molecule) to a subject, e.g., a human.
[0288] This disclosure provides an isolated nucleic acid molecule
encoding the above antibody molecule, vectors and host cells
thereof. The nucleic acid molecule includes, but is not limited to,
RNA, genomic DNA and cDNA.
[0289] Exemplary sequences of antibody molecules are described in
Tables 1, 2 and 8 below.
TABLE-US-00001 TABLE 1 Amino acid sequences of heavy chain variable
regions (VHs), light chain variable regions (VLs), heavy chain CDRs
(HCDRs), and light chain CDRs (LCDRs) of exemplary antibody
molecules. Heavy and light chain CDRs defined according to Chothia
system and Kabat system are shown. SEQ ID SEQ ID SEQ ID Antibody VH
NO Chothia CDR NO Kabat CDR NO A001-25
EVQLQQSGPVLVKPGASVKMSCKASGYTFTDHYINWVKQSH 1 HCDR1 GYTFTDH 14 HCDR1
DHYIN 17 GKSLEWIGGIYPYHGITKYNRNFKDKATLTVDKSSSTAYME HCDR2 YPYHGI 15
HCDR2 GIYPYHGITKYNRNFKD 18 LNSLTSELSAVYYCASGGSRRYFDVWGTGTTVTVSS
HCDR3 GGSRRYFDV 16 HCDR3 GGSRRYFDV 16 hWN01
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAP 2 HCDR1 GFTFSDY 19 HCDR1
DYYMT 22 GKGLEWVGLIRNKRNGDTAEYSASVKGRFTISRDDSKNSLY HCDR2 RNKRNGDT
20 HCDR2 LIRNKRNGDTAEYSASVKG 23
LQMNSLKTEDTAVYYCARQGRGYTLDYWGQGTLVTVSS HCDR3 QGRGYTLDY 21 HCDR3
QGRGYTLDY 21 hWNv1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAP 3
HCDR1 GFTFSDY 19 HCDR1 DYWMT 27
GKGLEWVGLIRAKANGDTAEYSASVKGRFTISRDDSKNSLY HCDR2 RAKANGDT 25 HCDR2
LIRAKANGDTAEYSASVKG 28 LQMNSLKTEDTAVYYCARQGRGYTLDYWGQGTLVTVSS HCDR3
QGRGYTLDY 21 HCDR3 QGRGYTLDY 21 3E7
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVKQRP 4 HCDR1 GYTFTSY 29 HCDR1
SYWIT 32 GQGLEWIGDIYPGSGSTNYNEKFKSKATLTVDTSSSTAYMQ HCDR2 YPGSGS 30
HCDR2 DIYPGSGSTNYNEKFKS 33 LSSLTSEDSAVYYCARGSYSLDYWGQGTTLTVSS HCDR3
GSYSLDY 31 HCDR3 GSYSLDY 31 3G1
EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKQRP 5 HCDR1 GFNIKNT 34 HCDR1
NTYMH 37 EQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQ HCDR2 DPANGN 35
HCDR2 RIDPANGNTKYAPKFQG 38 LSSLTSEDTAIYYCAPSNYHAMDYWGQGTSVTVSS
HCDR3 SNYHAMDY 36 HCDR3 SNYHAMDY 36 2C7
DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQF 6 HCDR1 GYSITSGY 39 HCDR1
SGYYWN 42 PGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLK HCDR2 SYDGS 40
HCDR2 YISYDGSNNYNPSLKN 43 LNSVTTEDTATYYCARWNGNYFDYWGQGTTLTVSS HCDR3
WNGNYFDY 41 HCDR3 WNGNYFDY 41 3D6
EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSH 7 HCDR1 GYTFTDY 44 HCDR1
DYYMN 47 GKSLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTAYME HCDR2 NPYNGG 45
HCDR2 VINPYNGGTSYNQKFKG 48 LNSLTSEDSAVYYCARTRQLGLRWFAYWGQGTLVTVSA
HCDR3 TRQLGLRWFAY 46 HCDR3 TRQLGLRWFAY 46 mAb001
EVKLVESGGDLVKPGGSLRLSCAASEFTFSDYAMSWVRQTP 103 HCDR1 EFTFSDY 105
HCDR1 DYAMS 108 AKRLEWVAYISSDGDSTYYPDNIKGRFTISRDNAKNTLYLQ HCDR2
SSDGDS 106 HCDR2 YISSDGDSTYYPDNIKG 109
MNSLRSEDTAMYFCAREIRLRGYFDVWGAGTTVTVSS HCDR3 EIRLRGYFDV 107 HCDR3
EIRLRGYFDV 107 SEQ ID SEQ ID SEQ ID Antibody VL NO Chothia CDR NO
Kabat CDR NO A001-25 DVVMTQTPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWY 8
LCDR1 RSSQRLVHSNGNTYLH 49 LCDR1 RSSQRLVHSNGNTYLH 49
LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR LCDR2 KVSNRFS 50 LCDR2
KVSNRFS 50 VEAEDLGVYFCSQSTHVPYTFGGGTKLEIK LCDR3 SQSTHVPYT 51 LCDR3
SQSTHVPYT 51 hWN01 DIQMTQSPSSVSASVGDRVTITCRASQNINIWLSWYQQKPG 9
LCDR1 RASQNINIWLS 52 LCDR1 RASQNINIWLS 52
KAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPED LCDR2 KASNLHT 53 LCDR2
KASNLHT 53 FATYYCLQGQSYPRTFGGGTKVEIK LCDR3 LQGQSYPRT 54 LCDR3
LQGQSYPRT 54 hWNv1 DIQMTQSPSSVSASVGDRVTITCRASQNINIWLSWYQQKPG 9
LCDR1 RASQNINIWLS 52 LCDR1 RASQNINIWLS 52
KAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPED LCDR2 KASNLHT 53 LCDR2
KASNLHT 53 FATYYCLQGQSYPRTFGGGTKVEIK LCDR3 LQGQSYPRT 54 LCDR3
LQGQSYPRT 54 3E7 DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWF 10 LCDR1
RSSKSLLHSNGNTYLY 55 LCDR1 RSSKSLLHSNGNTYLY 55
LQRPGQSPQRLIYYMSNLASGVPDRFSGRGSGTDFTLRISR LCDR2 YMSNLAS 56 LCDR2
YMSNLAS 56 VEAEDVGVYYCMQSLEYPLTFGAGTKLELK LCDR3 MQSLEYPLT 57 LCDR3
MQSLEYPLT 57 3G1 DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWF 11 LCDR1
RSSKSLLHSNGNTYLY 58 LCDR1 RSSKSLLHSNGNTYLY 58
LQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISR LCDR2 RMSNLAS 59 LCDR2
RMSNLAS 59 VEAEDVGVYYCMQHLEYPYTFGGGTKLEIK LCDR3 MQHLEYPYT 60 LCDR3
MQHLEYPYT 60 2C7 DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAW 12 LCDR1
KSSQSLLNSRTRKNYLA 61 LCDR1 KSSQSLLNSRTRKNYLA 61
YQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIS LCDR2 WASTRES 62 LCDR2
WASTRES 62 SVQAEDLAVYYCKQSYNLWTFGGGTKLEIK LCDR3 KQSYNLWT 63 LCDR3
KQSYNLWT 63 3D6 DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPG 13 LCDR1
LASQTIGTWLA 64 LCDR1 LASQTIGTWLA 64
KSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAED LCDR2 AATSLAD 65 LCDR2
AATSLAD 65 FVSYYCQQLYSTPWTFGGGTKLEIK LCDR3 QQLYSTPWT 66 LCDR3
QQLYSTPWT 66 mAb001 DIVLTQSPASLAVSLGQRATISCRASESVFGHGISPMHWYQ 104
LCDR1 RASESVFGHGISPMH 110 LCDR1 RASESVFGHGISPMH 110
QKPGQPPKLLIYRASNLKFGIPARFSGSGSRTDFTLTINPV LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 EADDVATYYCQQSNEYPRTFGGGTKLEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112
TABLE-US-00002 TABLE 2 Nucleotide sequences of heavy chain variable
regions (VHs) and light chain variable regions (VLs) of exemplary
antibody molecules. Antibody VH SEQ ID NO A001-25
GAGGTCCAGCTGCAGCAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGT-
AAGGCTTCTGGATACACATTCACTGACC 81
ACTATATAAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTTATCCTTACCACGG-
TATTACTAAGTACAACCGGAA
TTTCAAGGACAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACA-
TCTGAACTCTCTGCAGTCTAT
TACTGTGCAAGCGGGGGAAGTCGCCGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA
hWN01
GAAGTGCAGCTCGTGGAATCTGGAGGAGGACTTGTGCAACCTGGAGGTTCCCTGCGACTGTCGTGTGC-
CGCATCCGGTTTCACCTTTTCCGACT 82
ACTACATGACCTGGGTCAGACAGGCGCCGGGGAAGGGACTGGAGTGGGTCGGCTTGATCCGCAACAAGAGGAA-
CGGCGATACTGCTGAATACTC
GGCCAGCGTGAAGGGGCGGTTCACCATCTCGAGAGATGACAGCAAGAACTCCCTGTACCTCCAAATGAACTCC-
CTGAAAACCGAGGACACTGCG
GTGTACTACTGCGCCCGCCAGGGTCGCGGCTACACGCTGGACTATTGGGGCCAGGGCACCCTGGTCACTGTGT-
CAAGC hWNv1
GAAGTGCAGCTCGTGGAATCTGGAGGAGGACTTGTGCAACCTGGAGGTTCCCTGCGACTGTCGTGTGC-
CGCATCCGGTTTCACCTTTTCCGACT 83
ACTGGATGACCTGGGTCAGACAGGCGCCGGGGAAGGGACTGGAGTGGGTCGGCTTGATCCGCGCCAAGGCGAA-
CGGCGATACTGCTGAATACTC
GGCCAGCGTGAAGGGGCGGTTCACCATCTCGAGAGATGACAGCAAGAACTCCCTGTACCTCCAAATGAACTCC-
CTGAAAACCGAGGACACTGCG
GTGTACTACTGCGCCCGCCAGGGTCGCGGCTACACGCTGGACTATTGGGGCCAGGGCACCCTGGTCACTGTGT-
CAAGC 3E7
CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGG-
CTTCTGGCTACACCTTCACCAGCT 84
ACTGGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGG-
TAGTACTAACTACAATGAGAA
GTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACA-
TCTGAGGACTCTGCGGTCTAT
TACTGTGCAAGAGGTAGCTACTCCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
3G1
GAGGTTCAGCTGCAGCAGTCTGTGGCAGAGCTTGTGAGGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAG-
CTTCTGGCTTCAACATTAAAAACA 85
CCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGG-
TAATACTAAATATGCCCCGAA
GTTCCAGGGCAAGGCCACTATAACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACA-
TCTGAGGACACTGCCATCTAT
TACTGTGCTCCTAGTAACTACCATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
2C7
GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTG-
TCACTGGCTACTCCATCACCAGTG 86
GTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGATGG-
TAGCAATAACTACAACCCATC
TCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACT-
ACTGAGGACACAGCCACATAT
TACTGTGCAAGATGGAATGGTAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
3D6
GAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGG-
CTTCTGGATACACATTCACTGACT 87
ACTATATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAATCCTTACAACGG-
TGGTACTAGCTACAACCAGAA
GTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACA-
TCTGAGGACTCTGCAGTCTAT
TACTGTGCAAGAACCAGACAGCTCGGGCTACGTTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCT-
CTGCA mAb001
GAAGTGAAGTTGGTGGAGTCTGGGGGAGACTTGGTGAAACCTGGAGGGTCCCTGAGACTCTCCTGTG-
CAGCCTCTGAATTCACTTTCAGTGATT 113
ATGCCATGTCTTGGGTTCGCCAGACTCCGGCGAAGAGGCTGGAGTGGGTCGCATACATTAGTAGTGATGGTGA-
TAGTACCTACTATCCGGACAA
TATTAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTATACCTGCAAATGAACAGTCTGAGG-
TCTGAGGACACGGCCATGTAT
TTTTGTGCAAGAGAAATACGGCTAAGGGGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCT-
CA Antibody VL SEQ ID NO A001-25
GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCT-
TGCAGATCTAGTCAGAGACTTGTACACA 88
GTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGT-
TTCCAACCGATTTTCTGGGGT
CCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGAT-
CTGGGAGTTTATTTCTGCTCT
CAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA hWN01
GACATCCAGATGACTCAGTCCCCGTCCTCAGTCTCCGCATCCGTGGGAGATCGCGTGACGATTACTTG-
CCGGGCGTCGCAGAACATCAACATCT 89
GGCTGTCGTGGTACCAGCAGAAGCCCGGGAAGGCTCCGAAGCTGCTGATCTACAAGGCCTCAAACTTGCACAC-
CGGCGTGCCTTCCCGCTTTTC
TGGTTCGGGCTCCGGGACTGACTTCACCCTGACCATCAGCAGCCTGCAACCCGAGGACTTCGCCACCTATTAC-
TGCCTCCAAGGACAGTCCTAC CCAAGAACCTTCGGCGGAGGAACCAAGGTCGAAATCAAA hWNv1
GACATCCAGATGACTCAGTCCCCGTCCTCAGTCTCCGCATCCGTGGGAGATCGCGTGACGATTACTTG-
CCGGGCGTCGCAGAACATCAACATCT 89
GGCTGTCGTGGTACCAGCAGAAGCCCGGGAAGGCTCCGAAGCTGCTGATCTACAAGGCCTCAAACTTGCACAC-
CGGCGTGCCTTCCCGCTTTTC
TGGTTCGGGCTCCGGGACTGACTTCACCCTGACCATCAGCAGCCTGCAACCCGAGGACTTCGCCACCTATTAC-
TGCCTCCAAGGACAGTCCTAC CCAAGAACCTTCGGCGGAGGAACCAAGGTCGAAATCAAA 3E7
GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCA-
GGTCTAGTAAGAGTCTTCTGCATA 90
GTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCGCCTGATATATTATAT-
GTCCAACCTTGCCTCAGGAGT
CCCAGACAGGTTCAGTGGCAGAGGGTCAGGAACTGATTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGAT-
GTGGGTGTTTATTACTGTATG
CAAAGTCTAGAATATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA 3G1
GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCA-
GGTCTAGTAAGAGTCTCCTGCATA 91
GTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGAT-
GTCCAACCTTGCCTCAGGAGT
CCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGAT-
GTGGGTGTTTATTACTGTATG
CAACATCTAGAATATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 2C7
GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCA-
AATCCAGTCAGAGTCTGCTCAACA 92
GTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTG-
GGCATCCACTAGGGAATCTGG
GGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAA-
GACCTGGCAGTTTATTACTGC
AAGCAATCTTATAATCTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 3D6
GACATTCAGATGACCCAGTCTCCTGCCTCCCAGTCTGCATCTCTGGGAGAAAGTGTCACCATCACATGCC-
TGGCAAGTCAGACCATTGGTACAT 93
GGTTAGCATGGTATCAGCAGAAACCAGGGAAATCTCCTCAGCTCCTGATTTATGCTGCAACCAGCTTGGCAGA-
TGGGGTCCCATCAAGGTTCAG
TGGTAGTGGATCTGGCACAAAATTTTCTTTCAAGATCAGCAGCCTACAGGCTGAAGATTTTGTAAGTTATTAC-
TGTCAACAACTTTACAGTACT CCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA
mAb001
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCT-
GCAGAGCCAGTGAAAGTGTTTTTGGTC 114
ATGGCATTAGTCCTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATC-
CAACCTAAAATTTGGGATCCC
TGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTT-
GCAACCTATTACTGTCAGCAA
AGTAATGAATATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAA
TABLE-US-00003 TABLE 8 Amino acid sequences of heavy chain variable
regions (VHs), light chain variable regions (VLs), heavy chain CDRs
(HCDRs), and light chain CDRs (LCDRs) of exemplary humanized
antibody molecules. Heavy and light chain CDRs defined according to
Chothia system and Kabat system are shown. Antibody/ SEQ ID SEQ ID
SEQ ID Chain VH NO Chothia CDR NO Kabat CDR NO mAb001_
EVKLVESGGGLVQPGGSLRLSCSASEFTFSDYAMSWVRQAP 115 HCDR1 EFTFSDY 105
HCDR1 DYAMS 108 VH1 GKGLEWVSYISSDGDSTYYPDNIKGRFTISRDNSKNTLYVQ HCDR2
SSDGDS 106 HCDR2 YISSDGDSTYYPDNIKG 109
MSSLRAEDTAVYFCAREIRLRGYFDVWGQGTTVTVSS HCDR3 EIRLRGYFDV 107 HCDR3
EIRLRGYFDV 107 mAb001_ EVKLVESGGGLVKPGGSLRLSCAASEFTFSDYAMSWVRQAP
116 HCDR1 EFTFSDY 105 HCDR1 DYAMS 108 VH2
GKRLEWVAYISSDGDSIYYPDNIKGRFTISRDNAKNSLYLQ HCDR2 SSDGDS 106 HCDR2
YISSDGDSIYYPDNIKG 145 MNSLRAEDTAMYFCAREIRLRGYFDVWGQGTTVTVSS HCDR3
EIRLRGYFDV 107 HCDR3 EIRLRGYFDV 107 mAb001_
EVKLVESGGGLVQPGGSLRLSCAASEFTFSDYAMSWVRQAP 117 HCDR1 EFTFSDY 105
HCDR1 DYAMS 108 VH3 GKRLEWVAYISSDGDSTYYPDSVKGRFTISRDNAKNSLYLQ HCDR2
SSDGDS 106 HCDR2 YISSDGDSTYYPDSVKG 146
MNSLRAEDTAMYFCAREIRLRGYFDVWGQGTTVTVSS HCDR3 EIRLRGYFDV 107 HCDR3
EIRLRGYFDV 107 mAb001_ EVKLVESGEGLVQPGGSLRLSCAASEFTFSDYAMSWVRQAP
118 HCDR1 EFTFSDY 105 HCDR1 DYAMS 108 VH4
GKRLEWVAYISSDGDSTYYPDNIKGRFTISRDNSKNTLYLQ HCDR2 SSDGDS 106 HCDR2
YISSDGDSTYYPDNIKG 109 MGSLRAEDMAMYFCAREIRLRGYFDVWGQGTTVTVSS HCDR3
EIRLRGYFDV 107 HCDR3 EIRLRGYFDV 107 Antibody/ SEQ ID SEQ ID SEQ ID
Chain VL NO Chothia CDR NO Kabat CDR NO mAb001_
EIVMTQSPATLSVSPGERATLSCRASESVFGHGISPLHWYQ 119 LCDR1 RASESVFGHGISPLH
138 LCDR1 RASESVFGHGISPLH 138 VL1
QKPGQAPKLLIYRASNRKTGIPARFSGSGSGTEFTLTISSL LCDR2 RASNRKT 139 LCDR2
RASNRKT 139 QSEDFAVYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 120
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VL2
QKPGKAPKLLIYRASNLKFGVPSRFSGSGSGTEFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ EIVMTQSPATLSVSPGERATLSCRASESVFGHGISPLHWYQ 121
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VL3
QKPGQPPRLLIYRASNRKTGIPARFSGSGSGTEFTLTISSL LCDR2 RASNRKT 139 LCDR2
RASNRKT 139 QSEDFAVYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 122
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_1
QKPGKAPKLLIYRASNLKFGVPSRFSGSGSRTDFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 123
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_2
QKPGKAPKLLIYRASNLKFGIPSRFSGSGSRTDFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 124
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_3
QKPGQPPKLLIYRASNLKFGIPSRFSGSGSGTEFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIVLTQSPSTLSASVGDRVTITCRASESVFGHGISPMHWYQ 125
LCDR1 RASESVFGHGISPMH 110 LCDR1 RASESVFGHGISPMH 110 VLr2_4
QKPGKAPKLLIYRASNLKFGVPSRFSGSGSGTEFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001- DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 126
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_5
QKPGKAPKLLIYRASNLKFGIPARFSGSGSGTEFTLTISSL LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001- DIVLTQSPASLAVSLGQRATISCRASESIFGHGISPMHWYQ 127
LCDR1 RASESIFGHGISPMH 140 LCDR1 RASESIFGHGISPMH 140 VLr2_6
QKPGQPPKLLIYRASNLKFGIPARFSGSGSRTDFTLTINPV LCDR2 RASNLKF 111 LCDR2
RASNLKF 111 EADDVATYYCQQSNEYPRTFGGGTKLEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIVLTQSPASLAVSLGQRATISCRASESVFGHGISPMHWYQ 128
LCDR1 RASESVFGHGISPMH 110 LCDR1 RASESVFGHGISPMH 110 VLr2_7
QKPGQPPKLLIYRASSLKFGIPARFSGSGSRTDFTLTINPV LCDR2 RASSLKF 141 LCDR2
RASSLKF 141 EADDVATYYCQQSNEYPRTFGGGTKLEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIVLTQSPASLAVSLGQRATISCRASESVFGHGISPMHWYQ 129
LCDR1 RASESVFGHGISPMH 110 LCDR1 RASESVFGHGISPMH 110 VLr2_8
QKPGQPPKLLIYRASNLKSGIPARFSGSGSRTDFTLTINPV LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 EADDVATYYCQQSNEYPRTFGGGTKLEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIVMTQSPATLSVSPGERATLSCRASESVFGHGISPLHWYQ 130
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_9
QKPGQAPKLLIYRASNLKTGIPARFSGSGSRTDFTLTISSL LCDR2 RASNLKT 143 LCDR2
RASNLKT 143 QSEDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ EIVMTQSPATLSVSPGERATISCRASESVFGHGISPLHWYQ 131
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr2_10
QKPGQAPKLLIYRASNRKTGIPARFSGSGSGTEFTLTISPV LCDR2 RASNRKT 139 LCDR2
RASNRKT 139 QSEDFAVYYCQQSNEYPRTFGGGTKLEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001- DIQMTQSPSTLSASVGDRVTITCRASESIFGHGISPLHWYQ 132
LCDR1 RASESIFGHGISPLH 144 LCDR1 RASESIFGHGISPLH 144 VLr3_1
QKPGKAPKLLIYRASNLKSGIPSRFSGSGSRTEFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESIFGHGISPLHWYQ 133
LCDR1 RASESIFGHGISPLH 144 LCDR1 RASESIFGHGISPLH 144 VLr3_2
QKPGKAPKLLIYRASNLKSGVPSRFSGSGSRTEFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001- DIQMTQSPSTLSASVGDRVTITCRASESIFGHGISPLHWYQ 134
LCDR1 RASESIFGHGISPLH 144 LCDR1 RASESIFGHGISPLH 144 VLr3_3
QKPGKAPKLLIYRASNLKSGVPARFSGSGSRTEFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESVFGHGISPLHWYQ 135
LCDR1 RASESVFGHGISPLH 138 LCDR1 RASESVFGHGISPLH 138 VLr3_4
QKPGKAPKLLIYRASNLKSGVPSRFSGSGSRTEFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESIFGHGISPLHWYQ 136
LCDR1 RASESIFGHGISPLH 144 LCDR1 RASESIFGHGISPLH 144 VLr3_5
QKPGKAPKLLIYRASNLKSGVPSRFSGSGSRTDFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112 mAb001_ DIQMTQSPSTLSASVGDRVTITCRASESIFGHGISPLHWYQ 137
LCDR1 RASESIFGHGISPLH 144 LCDR1 RASESIFGHGISPLH 144 VLr3_6
QKPGKAPKLLIYRASNLKSGIPSRFSGSGSRTDFTLTISSL LCDR2 RASNLKS 142 LCDR2
RASNLKS 142 QPDDFATYYCQQSNEYPRTFGGGTKVEIK LCDR3 QQSNEYPRT 112 LCDR3
QQSNEYPRT 112
[0290] In an embodiment, the antibody molecule comprises one, two,
or three CDRs of the VH region of an antibody molecule described in
Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7,
or 3D6, or any of the humanized mAb001), using the Kabat or Chothia
definitions of CDRs. In an embodiment, the antibody molecule
comprises one, two, or three CDRs of the VL region of an antibody
molecule described in Table 1 or 8 (e.g., mAb001, A001-25, hWN01,
hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the humanized mAb001),
using the Kabat or Chothia definitions of CDRs. In an embodiment,
the antibody molecule comprises one or more (e.g., two or three)
CDRs of the VH region and/or one or more (e.g., two or three) CDRs
of the VL region of an antibody molecule described in Table 1 or 8
(e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any
of the humanized mAb001), using the Kabat or Chothia definitions of
CDRs.
[0291] In an embodiment, the antibody molecule comprises one, two,
or three HCDRs described in Table 1 or 8. In an embodiment, the
antibody molecule comprises one, two, or three LCDRs described in
Table 1 or 8. In an embodiment, the antibody molecule comprises one
or more (e.g., two or three) HCDRs and/or one or more (e.g., two or
three) LCDRs described in Table 1 or 8.
[0292] In an embodiment, the antibody molecule comprises one, two,
three, or four frameworks of the VH region of an antibody molecule
described in Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1,
3E7, 3G1, 2C7, or 3D6, or any of the humanized mAb001). In an
embodiment, the antibody molecule comprises one, two, three, or
four frameworks of the VL region of an antibody molecule described
in Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1,
2C7, or 3D6, or any of the humanized mAb001). In an embodiment, the
antibody molecule comprises one or more (e.g., two, three, or four)
frameworks of the VH region and/or one or more (e.g., two, three,
or four) frameworks of the VL region of an antibody molecule
described in Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1,
3E7, 3G1, 2C7, or 3D6, or any of the humanized mAb001).
[0293] In an embodiment, the antibody molecule comprises a heavy
chain variable region of an antibody molecule described in Table 1
or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
or any of the humanized mAb001). In an embodiment, the antibody
molecule comprises a light chain variable region of an antibody
molecule described in Table 1 or 8 (e.g., mAb001, A001-25, hWN01,
hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the humanized mAb001). In
an embodiment, the antibody molecule comprises a heavy chain
variable region and a light chain variable region of an antibody
molecule described in Table 1 or 8 (e.g., mAb001, A001-25, hWN01,
hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the humanized mAb001).
[0294] In an embodiment, the antibody molecule comprises a heavy
chain variable region having an amino acid sequence described in
Table 1 or 8. In an embodiment, the antibody molecule comprises a
light chain variable region having an amino acid sequence described
in Table 1 or 8. In an embodiment, the antibody molecule comprises
a heavy chain variable region having an amino acid sequence
described in Table 1 or 8 and a light chain variable region having
an amino acid sequences described in Table 1 or 8.
[0295] In an embodiment, the antibody molecule comprises a heavy
chain variable region encoded by a nucleotide sequence described in
Table 2. In an embodiment, the antibody molecule comprises a light
chain variable region encoded by a nucleotide sequence described in
Table 2. In an embodiment, the antibody molecule comprises a heavy
chain variable region encoded by a nucleotide sequence described in
Table 2 and a light chain variable region encoded by a nucleotide
sequence described in Table 2.
[0296] In an embodiment, the antibody molecule further comprises a
heavy chain constant region. In an embodiment, the antibody
molecule further comprises a light chain constant region. In an
embodiment, the antibody molecule further comprises a heavy chain
constant region and a light chain constant region. In an
embodiment, the antibody molecule comprises a heavy chain constant
region, a light chain constant region, and heavy and light chain
variable regions of an antibody molecule described in Table 1 or 8.
In certain embodiments, the antibody molecule comprises a heavy
chain constant region, a light chain constant region, and variable
regions that comprise one, two, three, four, five, or six CDRs of
an antibody molecule described in Table 1 or 8.
[0297] In an embodiment, the antibody molecule binds to a core
pentasaccharide region of the LPS. In an embodiment, the core
pentasaccharide region comprises one or more (e.g., two) Kdo
residues and one or more (e.g., two or three) Hep residues. In an
embodiment, the antibody molecule binds to one or more (e.g., two)
Kdo residues, or one or more (e.g., two or three) Hep residues, or
any combination thereof.
[0298] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable
region comprises one, two, or all of the following: an HCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 108); an HCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR2 of antibody mAb001
(e.g., SEQ ID NO: 109); or an HCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody mAb001
(e.g., SEQ ID NO: 107).
[0299] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
108); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 109); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 107).
[0300] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 108); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody mAb001 (e.g., SEQ ID
NO: 109); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 (e.g., SEQ ID NO: 107).
[0301] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody mAb001
(e.g., SEQ ID NO: 111); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody mAb001
(e.g., SEQ ID NO: 112).
[0302] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO:
110); an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 111); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 112).
[0303] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising the
amino acid sequence of the LCDR2 of antibody mAb001 (e.g., SEQ ID
NO: 111); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody mAb001 (e.g., SEQ ID NO: 112).
[0304] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3), wherein the heavy chain variable region comprises one, two,
or all of the following: an HCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
108); an HCDR2 comprising an amino acid sequence that differs by no
more than 1, 2, or 3 amino acid residues from, or has at least 85,
90, 95, 99 or 100% homology with, the amino acid sequence of the
HCDR2 of antibody mAb001 (e.g., SEQ ID NO: 109); or an HCDR3
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR3 of
antibody mAb001 (e.g., SEQ ID NO: 107), and wherein the light chain
variable region comprises one, two, or all of the following: an
LCDR1 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR1
of antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody mAb001
(e.g., SEQ ID NO: 111); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody mAb001
(e.g., SEQ ID NO: 112).
[0305] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
108); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 109); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 107), and the light chain variable region comprises one, two,
or all of the following: an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO: 110); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
mAb001 (e.g., SEQ ID NO: 111); or an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID NO:
112).
[0306] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 108); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody mAb001 (e.g., SEQ ID
NO: 109); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 (e.g., SEQ ID NO: 107), and the light
chain variable region comprises an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO: 110); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
mAb001 (e.g., SEQ ID NO: 111); and an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID NO:
112).
[0307] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable
region comprises one, two, or all of the following: an HCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 105); an HCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR2 of antibody mAb001
(e.g., SEQ ID NO: 106); or an HCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody mAb001
(e.g., SEQ ID NO: 107).
[0308] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
105); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 106); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 107).
[0309] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 105); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody mAb001 (e.g., SEQ ID
NO: 106); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 (e.g., SEQ ID NO: 107).
[0310] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody mAb001
(e.g., SEQ ID NO: 111); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody mAb001
(e.g., SEQ ID NO: 112).
[0311] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO:
110); an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 111); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 112).
[0312] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising the
amino acid sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID
NO: 111); and an LCDR3 comprising the amino acid sequence of the
LCDR1 of antibody mAb001 (e.g., SEQ ID NO: 112).
[0313] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3), wherein the heavy chain variable region comprises one, two,
or all of the following: an HCDR1 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
105); an HCDR2 comprising an amino acid sequence that differs by no
more than 1, 2, or 3 amino acid residues from, or has at least 85,
90, 95, 99 or 100% homology with, the amino acid sequence of the
HCDR2 of antibody mAb001 (e.g., SEQ ID NO: 106); or an HCDR3
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the HCDR3 of
antibody mAb001 (e.g., SEQ ID NO: 107), and wherein the light chain
variable region comprises one, two, or all of the following: an
LCDR1 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR1
of antibody mAb001 (e.g., SEQ ID NO: 110); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody mAb001
(e.g., SEQ ID NO: 111); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody mAb001
(e.g., SEQ ID NO: 112).
[0314] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody mAb001 (e.g., SEQ ID NO:
105); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody mAb001 (e.g., SEQ ID NO: 106); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody mAb001 (e.g., SEQ ID
NO: 107), and the light chain variable region comprises one, two,
or all of the following: an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO: 110); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
mAb001 (e.g., SEQ ID NO: 111); or an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID NO:
112).
[0315] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody mAb001 (e.g., SEQ ID NO: 105); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody mAb001 (e.g., SEQ ID
NO: 106); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody mAb001 (e.g., SEQ ID NO: 107), and the light
chain variable region comprises an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody mAb001 (e.g., SEQ ID NO: 110); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
mAb001 (e.g., SEQ ID NO: 111); and an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody mAb001 (e.g., SEQ ID NO:
112).
[0316] In an embodiment, the ADC or antibody molecule further
comprises one or more human or human derived heavy or light chain
variable region frameworks.
[0317] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises an amino acid sequence that differs by no more
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino
acid residues from, or has at least 85, 90, 95, 96, 97, 98, 99, or
100% homology with, the amino acid sequence of the VH of antibody
mAb001 (e.g., SEQ ID NO: 103). In an embodiment, the heavy chain
variable region comprises the amino acid sequence of the VH of
antibody mAb001 (e.g., SEQ ID NO: 103).
[0318] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises an amino acid sequence that differs by no more
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino
acid residues from, or has at least 85, 90, 95, 96, 97, 98, 99, or
100% homology with, the amino acid sequence of the VL of antibody
mAb001 (e.g., SEQ ID NO: 104). In an embodiment, the light chain
variable region comprises the amino acid sequence of the VL of
antibody mAb001 (e.g., SEQ ID NO: 104).
[0319] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein the heavy chain variable region comprises an amino
acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 amino acid residues from, or has at
least 85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino
acid sequence of the VH of antibody mAb001 (e.g., SEQ ID NO: 103),
and wherein the light chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VL of antibody mAb001 (e.g., SEQ ID NO: 104).
[0320] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of antibody mAb001 (e.g., SEQ ID
NO: 103) and the light chain variable region comprises the amino
acid sequence of the VL of antibody mAb001 (e.g., SEQ ID NO:
104).
[0321] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 113). In an embodiment, the light chain
variable region comprises and amino acid sequence encoded by a
nucleotide sequence from Table 2 (e.g., SEQ ID NO: 114).
[0322] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody A001-25
(e.g., SEQ ID NO: 17); an HCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID NO:
18); or an HCDR3 comprising an amino acid sequence that differs by
no more than 1, 2, or 3 amino acid residues from, or has at least
85, 90, 95, 99 or 100% homology with, the amino acid sequence of
the HCDR3 of antibody A001-25 (e.g., SEQ ID NO: 16).
[0323] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO:
17); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 18); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 16).
[0324] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 17); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID
NO: 18); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody A001-25 (e.g., SEQ ID NO: 16).
[0325] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody A001-25
(e.g., SEQ ID NO: 49); an LCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR2 of antibody A001-25 (e.g., SEQ ID NO:
50); or an LCDR3 comprising an amino acid sequence that differs by
no more than 1, 2, or 3 amino acid residues from, or has at least
85, 90, 95, 99 or 100% homology with, the amino acid sequence of
the LCDR3 of antibody A001-25 (e.g., SEQ ID NO: 51).
[0326] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO:
49); an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 50); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 51).
[0327] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2 comprising the
amino acid sequence of the LCDR2 of antibody A001-25 (e.g., SEQ ID
NO: 50); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody A001-25 (e.g., SEQ ID NO: 51).
[0328] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO: 17); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody A001-25 (e.g., SEQ ID NO: 18); or an HCDR3 comprising
an amino acid sequence that differs by no more than 1, 2, or 3
amino acid residues from, or has at least 85, 90, 95, 99 or 100%
homology with, the amino acid sequence of the HCDR3 of antibody
A001-25 (e.g., SEQ ID NO: 16), and wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody A001-25
(e.g., SEQ ID NO: 50); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody A001-25
(e.g., SEQ ID NO: 51).
[0329] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO:
17); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 18); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 16), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody A001-25
(e.g., SEQ ID NO: 50); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID NO:
51).
[0330] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 17); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID
NO: 18); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody A001-25 (e.g., SEQ ID NO: 16), and the light
chain variable region comprises an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO: 49); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
A001-25 (e.g., SEQ ID NO: 50); and an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID NO:
51).
[0331] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody A001-25
(e.g., SEQ ID NO: 14); an HCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID NO:
15); or an HCDR3 comprising an amino acid sequence that differs by
no more than 1, 2, or 3 amino acid residues from, or has at least
85, 90, 95, 99 or 100% homology with, the amino acid sequence of
the HCDR1 of antibody A001-25 (e.g., SEQ ID NO: 16).
[0332] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO:
14); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 15); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 16).
[0333] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 14); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID
NO: 15); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody A001-25 (e.g., SEQ ID NO: 16).
[0334] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody A001-25
(e.g., SEQ ID NO: 49); an LCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR2 of antibody A001-25 (e.g., SEQ ID NO:
50); or an LCDR3 comprising an amino acid sequence that differs by
no more than 1, 2, or 3 amino acid residues from, or has at least
85, 90, 95, 99 or 100% homology with, the amino acid sequence of
the LCDR3 of antibody A001-25 (e.g., SEQ ID NO: 51).
[0335] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO:
49); an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 50); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 51).
[0336] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2 comprising the
amino acid sequence of the LCDR1 of antibody A001-25 (e.g., SEQ ID
NO: 50); and an LCDR3 comprising the amino acid sequence of the
LCDR1 of antibody A001-25 (e.g., SEQ ID NO: 51).
[0337] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO: 14); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody A001-25 (e.g., SEQ ID NO: 15); or an HCDR3 comprising
an amino acid sequence that differs by no more than 1, 2, or 3
amino acid residues from, or has at least 85, 90, 95, 99 or 100%
homology with, the amino acid sequence of the HCDR3 of antibody
A001-25 (e.g., SEQ ID NO: 16), and wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising an amino acid sequence that differs by no more than 1,
2, or 3 amino acid residues from, or has at least 85, 90, 95, 99 or
100% homology with, the amino acid sequence of the LCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR2 of antibody A001-25
(e.g., SEQ ID NO: 50); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of antibody A001-25
(e.g., SEQ ID NO: 51).
[0338] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody A001-25 (e.g., SEQ ID NO:
14); an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody A001-25 (e.g., SEQ ID NO: 15); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody A001-25 (e.g., SEQ ID
NO: 16), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO: 49); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody A001-25
(e.g., SEQ ID NO: 50); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID NO:
51).
[0339] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody A001-25 (e.g., SEQ ID NO: 14); an HCDR2 comprising the
amino acid sequence of the HCDR2 of antibody A001-25 (e.g., SEQ ID
NO: 15); and an HCDR3 comprising the amino acid sequence of the
HCDR3 of antibody A001-25 (e.g., SEQ ID NO: 16), and the light
chain variable region comprises an LCDR1 comprising the amino acid
sequence of the LCDR1 of antibody A001-25 (e.g., SEQ ID NO: 49); an
LCDR2 comprising the amino acid sequence of the LCDR2 of antibody
A001-25 (e.g., SEQ ID NO: 50); and an LCDR3 comprising the amino
acid sequence of the LCDR3 of antibody A001-25 (e.g., SEQ ID NO:
51).
[0340] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0341] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of antibody A001-25 (e.g.,
SEQ ID NO: 1). In an embodiment, the heavy chain variable region
comprises the amino acid sequence of the VH of antibody A001-25
(e.g., SEQ ID NO: 1).
[0342] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of antibody A001-25 (e.g.,
SEQ ID NO: 8). In an embodiment, the light chain variable region
comprises the amino acid sequence of the VL of antibody A001-25
(e.g., SEQ ID NO: 8).
[0343] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of antibody A001-25 (e.g., SEQ ID NO: 1), and
wherein the light chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VL of antibody A001-25 (e.g., SEQ ID NO: 8).
[0344] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of antibody A001-25 (e.g., SEQ ID
NO: 1) and the light chain variable region comprises the amino acid
sequence of the VL of antibody A001-25 (e.g., SEQ ID NO: 8).
[0345] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 81). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 88).
[0346] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 22); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody hWN01 (e.g., SEQ ID NO: 23); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody hWN01 (e.g., SEQ ID NO: 21).
[0347] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 22);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 23); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 21).
[0348] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 22); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWN01 (e.g., SEQ ID NO: 23);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 21).
[0349] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWN01 (e.g., SEQ ID NO: 54).
[0350] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody hWN01 (e.g., SEQ ID NO: 52;
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 53; or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 54.
[0351] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 54).
[0352] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 22); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody hWN01 (e.g., SEQ ID NO: 23); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody hWN01 (e.g.,
SEQ ID NO: 21), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWN01 (e.g., SEQ ID NO: 54).
[0353] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 22);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 23); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 21), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody hWN01
(e.g., SEQ ID NO: 53); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody hWN01 (e.g., SEQ ID NO: 54).
[0354] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 22); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWN01 (e.g., SEQ ID NO: 23);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 21), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody hWN01 (e.g., SEQ
ID NO: 53); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody hWN01 (e.g., SEQ ID NO: 54).
[0355] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 19); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody hWN01 (e.g., SEQ ID NO: 20); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody hWN01 (e.g., SEQ ID NO: 21).
[0356] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 19);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 20); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 21).
[0357] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the hCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 19); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWN01 (e.g., SEQ ID NO: 20);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 21).
[0358] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWN01 (e.g., SEQ ID NO: 54).
[0359] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody hWN01 (e.g., SEQ ID NO: 52);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 53); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 54).
[0360] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 54).
[0361] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 19); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody hWN01 (e.g., SEQ ID NO: 20); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody hWN01 (e.g.,
SEQ ID NO: 21), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWN01 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody hWN01 (e.g., SEQ ID NO: 54).
[0362] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWN01 (e.g., SEQ ID NO: 19);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWN01 (e.g., SEQ ID NO: 20); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 21), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody hWN01
(e.g., SEQ ID NO: 53); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody hWN01 (e.g., SEQ ID NO: 54).
[0363] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of SEQ ID NO: 19; an
HCDR2 comprising the amino acid sequence of SEQ ID NO: 20; and an
HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the
light chain variable region comprises an LCDR1 comprising the amino
acid sequence of SEQ ID NO: 52; an LCDR2 comprising the amino acid
sequence of SEQ ID NO: 53; and an LCDR3 comprising the amino acid
sequence of SEQ ID NO: 54.
[0364] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0365] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of antibody hWN01 (e.g.,
SEQ ID NO: 2). In an embodiment, the heavy chain variable region
comprises the amino acid sequence of the VH of antibody hWN01
(e.g., SEQ ID NO: 2).
[0366] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of antibody hWN01 (e.g.,
SEQ ID NO: 9). In an embodiment, the light chain variable region
comprises the amino acid sequence of the VL of antibody hWN01
(e.g., SEQ ID NO: 9).
[0367] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of antibody hWN01 (e.g., SEQ ID NO: 2), and
wherein the light chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VL of antibody hWN01 (e.g., SEQ ID NO: 9). In an
embodiment, the heavy chain variable region comprises the amino
acid sequence of the VH of antibody hWN01 (e.g., SEQ ID NO: 2), and
the light chain variable region comprises the amino acid sequence
of the VL of antibody hWN01 (e.g., SEQ ID NO: 9).
[0368] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 82). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 89).
[0369] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 27); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 28); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 21).
[0370] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 27);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 28); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWNv1 (e.g., SEQ ID
NO: 21).
[0371] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWNv1 (e.g., SEQ ID NO: 27); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 28);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWNv1 (e.g., SEQ ID NO: 21).
[0372] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0373] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 53); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody hWNv1 (e.g., SEQ ID
NO: 54).
[0374] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody hWNv1 (e.g., SEQ ID NO: 52); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 53);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody hWNv1 (e.g., SEQ ID NO: 54).
[0375] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 27); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody hWNv1 (e.g., SEQ ID NO: 28); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody hWNv1 (e.g.,
SEQ ID NO: 21), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0376] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 27);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 28); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWNv1 (e.g., SEQ ID
NO: 21), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody hWNv1
(e.g., SEQ ID NO: 53); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0377] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWNv1 (e.g., SEQ ID NO: 27); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 28);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWNv1 (e.g., SEQ ID NO: 21), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ
ID NO: 53); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0378] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 19); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 25); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 21).
[0379] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 19);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 25); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWNv1 (e.g., SEQ ID
NO: 21).
[0380] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWNv1 (e.g., SEQ ID NO: 19); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 25);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWNv1 (e.g., SEQ ID NO: 21).
[0381] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0382] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 53); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody hWN01 (e.g., SEQ ID
NO: 54).
[0383] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody hWN01 (e.g., SEQ ID NO: 52); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody hWN01 (e.g., SEQ ID NO: 53);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody hWN01 (e.g., SEQ ID NO: 54).
[0384] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 19); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody hWNv1 (e.g., SEQ ID NO: 25); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody hWNv1 (e.g.,
SEQ ID NO: 21), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody hWNv1 (e.g.,
SEQ ID NO: 52); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 53); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0385] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 19);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody hWNv1 (e.g., SEQ ID NO: 25); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody hWNv1 (e.g., SEQ ID
NO: 21), and the light chain variable region comprises one, two, or
all of the following: an LCDR1 comprising the amino acid sequence
of the LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody hWNv1
(e.g., SEQ ID NO: 53); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0386] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody hWNv1 (e.g., SEQ ID NO: 19); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody hWNv1 (e.g., SEQ ID NO: 25);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody hWNv1 (e.g., SEQ ID NO: 21), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody hWNv1 (e.g., SEQ ID NO: 52); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody hWNv1 (e.g., SEQ
ID NO: 53); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody hWNv1 (e.g., SEQ ID NO: 54).
[0387] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0388] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of hWNv1 (e.g., SEQ ID NO:
3). In an embodiment, the heavy chain variable region comprises the
amino acid sequence of the VH of hWNv1 (e.g., SEQ ID NO: 3).
[0389] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of hWNv1 (e.g., SEQ ID NO:
9). In an embodiment, the light chain variable region comprises the
amino acid sequence of the VL of hWNv1 (e.g., SEQ ID NO: 9).
[0390] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of hWNv1 (e.g., SEQ ID NO: 3), and wherein the
light chain variable region comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of the
VL of hWNv1 (e.g., SEQ ID NO: 9).
[0391] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of hWNv1 (e.g., SEQ ID NO: 3),
and wherein the light chain variable region comprises the amino
acid sequence of the VL of hWNv1 (e.g., SEQ ID NO: 9).
[0392] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 83). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 89).
[0393] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 32); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 33); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3E7 (e.g., SEQ ID NO: 31).
[0394] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 32);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 33); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3E7 (e.g., SEQ ID NO:
31).
[0395] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3E7 (e.g., SEQ ID NO: 32); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 33);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3E7 (e.g., SEQ ID NO: 31).
[0396] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 55); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3E7 (e.g., SEQ ID NO: 57).
[0397] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 56); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3E7 (e.g., SEQ ID NO:
57).
[0398] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3E7 (e.g., SEQ ID NO: 57).
[0399] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 32); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3E7 (e.g., SEQ ID NO: 33); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3E7 (e.g.,
SEQ ID NO: 31), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 55); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3E7 (e.g., SEQ ID NO: 57).
[0400] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 32);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 33); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3E7 (e.g., SEQ ID NO:
31), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3E7
(e.g., SEQ ID NO: 56); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3E7 (e.g., SEQ ID NO: 57).
[0401] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3E7 (e.g., SEQ ID NO: 32); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 33);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3E7 (e.g., SEQ ID NO: 31), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID
NO: 56); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3E7 (e.g., SEQ ID NO: 57).
[0402] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 29); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 30); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3E7 (e.g., SEQ ID NO: 31).
[0403] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 29);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 30); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3E7 (e.g., SEQ ID NO:
31). In an embodiment, the heavy chain variable region comprises an
HCDR1 comprising the amino acid sequence of the HCDR1 of antibody
3E7 (e.g., SEQ ID NO: 29); an HCDR2 comprising the amino acid
sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 30); and an
HCDR3 comprising the amino acid sequence of the HCDR3 of antibody
3E7 (e.g., SEQ ID NO: 31).
[0404] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 55); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3E7 (e.g., SEQ ID NO: 57).
[0405] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 56); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3E7 (e.g., SEQ ID NO:
57).
[0406] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3E7 (e.g., SEQ ID NO: 57).
[0407] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 29); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3E7 (e.g., SEQ ID NO: 30); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3E7 (e.g.,
SEQ ID NO: 31), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3E7 (e.g.,
SEQ ID NO: 55); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID NO: 56); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3E7 (e.g., SEQ ID NO: 57).
[0408] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3E7 (e.g., SEQ ID NO: 29);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3E7 (e.g., SEQ ID NO: 30); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3E7 (e.g., SEQ ID NO:
31), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3E7
(e.g., SEQ ID NO: 56); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3E7 (e.g., SEQ ID NO: 57).
[0409] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3E7 (e.g., SEQ ID NO: 29); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3E7 (e.g., SEQ ID NO: 30);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3E7 (e.g., SEQ ID NO: 31), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3E7 (e.g., SEQ ID NO: 55); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3E7 (e.g., SEQ ID
NO: 56); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3E7 (e.g., SEQ ID NO: 57).
[0410] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0411] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of 3E7 (e.g., SEQ ID NO:
4). In an embodiment, the heavy chain variable region comprises the
amino acid sequence of the VH of 3E7 (e.g., SEQ ID NO: 4).
[0412] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of 3E7 (e.g., SEQ ID NO:
10). In an embodiment, the light chain variable region comprises
the amino acid sequence of the VL of 3E7 (e.g., SEQ ID NO: 10).
[0413] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of 3E7 (e.g., SEQ ID NO: 4), and wherein the
light chain variable region comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of the
VL of 3E7 (e.g., SEQ ID NO: 10).
[0414] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of 3E7 (e.g., SEQ ID NO: 4), and
wherein the light chain variable region comprises the amino acid
sequence of the VL of 3E7 (e.g., SEQ ID NO: 10).
[0415] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 84). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 90).
[0416] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 37); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 38); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3G1 (e.g., SEQ ID NO: 36).
[0417] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 37);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 38); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3G1 (e.g., SEQ ID NO:
36).
[0418] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 37); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 38);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 36).
[0419] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 58); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3G1 (e.g., SEQ ID NO: 60).
[0420] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 59); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3G1 (e.g., SEQ ID NO:
60).
[0421] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 60).
[0422] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 37); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3G1 (e.g., SEQ ID NO: 38); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3G1 (e.g.,
SEQ ID NO: 36), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 58); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3G1 (e.g., SEQ ID NO: 60).
[0423] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 37);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 38); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3G1 (e.g., SEQ ID NO:
36), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3G1
(e.g., SEQ ID NO: 59); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3G1 (e.g., SEQ ID NO: 60).
[0424] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 37); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 38);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 36), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID
NO: 59); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3G1 (e.g., SEQ ID NO: 60).
[0425] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 34); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 35); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3G1 (e.g., SEQ ID NO: 36).
[0426] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 34);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 35); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3G1 (e.g., SEQ ID NO:
36).
[0427] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 34); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 35);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 36).
[0428] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 58); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3G1 (e.g., SEQ ID NO: 60).
[0429] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 59); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3G1 (e.g., SEQ ID NO:
60).
[0430] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 60).
[0431] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 34); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3G1 (e.g., SEQ ID NO: 35); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3G1 (e.g.,
SEQ ID NO: 36), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3G1 (e.g.,
SEQ ID NO: 58); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID NO: 59); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3G1 (e.g., SEQ ID NO: 60).
[0432] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3G1 (e.g., SEQ ID NO: 34);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3G1 (e.g., SEQ ID NO: 35); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3G1 (e.g., SEQ ID NO:
36), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3G1
(e.g., SEQ ID NO: 59); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3G1 (e.g., SEQ ID NO: 60).
[0433] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3G1 (e.g., SEQ ID NO: 34); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3G1 (e.g., SEQ ID NO: 35);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3G1 (e.g., SEQ ID NO: 36), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3G1 (e.g., SEQ ID NO: 58); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3G1 (e.g., SEQ ID
NO: 59); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3G1 (e.g., SEQ ID NO: 60).
[0434] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0435] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of 3G1 (e.g., SEQ ID NO:
5). In an embodiment, the heavy chain variable region comprises the
amino acid sequence of the VH of 3G1 (e.g., SEQ ID NO: 5).
[0436] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of 3G1 (e.g., SEQ ID NO:
11). In an embodiment, the light chain variable region comprises
the amino acid sequence of the VL of 3G1 (e.g., SEQ ID NO: 11).
[0437] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of 3G1 (e.g., SEQ ID NO: 5), and wherein the
light chain variable region comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of the
VL of 3G1 (e.g., SEQ ID NO: 11).
[0438] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of 3G1 (e.g., SEQ ID NO: 5), and
wherein the light chain variable region comprises the amino acid
sequence of the VL of 3G1 (e.g., SEQ ID NO: 11).
[0439] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 85). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 91).
[0440] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 42); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 43); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 2C7 (e.g., SEQ ID NO: 41).
[0441] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 2C7 (e.g., SEQ ID NO: 42);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 2C7 (e.g., SEQ ID NO: 43); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 2C7 (e.g., SEQ ID NO:
41).
[0442] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 42); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 43);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 41).
[0443] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 61); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 2C7 (e.g., SEQ ID NO: 63).
[0444] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 2C7 (e.g., SEQ ID NO: 61);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 2C7 (e.g., SEQ ID NO: 62); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 2C7 (e.g., SEQ ID NO:
63).
[0445] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 61); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 63).
[0446] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 2C7 (e.g., SEQ ID NO: 42); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 2C7 (e.g., SEQ ID NO: 43); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 2C7 (e.g.,
SEQ ID NO: 41), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 61); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 2C7 (e.g., SEQ ID NO: 63). In an embodiment, the heavy
chain variable region comprises one, two, or all of the following:
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 42); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 43);
or an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 41), and the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising the amino acid sequence of the LCDR1 of antibody 2C7
(e.g., SEQ ID NO: 61); an LCDR2 comprising the amino acid sequence
of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62); or an LCDR3
comprising the amino acid sequence of the LCDR3 of antibody 2C7
(e.g., SEQ ID NO: 63).
[0447] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 42); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 43);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 41), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 2C7 (e.g., SEQ ID NO: 61); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID
NO: 62); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 2C7 (e.g., SEQ ID NO: 63).
[0448] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 39); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 40); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 2C7 (e.g., SEQ ID NO: 41).
[0449] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 2C7 (e.g., SEQ ID NO: 39);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 2C7 (e.g., SEQ ID NO: 40); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 2C7 (e.g., SEQ ID NO:
41).
[0450] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 39); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 40);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 41).
[0451] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 61); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 2C7 (e.g., SEQ ID NO: 63).
[0452] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 2C7 (e.g., SEQ ID NO: 61);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 2C7 (e.g., SEQ ID NO: 62); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 2C7 (e.g., SEQ ID NO:
63).
[0453] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 61); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 63).
[0454] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 2C7 (e.g., SEQ ID NO: 39); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 2C7 (e.g., SEQ ID NO: 40); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 2C7 (e.g.,
SEQ ID NO: 41), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 2C7 (e.g.,
SEQ ID NO: 61); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID NO: 62); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 2C7 (e.g., SEQ ID NO: 63).
[0455] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 2C7 (e.g., SEQ ID NO: 39);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 2C7 (e.g., SEQ ID NO: 40); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 2C7 (e.g., SEQ ID NO:
41), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 2C7 (e.g., SEQ ID NO: 61); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 2C7
(e.g., SEQ ID NO: 62); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 2C7 (e.g., SEQ ID NO: 63).
[0456] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 2C7 (e.g., SEQ ID NO: 39); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 2C7 (e.g., SEQ ID NO: 40);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 2C7 (e.g., SEQ ID NO: 41), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 2C7 (e.g., SEQ ID NO: 61); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 2C7 (e.g., SEQ ID
NO: 62); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 2C7 (e.g., SEQ ID NO: 63).
[0457] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0458] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of 2C7 (e.g., SEQ ID NO:
6). In an embodiment, the heavy chain variable region comprises the
amino acid sequence of the VH of 2C7 (e.g., SEQ ID NO: 6).
[0459] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of 2C7 (e.g., SEQ ID NO:
12). In an embodiment, the light chain variable region comprises
the amino acid sequence of the VL of 2C7 (e.g., SEQ ID NO: 12).
[0460] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of 2C7 (e.g., SEQ ID NO: 6), and wherein the
light chain variable region comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of the
VL of 2C7 (e.g., SEQ ID NO: 12).
[0461] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of 2C7 (e.g., SEQ ID NO: 6), and
wherein the light chain variable region comprises the amino acid
sequence of the VL of 2C7 (e.g., SEQ ID NO: 12).
[0462] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 86). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 92).
[0463] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 47); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 48); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3D6 (e.g., SEQ ID NO: 46).
[0464] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 47);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 48); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3D6 (e.g., SEQ ID NO:
46).
[0465] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3D6 (e.g., SEQ ID NO: 47); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 48);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3D6 (e.g., SEQ ID NO: 46).
[0466] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 64); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3D6 (e.g., SEQ ID NO: 66).
[0467] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 65); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3D6 (e.g., SEQ ID NO:
66).
[0468] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3D6 (e.g., SEQ ID NO: 66).
[0469] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 47); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3D6 (e.g., SEQ ID NO: 48); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3D6 (e.g.,
SEQ ID NO: 46), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 64); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3D6 (e.g., SEQ ID NO: 66).
[0470] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 47);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 48); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3D6 (e.g., SEQ ID NO:
46), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3D6
(e.g., SEQ ID NO: 65); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3D6 (e.g., SEQ ID NO: 66).
[0471] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3D6 (e.g., SEQ ID NO: 47); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 48);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3D6 (e.g., SEQ ID NO: 46), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID
NO: 65); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3D6 (e.g., SEQ ID NO: 66).
[0472] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises three heavy chain complementarity determining regions
(HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable region
comprises one, two, or all of the following: an HCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 44); an HCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 45); or an
HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of antibody 3D6 (e.g., SEQ ID NO: 46).
[0473] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 44);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 45); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3D6 (e.g., SEQ ID NO:
46). In an embodiment, the heavy chain variable region comprises an
HCDR1 comprising the amino acid sequence of the HCDR1 of antibody
3D6 (e.g., SEQ ID NO: 44); an HCDR2 comprising the amino acid
sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 45); and an
HCDR3 comprising the amino acid sequence of the HCDR3 of antibody
3D6 (e.g., SEQ ID NO: 46).
[0474] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises three light chain complementarity determining regions
(LCDR1, LCDR2, and LCDR3), wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 64); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3D6 (e.g., SEQ ID NO: 66).
[0475] In an embodiment, the light chain variable region comprises
one, two, or all of the following: an LCDR1 comprising the amino
acid sequence of the LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64);
an LCDR2 comprising the amino acid sequence of the LCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 65); or an LCDR3 comprising the
amino acid sequence of the LCDR3 of antibody 3D6 (e.g., SEQ ID NO:
66).
[0476] In an embodiment, the light chain variable region comprises
an LCDR1 comprising the amino acid sequence of the LCDR1 of
antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2 comprising the amino
acid sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65);
and an LCDR3 comprising the amino acid sequence of the LCDR3 of
antibody 3D6 (e.g., SEQ ID NO: 66).
[0477] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the light chain variable region comprises three light chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein the heavy chain variable region comprises one, two, or all
of the following: an HCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 44); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of antibody 3D6 (e.g., SEQ ID NO: 45); or an HCDR3 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the HCDR3 of antibody 3D6 (e.g.,
SEQ ID NO: 46), and wherein the light chain variable region
comprises one, two, or all of the following: an LCDR1 comprising an
amino acid sequence that differs by no more than 1, 2, or 3 amino
acid residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR1 of antibody 3D6 (e.g.,
SEQ ID NO: 64); an LCDR2 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID NO: 65); or an
LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of antibody 3D6 (e.g., SEQ ID NO: 66).
[0478] In an embodiment, the heavy chain variable region comprises
one, two, or all of the following: an HCDR1 comprising the amino
acid sequence of the HCDR1 of antibody 3D6 (e.g., SEQ ID NO: 44);
an HCDR2 comprising the amino acid sequence of the HCDR2 of
antibody 3D6 (e.g., SEQ ID NO: 45); or an HCDR3 comprising the
amino acid sequence of the HCDR3 of antibody 3D6 (e.g., SEQ ID NO:
46), and the light chain variable region comprises one, two, or all
of the following: an LCDR1 comprising the amino acid sequence of
the LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2
comprising the amino acid sequence of the LCDR2 of antibody 3D6
(e.g., SEQ ID NO: 65); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of antibody 3D6 (e.g., SEQ ID NO: 66).
[0479] In an embodiment, the heavy chain variable region comprises
an HCDR1 comprising the amino acid sequence of the HCDR1 of
antibody 3D6 (e.g., SEQ ID NO: 44); an HCDR2 comprising the amino
acid sequence of the HCDR2 of antibody 3D6 (e.g., SEQ ID NO: 45);
and an HCDR3 comprising the amino acid sequence of the HCDR3 of
antibody 3D6 (e.g., SEQ ID NO: 46), and the light chain variable
region comprises an LCDR1 comprising the amino acid sequence of the
LCDR1 of antibody 3D6 (e.g., SEQ ID NO: 64); an LCDR2 comprising
the amino acid sequence of the LCDR2 of antibody 3D6 (e.g., SEQ ID
NO: 65); and an LCDR3 comprising the amino acid sequence of the
LCDR3 of antibody 3D6 (e.g., SEQ ID NO: 66).
[0480] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0481] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VH of 3D6 (e.g., SEQ ID NO:
7). In an embodiment, the heavy chain variable region comprises the
amino acid sequence of the VH of 3D6 (e.g., SEQ ID NO: 7).
[0482] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
comprises an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
from, or has at least 85, 90, 95, 96, 97, 98, 99, or 100% homology
with, the amino acid sequence of the VL of 3D6 (e.g., SEQ ID NO:
13). In an embodiment, the light chain variable region comprises
the amino acid sequence of the VL of 3D6 (e.g., SEQ ID NO: 13).
[0483] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the heavy chain variable region comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of the VH of 3D6 (e.g., SEQ ID NO: 7), and wherein the
light chain variable region comprises an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid residues from, or has at least 85, 90, 95, 96,
97, 98, 99, or 100% homology with, the amino acid sequence of the
VL of 3D6 (e.g., SEQ ID NO: 13).
[0484] In an embodiment, the heavy chain variable region comprises
the amino acid sequence of the VH of 3D6 (e.g., SEQ ID NO: 7), and
wherein the light chain variable region comprises the amino acid
sequence of the VL of 3D6 (e.g., SEQ ID NO: 13).
[0485] In an embodiment, the heavy chain variable region comprises
an amino acid sequence encoded by a nucleotide sequence from Table
2 (e.g., SEQ ID NO: 87). In an embodiment, the light chain variable
region comprises and amino acid sequence encoded by a nucleotide
sequence from Table 2 (e.g., SEQ ID NO: 93).
[0486] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the VH comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), wherein the VH comprises one, two, or all of the following:
an HCDR1 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR1
of a VH described in Table 8 (e.g., any of SEQ ID NOS: 115-118); an
HCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR2
of a VH described in Table 8 (e.g., any of SEQ ID NOS: 115-118); or
an HCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the HCDR3
of a VH described in Table 8 (e.g., any of SEQ ID NOS:
115-118).
[0487] In an embodiment, the VH comprises one, two, or all of the
following: an HCDR1 comprising the amino acid sequence of the HCDR1
of a VH described in Table 8 (e.g., any of SEQ ID NOS: 115-118); an
HCDR2 comprising the amino acid sequence of the HCDR2 of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118); or an
HCDR3 comprising the amino acid sequence of the HCDR3 of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118).
[0488] In an embodiment, the VH comprises: an HCDR1 comprising the
amino acid sequence of the HCDR1 of a VH described in Table 8
(e.g., any of SEQ ID NOS: 115-118); an HCDR2 comprising the amino
acid sequence of the HCDR2 of a VH described in Table 8 (e.g., any
of SEQ ID NOS: 115-118); and an HCDR3 comprising the amino acid
sequence of the HCDR3 of a VH described in Table 8 (e.g., any of
SEQ ID NOS: 115-118).
[0489] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the VL comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3), wherein the VL comprises one, two, or all of the following:
an LCDR1 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR1
of a VL described in Table 8 (e.g., any of SEQ ID NOS: 119-137); an
LCDR2 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR2
of a VL described in Table 8 (e.g., any of SEQ ID NOS: 119-137); or
an LCDR3 comprising an amino acid sequence that differs by no more
than 1, 2, or 3 amino acid residues from, or has at least 85, 90,
95, 99 or 100% homology with, the amino acid sequence of the LCDR3
of a VL described in Table 8 (e.g., any of SEQ ID NOS:
119-137).
[0490] In an embodiment, the VL comprises one, two, or all of the
following: an LCDR1 comprising the amino acid sequence of the LCDR1
of a VL described in Table 8 (e.g., any of SEQ ID NOS: 119-137); an
LCDR2 comprising the amino acid sequence of the LCDR2 of a VL
described in Table 8 (e.g., any of SEQ ID NOS: 119-137); or an
LCDR3 comprising the amino acid sequence of the LCDR3 of a VL
described in Table 8 (e.g., any of SEQ ID NOS: 119-137).
[0491] In an embodiment, the VL comprises: an LCDR1 comprising the
amino acid sequence of the LCDR1 of a VL described in Table 8
(e.g., any of SEQ ID NOS: 119-137); an LCDR2 comprising the amino
acid sequence of the LCDR2 of a VL described in Table 8 (e.g., any
of SEQ ID NOS: 119-137); and an LCDR3 comprising the amino acid
sequence of the LCDR3 of a VL described in Table 8 (e.g., any of
SEQ ID NOS: 119-137).
[0492] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the VH comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the VL comprises
three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), wherein the VH comprises one, two, or all of the
following: an HCDR1 comprising an amino acid sequence that differs
by no more than 1, 2, or 3 amino acid residues from, or has at
least 85, 90, 95, 99 or 100% homology with, the amino acid sequence
of the HCDR1 of a VH described in Table 8 (e.g., any of SEQ ID NOS:
115-118); an HCDR2 comprising an amino acid sequence that differs
by no more than 1, 2, or 3 amino acid residues from, or has at
least 85, 90, 95, 99 or 100% homology with, the amino acid sequence
of the HCDR2 of a VH described in Table 8 (e.g., any of SEQ ID NOS:
115-118); or an HCDR3 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the HCDR3 of a VH described in Table 8 (e.g., any of
SEQ ID NOS: 115-118); and wherein the VL comprises one, two, or all
of the following: an LCDR1 comprising an amino acid sequence that
differs by no more than 1, 2, or 3 amino acid residues from, or has
at least 85, 90, 95, 99 or 100% homology with, the amino acid
sequence of the LCDR1 of a VL described in Table 8 (e.g., any of
SEQ ID NOS: 119-137); an LCDR2 comprising an amino acid sequence
that differs by no more than 1, 2, or 3 amino acid residues from,
or has at least 85, 90, 95, 99 or 100% homology with, the amino
acid sequence of the LCDR2 of a VL described in Table 8 (e.g., any
of SEQ ID NOS: 119-137); or an LCDR3 comprising an amino acid
sequence that differs by no more than 1, 2, or 3 amino acid
residues from, or has at least 85, 90, 95, 99 or 100% homology
with, the amino acid sequence of the LCDR3 of a VL described in
Table 8 (e.g., any of SEQ ID NOS: 119-137).
[0493] In an embodiment, the VH comprises one, two, or all of the
following: an HCDR1 comprising the amino acid sequence of the HCDR1
of a VH described in Table 8 (e.g., any of SEQ ID NOS: 115-118); an
HCDR2 comprising the amino acid sequence of the HCDR2 of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118); or an
HCDR3 comprising the amino acid sequence of the HCDR3 of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118); and the VL
comprises one, two, or all of the following: an LCDR1 comprising
the amino acid sequence of the LCDR1 of a VL described in Table 8
(e.g., any of SEQ ID NOS: 119-137); an LCDR2 comprising the amino
acid sequence of the LCDR2 of a VL described in Table 8 (e.g., any
of SEQ ID NOS: 119-137); or an LCDR3 comprising the amino acid
sequence of the LCDR3 of a VL described in Table 8 (e.g., any of
SEQ ID NOS: 119-137).
[0494] In an embodiment, the VH comprises: an HCDR1 comprising the
amino acid sequence of the HCDR1 of a VH described in Table 8
(e.g., any of SEQ ID NOS: 115-118); an HCDR2 comprising the amino
acid sequence of the HCDR2 of a VH described in Table 8 (e.g., any
of SEQ ID NOS: 115-118); and an HCDR3 comprising the amino acid
sequence of the HCDR3 of a VH described in Table 8 (e.g., any of
SEQ ID NOS: 115-118); and the VL comprises: an LCDR1 comprising the
amino acid sequence of the LCDR1 of a VL described in Table 8
(e.g., any of SEQ ID NOS: 119-137); an LCDR2 comprising the amino
acid sequence of the LCDR2 of a VL described in Table 8 (e.g., any
of SEQ ID NOS: 119-137); and an LCDR3 comprising the amino acid
sequence of the LCDR3 of a VL described in Table 8 (e.g., any of
SEQ ID NOS: 119-137).
[0495] In an embodiment, the antibody molecule further comprises
one or more human or human derived heavy or light chain variable
region frameworks.
[0496] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the VH comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of a VH described in Table 8 (e.g., any of SEQ ID NOS:
115-118). In an embodiment, the VH comprises the amino acid
sequence of a VH described in Table 8 (e.g., any of SEQ ID NOS:
115-118).
[0497] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the VL comprises an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 amino acid residues from, or has at least
85, 90, 95, 96, 97, 98, 99, or 100% homology with, the amino acid
sequence of a VL described in Table 8 (e.g., any of SEQ ID NOS:
119-137). In an embodiment, the VL comprises the amino acid
sequence of a VL described in Table 8 (e.g., any of SEQ ID NOS:
119-137).
[0498] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the VH comprises an amino acid sequence that differs by no
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues from, or has at least 85, 90, 95, 96, 97, 98,
99, or 100% homology with, the amino acid sequence of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118), and
wherein the VL comprises an amino acid sequence that differs by no
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues from, or has at least 85, 90, 95, 96, 97, 98,
99, or 100% homology with, the amino acid sequence of a VL
described in Table 8 (e.g., any of SEQ ID NOS: 119-137). In an
embodiment, the VH comprises the amino acid sequence of a VH
described in Table 8 (e.g., any of SEQ ID NOS: 115-118), and the VL
comprises the amino acid sequence of a VL described in Table 8
(e.g., any of SEQ ID NOS: 119-137).
[0499] In an embodiment, the HCCDR1, HCCDR2, and HCCDR3 are from
the same VH described in Table 8. In an embodiment, the LCCDR1,
LCCDR2, and LCCDR3 are from the same VL described in Table 8.
[0500] Any of the VH amino acid sequences (or the amino acid
sequences of HCDR1, HCDR2, and HCDR3 thereof) disclosed in Table 8
(or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 amino acid residues therefrom, or has
at least 85, 90, 95, 96, 97, 98, 99, or 100% homology therewith)
can be combined with any of the VL amino acid sequences (or the
amino acid sequences of LCDR1, LCDR2, and LCDR3 thereof) disclosed
in Table 8 (or a sequence that differs by no more than 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues
therefrom, or has at least 85, 90, 95, 96, 97, 98, 99, or 100%
homology therewith), e.g., to form a humanized antibody molecule.
Exemplary combinations include:
[0501] SEQ ID NOS: 103 and 104; SEQ ID NOS: 103 and 119; SEQ ID
NOS: 103 and 120; SEQ ID NOS: 103 and 121; SEQ ID NOS: 103 and 122;
SEQ ID NOS: 103 and 123; SEQ ID NOS: 103 and 124; SEQ ID NOS: 103
and 125; SEQ ID NOS: 103 and 126; SEQ ID NOS: 103 and 127; SEQ ID
NOS: 103 and 128; SEQ ID NOS: 103 and 129; SEQ ID NOS: 103 and 130;
SEQ ID NOS: 103 and 131; SEQ ID NOS: 103 and 132; SEQ ID NOS: 103
and 133; SEQ ID NOS: 103 and 134; SEQ ID NOS: 103 and 135; SEQ ID
NOS: 103 and 136; SEQ ID NOS: 103 and 137; SEQ ID NOS: 115 and 104;
SEQ ID NOS: 115 and 119; SEQ ID NOS: 115 and 120; SEQ ID NOS: 115
and 121; SEQ ID NOS: 115 and 122; SEQ ID NOS: 115 and 123; SEQ ID
NOS: 115 and 124; SEQ ID NOS: 115 and 125; SEQ ID NOS: 115 and 126;
SEQ ID NOS: 115 and 127; SEQ ID NOS: 115 and 128; SEQ ID NOS: 115
and 129; SEQ ID NOS: 115 and 130; SEQ ID NOS: 115 and 131; SEQ ID
NOS: 115 and 132; SEQ ID NOS: 115 and 133; SEQ ID NOS: 115 and 134;
SEQ ID NOS: 115 and 135; SEQ ID NOS: 115 and 136; SEQ ID NOS: 115
and 137; SEQ ID NOS: 116 and 104; SEQ ID NOS: 116 and 119; SEQ ID
NOS: 116 and 120; SEQ ID NOS: 116 and 121; SEQ ID NOS: 116 and 122;
SEQ ID NOS: 116 and 123; SEQ ID NOS: 116 and 124; SEQ ID NOS: 116
and 125; SEQ ID NOS: 116 and 126; SEQ ID NOS: 116 and 127; SEQ ID
NOS: 116 and 128; SEQ ID NOS: 116 and 129; SEQ ID NOS: 116 and 130;
SEQ ID NOS: 116 and 131; SEQ ID NOS: 116 and 132; SEQ ID NOS: 116
and 133; SEQ ID NOS: 116 and 134; SEQ ID NOS: 116 and 135; SEQ ID
NOS: 116 and 136; SEQ ID NOS: 116 and 137; SEQ ID NOS: 117 and 104;
SEQ ID NOS: 117 and 119; SEQ ID NOS: 117 and 120; SEQ ID NOS: 117
and 121; SEQ ID NOS: 117 and 122; SEQ ID NOS: 117 and 123; SEQ ID
NOS: 117 and 124; SEQ ID NOS: 117 and 125; SEQ ID NOS: 117 and 126;
SEQ ID NOS: 117 and 127; SEQ ID NOS: 117 and 128; SEQ ID NOS: 117
and 129; SEQ ID NOS: 117 and 130; SEQ ID NOS: 117 and 131; SEQ ID
NOS: 117 and 132; SEQ ID NOS: 117 and 133; SEQ ID NOS: 117 and 134;
SEQ ID NOS: 117 and 135; SEQ ID NOS: 117 and 136; SEQ ID NOS: 117
and 137; SEQ ID NOS: 118 and 104; SEQ ID NOS: 118 and 119; SEQ ID
NOS: 118 and 120; SEQ ID NOS: 118 and 121; SEQ ID NOS: 118 and 122;
SEQ ID NOS: 118 and 123; SEQ ID NOS: 118 and 124; SEQ ID NOS: 118
and 125; SEQ ID NOS: 118 and 126; SEQ ID NOS: 118 and 127; SEQ ID
NOS: 118 and 128; SEQ ID NOS: 118 and 129; SEQ ID NOS: 118 and 130;
SEQ ID NOS: 118 and 131; SEQ ID NOS: 118 and 132; SEQ ID NOS: 118
and 133; SEQ ID NOS: 118 and 134; SEQ ID NOS: 118 and 135; SEQ ID
NOS: 118 and 136; or SEQ ID NOS: 118 and 137.
[0502] In an embodiment, the ADC or antibody molecule comprises a
heavy chain variable region (VH), wherein the heavy chain variable
region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), wherein the heavy chain variable
region comprises one, two, or all of the following: an HCDR1
comprising the amino acid sequence of SEQ ID NO: 108; an HCDR2
comprising the amino acid sequence of YISSDGDSX.sub.1YYPD X.sub.2
X.sub.3KG (SEQ ID NO: 165), wherein X.sub.1 is I or T; X.sub.2 is N
or S; X.sub.3 is I or V; or an HCDR3 comprising the amino acid
sequence of SEQ ID NO: 107.
[0503] In an embodiment, the ADC or antibody molecule comprises a
light chain variable region (VL), wherein the light chain variable
region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein the light chain variable
region comprises one, two, or all of the following: an LCDR1
comprising the amino acid sequence of RASESX.sub.1FGHGISPX.sub.2H
(SEQ ID NO: 166), wherein X.sub.1 is V or I; X.sub.2 is M or L; an
LCDR2 comprising the amino acid sequence of
RASX.sub.1X.sub.2KX.sub.3 (SEQ ID NO: 167), wherein X.sub.1 is N or
S; X.sub.2 is L or R; X.sub.3 is F, T or S; or an LCDR3 comprising
the amino acid sequence of SEQ ID NO: 112.
[0504] In an embodiment, the ADC or antibody molecule comprises a
VH and a VL, wherein the VH comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the VL comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3),
[0505] wherein the VH comprises one, two, or all of the following:
an HCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an
HCDR2 comprising the amino acid sequence of YISSDGDSX.sub.1YYPD
X.sub.2 X.sub.3KG (SEQ ID NO: 165), wherein X.sub.1 is I or T;
X.sub.2 is N or S; X.sub.3 is I or V; or an HCDR3 comprising the
amino acid sequence of SEQ ID NO: 107, and
[0506] wherein the VL comprises one, two, or all of the following:
an LCDR1 comprising the amino acid sequence of
RASESX.sub.1FGHGISPX.sub.2H (SEQ ID NO: 166), wherein X.sub.1 is V
or I; X.sub.2 is M or L; an LCDR2 comprising the amino acid
sequence of RASX.sub.1X.sub.2KX.sub.3 (SEQ ID NO: 167), wherein
X.sub.1 is N or S; X.sub.2 is L or R; X.sub.3 is F, T or S; or an
LCDR3 comprising the amino acid sequence of SEQ ID NO: 112.
[0507] In an embodiment, the ADC or antibody molecule comprises a
VH and a VL, wherein the VH comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and
the VL comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3),
[0508] wherein the VH comprises: an HCDR1 comprising the amino acid
sequence of SEQ ID NO: 108; an HCDR2 comprising the amino acid
sequence of YISSDGDSX.sub.1YYPD X.sub.2 X.sub.3KG (SEQ ID NO: 165),
wherein X.sub.1 is I or T; X.sub.2 is N or S; X.sub.3 is I or V;
and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 107,
and
[0509] wherein the VL comprises: an LCDR1 comprising the amino acid
sequence of RASESX.sub.1FGHGISPX.sub.2H (SEQ ID NO: 166), wherein
X.sub.1 is V or I; X.sub.2 is M or L; an LCDR2 comprising the amino
acid sequence of RASX.sub.1X.sub.2KX.sub.3 (SEQ ID NO: 167),
wherein X.sub.1 is N or S; X.sub.2 is L or R; X.sub.3 is F, T or S;
and an LCDR3 comprising the amino acid sequence of SEQ ID NO:
112.
[0510] In an embodiment, the antibody molecule comprises or
consists of two heavy chain variable regions and two light chain
variable regions.
[0511] In an embodiment, the antibody molecule further comprises a
heavy chain constant region, a light chain constant region, or
both.
[0512] In an embodiment, the antibody molecule is an IgG antibody
molecule, e.g., IgG1, IgG2, IgG3, or IgG4 antibody molecule. In an
embodiment, the antibody molecule is not an IgM antibody
molecule.
[0513] In an embodiment, the antibody molecule comprises a light
chain constant region from a kappa or lambda light chain.
[0514] In an embodiment, the antibody molecule is capable of
binding to two or more Gram-negative strains. Antibody molecules
capable of binding to two or more Gram-negative bacterial strains
have several advantageous properties. For example, one therapy can
be used to treat, prevent, or diagnose multiple bacterial
infections. In addition, a physician need not determine which
bacterial strain infected a patient in order to determine the
appropriate therapy.
[0515] According, in an embodiment, the antibody molecule binds to
one or more bacteria, e.g., one or more Gram-negative bacteria,
e.g., of different genera, species, subspecies, and/or strains.
[0516] In an embodiment, the one or more Gram-negative bacteria are
selected from a species of Enterobacteriaceae (e.g., a species in
Klebsiella, Enterobacter, Shigella, Escherichia, Salmonella,
Yersinia, or Citrobacter, e.g., pan-resistant Enterobacteriaceae),
a species of Pseudomonas, a species of Acinetobacter, or any
combination thereof.
[0517] In an embodiment, the antibody molecule binds to one or more
of: Klebsiella pneumonia (e.g., Klebsiella pneumoniae subsp.
ozaenae, Klebsiella pneumoniae subsp. pneumoniae, or Klebsiella
pneumoniae subsp. rhinoscleromatis), Enterobacter cancerogenous,
Enterobacter cloacae, Enterobacter hormaechei, Enterobacter
asburiae, Shigella boydii, Shigella dysenteriae, Shigella flexneri,
Shigella sonnei, Escherichia coli (e.g., Escherichia coli ATCC
11775, Escherichia coli ATCC 25922, Escherichia coli ATCC 35401, or
Escherichia coli ATCC 43895), Escherichia fergusonii, Salmonella
choleraesuis, Salmonella choleraesuis subsp. indica, Salmonella
enteritidis, Salmonella virchow, Salmonella paratyphi B, Salmonella
typhimurium, Salmonella paratyphi A, Salmonella typhi, Salmonella
choleraesuis subsp. arizonae, Salmonella choleraesuis subsp.
diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonella
bongori, Citrobacter sedlakii, Citrobacter braakii, Citrobacter
werkmanii, Citrobacter freundii, Citrobacter youngae, Citrobacter
amalonaticus, Yersinia enterocolitica, Yersinia frederiksenii,
Yersinia pestis, Yersinia pseudotuberculosis, or any combination
thereof.
[0518] In an embodiment, the one or more bacteria are one or more
antibiotic-resistant bacteria, e.g., one or more
multidrug-resistant Gram-negative bacteria.
[0519] In an embodiment, the one or more antibiotic-resistant
bacteria are selected from Pseudomonas (e.g., P. aeruginosa),
Enterobacteriaceae (e.g., Klebsiella pneumonia or E. coli), or
Acinetobacter (e.g., A. baumannii).
[0520] In an embodiment, the antibody molecule binds to one or more
of: Enterococcus faecium (e.g., vancomycin-resistant (VRE)
Enterococcus faecium), Staphylococcus aureus (e.g.,
methicillin-resistant (MRSA) Staphylococcus aureus), Clostridium
difficile, Acinetobacter baumannii (e.g., multidrug resistant (MDR)
Acinetobacter), Pseudomonas aeruginosa (e.g., multidrug resistant
(MDR) P. aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis).
[0521] In an embodiment, the antibody molecule binds to one or more
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) P. aeruginosa strains in
Table 7. In another embodiment, the antibody molecule binds to one
or more (e.g., 2, 3, 4, 5, 6, or all) multidrug-resistant P.
aeruginosa strains in Table 7.
[0522] In an embodiment, the antibody molecule binds to LPS with
high affinity, e.g., with a K.sub.D that is less than about 10 nM,
e.g., measured by an ELISA method.
[0523] In an embodiment, the antibody molecule binds to LPS with a
K.sub.off slower than 1.times.10.sup.4, 5.times.10.sup.5, or
1.times.10.sup.-5 s.sup.-1. In an embodiment, the antibody molecule
binds to LPS with a K.sub.on faster than 1.times.10.sup.4,
5.times.10.sup.4, 1.times.10.sup.5, or 5.times.10.sup.5 M.sup.-1
s.sup.-1.
[0524] In an embodiment, the antibody molecule has opsonophagocytic
activity, e.g., as determined by an OPA assay, e.g., as described
herein.
[0525] In an embodiment, the antibody molecule binds to an epitope
comprising one or more (e.g., two) Kdo residues and/or one or more
(e.g., two or three) Hep residues in LPS.
[0526] In an embodiment, a) the antibody molecule that binds to
lipopolysaccharide (LPS) is coupled (e.g., fused) to b) the
antimicrobial peptide, e.g., an antimicrobial peptide described
herein, e.g., to form an antibody molecule-drug conjugate
(ADC).
[0527] In an embodiment, the antibody molecule comprises a heavy
chain variable region (VH), wherein the heavy chain variable region
is coupled (e.g., fused) to the antimicrobial peptide, e.g.,
wherein the heavy chain variable region is N-terminal to the
antimicrobial peptide.
[0528] In an embodiment, the heavy chain variable region is coupled
(e.g., fused) to the antimicrobial peptide indirectly, e.g.,
wherein the C-terminus of the heavy chain variable region is
coupled (e.g., fused) to the N-terminus of the antimicrobial
peptide via a constant region.
[0529] In an embodiment, the antibody molecule comprises a light
chain variable region (VL), wherein the light chain variable region
is coupled (e.g., fused) to the antimicrobial peptide, e.g.,
wherein the light chain variable region is N-terminal to the
antimicrobial peptide.
[0530] In an embodiment, the light chain variable region is coupled
(e.g., fused) to the antimicrobial peptide indirectly, e.g.,
wherein the C-terminus of the light chain variable region is
coupled (e.g., fused) to the N-terminus of the antimicrobial
peptide via a constant region.
[0531] In an embodiment, the antibody molecule is coupled (e.g.,
fused) to two or more (e.g., three, four, five, six, seven, eight,
or more, e.g., four) antimicrobial peptides, e.g., by enzymatic
conjugation or chemical conjugation. In an embodiment, at least two
of the antimicrobial peptides are identical. In an embodiment, at
least two of the antimicrobial peptides are different.
[0532] In an embodiment, the antibody molecule is coupled (e.g.,
fused) to two identical antimicrobial peptides, each is coupled
(e.g., fused) to a heavy chain variable region, e.g., indirectly,
e.g., via a constant region.
[0533] In an embodiment, the antimicrobial peptide coupled (e.g.,
fused) to the antibody molecule is more effective in inhibiting,
e.g., inhibiting the growth, virulence, or infectivity of, a
Gram-negative bacterium (e.g., a Gram-negative bacterium described
herein) than the antimicrobial peptide or antibody molecule alone,
e.g., having a minimum inhibitory concentration (MIC) that is
lower, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100 fold lower, than the MIC of the
antimicrobial peptide alone.
[0534] In an embodiment, the antimicrobial peptide coupled (e.g.,
fused) to the antibody molecule is more effective in reducing the
viability of, e.g., killing, a Gram-negative bacterium (e.g., a
Gram-negative bacterium described herein) than the antimicrobial
peptide or antibody molecule alone, e.g., having a minimum
bactericidal concentration (MBC) that is lower, e.g., at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100
fold lower, than the MBC of the antimicrobial peptide alone.
[0535] In an embodiment, the antibody molecule has opsonophagocytic
activity (e.g., is phagocytized when bound to the Fc receptor (FcR)
of a neutrophil), e.g., as determined by an OPA assay, e.g., as
described herein.
[0536] In an embodiment, the antimicrobial peptide coupled (e.g.,
fused) to the antibody molecule does not inhibit, e.g., does not
inhibit the growth, virulence, or infectivity of, a Gram-positive
bacterium (e.g., a Gram-positive bacterium described herein), e.g.,
having a minimum inhibitory concentration (MIC) for a Gram-negative
bacterium (e.g., a Gram-negative bacterium) that is lower, e.g., at
least 2, 5, 10, 20, 50, 100, 200, 500, or 1000 fold lower, than a
MIC for a Gram-positive bacterium (e.g., a Gram-positive
bacterium).
[0537] In an embodiment, the antimicrobial peptide coupled (e.g.,
fused) to the antibody molecule does not reduce the viability of,
e.g., does not kill, a Gram-positive bacterium (e.g., a
Gram-positive bacterium described herein), e.g., having a minimum
bactericidal concentration (MBC) for a Gram-negative bacterium
(e.g., a Gram-negative bacterium) that is lower, e.g., at least 2,
5, 10, 20, 50, 100, 200, 500, or 1000 fold lower fold lower, than a
MBC for a Gram-positive bacterium (e.g., a Gram-positive
bacterium).
[0538] In an embodiment, the Gram-positive bacterium is
Staphylococcus aureus.
[0539] In an embodiment, the antibody molecule binds to a linear or
conformational epitope on a Gram-negative bacterium or on LPS.
[0540] Antimicrobial Peptides
[0541] As used herein, the term "antimicrobial peptide" refers to a
peptide that has an antimicrobial activity, e.g., an antibacterial
activity. While not wishing to be bound by theory, it is believed
that, in an embodiment, the antimicrobial peptides can have
variable length, sequence and structure with broad spectrum
activity against a number of microorganisms, e.g., bacteria, and
optionally low levels of induced resistance. In an embodiment, the
antimicrobial peptide is an antibacterial peptide.
[0542] Antimicrobial peptides are a diverse group of molecules,
which are divided into subgroups on the basis of their amino acid
composition and structure. Typically, these peptides include two or
more positively charged residues provided by arginine, lysine or,
in acidic environments, histidine, and a large proportion (e.g.,
more than 50%) of hydrophobic residues. The secondary structures of
these molecules include, e.g., i) .alpha.-helical, ii)
.beta.-stranded due to the presence of 2 or more disulfide bonds,
iii) .beta.-hairpin or loop due to the presence of a single
disulfide bond and/or cyclization of the peptide chain, and iv)
extended. Some of these peptides can be unstructured in free
solution, and fold into their final configuration upon partitioning
into biological membranes. Antimicrobial peptides can contain
hydrophilic amino acid residues aligned along one side and
hydrophobic amino acid residues aligned along the opposite side of
a helical molecule. This amphipathicity of the antimicrobial
peptides allows them to partition into the membrane lipid bilayer.
While not wishing to be bound by theory, it is believed that in an
embodiment, the ability to associate with membranes is a typical
feature of antimicrobial peptides although membrane
permeabilization may not be required.
[0543] Types of antimicrobial peptides include, e.g., anionic
peptides (e.g., rich in glutamic and aspartic acids), linear
cationic .alpha.-helical peptides (e.g., lacking cysteine),
cationic peptides (e.g., rich in proline, arginine, phenylalanine,
glycine, or tryptophan), and anionic and cationic peptides that
contain cysteine and form disulfide bonds (e.g., containing 1-3
disulfide bonds).
[0544] The modes of action by which antimicrobial peptides kill
microbes are varied, and may differ for different bacterial
species. The cytoplasmic membrane is a frequent target, but
peptides may also interfere with DNA and protein synthesis, protein
folding, and cell wall synthesis. The initial contact between the
peptide and the target organism can be electrostatic, as most
bacterial surfaces are anionic, or hydrophobic. Their amino acid
composition, amphipathicity, cationic charge and size allow them to
attach to and insert into membrane bilayers to form pores by
"barrel-stave," "carpet" or "toroidal-pore" mechanisms.
Alternately, they may penetrate into the cell to bind intracellular
molecules which are crucial to cell living. Intracellular binding
models includes inhibition of cell wall synthesis, alteration of
the cytoplasmic membrane, activation of autolysin, inhibition of
DNA, RNA, and protein synthesis, and inhibition of certain enzymes.
These peptides can be bactericidal and/or bacteriostatic.
Typically, the antimicrobial (e.g., antibacterial) activity of
these peptides is determined by measuring the minimal inhibitory
concentration (MIC).
[0545] Antimicrobial peptides have been demonstrated to kill
Gram-negative and Gram-positive bacteria, viruses, fungi, and
transformed or cancerous cells (Reddy et al. (2004) International
Journal of Antimicrobial Agents 24 (6): 536-547). Antimicrobial
peptides may also have an immunomodulatory activity. For example,
the immunomodulatory activities may be involved in the clearance of
infection, e.g., the ability to alter host gene expression, act as
chemokines and/or induce chemokine production, inhibiting
lipopolysaccharide induced pro-inflammatory cytokine production,
promoting wound healing, or modulating the responses of dendritic
cells and cells of the adaptive immune response.
[0546] Several methods can be used to determine the mechanisms of
antimicrobial peptide activity.
[0547] These methods include, e.g., solid-state NMR spectroscopy,
microscopy, atomic emission spectroscopy, fluorescent dyes, ion
channel formation, circular dichroism and orientated circular
dichroism, dual polarization interferometry, or neutron and X-ray
diffraction.
[0548] In an embodiment, the antimicrobial peptide is under 10 kDa,
e.g., under 8 kDa, 6 kDa, 4 kDa, 2 kDa, or 1 kDa. In an embodiment,
the antimicrobial peptide comprises or consists of from about 6 to
about 100 amino acids, e.g., from about 6 to about 75 amino acids,
about 6 to about 50 amino acids, about 6 to about 25 amino acids,
about 25 to about 100 amino acids, about 50 to about 100 amino
acids, or about 75 to about 100 amino acids. In an embodiment, the
anti-bacterial peptide comprises or consists of from about 12 to
about 50 amino acids. In an embodiment, the anti-bacterial peptide
comprises or consists of from about 15 to about 45 amino acids. In
an embodiment, the antimicrobial peptide is substantially cationic.
In an embodiment, the antimicrobial peptide is substantially
amphipathic. In an embodiment, the antimicrobial peptide is
substantially cationic and amphipathic. In an embodiment, the
antimicrobial peptide is cytostatic to a Gram-negative bacterium.
In an embodiment, the antimicrobial peptide is cytotoxic to a
Gram-negative bacterium. In an embodiment, the antimicrobial
peptide is cytostatic and cytotoxic to a Gram-positive bacterium.
In an embodiment, the antimicrobial peptide is broad-spectrum
antimicrobial peptide, e.g., cytostatic and/or cytotoxic to two or
more bacteria of different genera, species, subspecies, and/or
strains. In an embodiment, the antimicrobial peptide is a secreted
polypeptide.
[0549] Antimicrobial peptides have been isolated and described from
a wide range of animals microorganisms, invertebrates, plants,
amphibians, birds, fish, and mammals (Wang et al., Nucleic Acids
Res. 2009; 37 (Database issue):D933-7). For example, antimicrobial
polypeptides are described in Antimicrobial Peptide Database
(http://aps.unmc.edu/AP/main.php; Wang et al., Nucleic Acids Res.
2009; 37 (Database issue):D933-7), CAMP: Collection of
Anti-Microbial Peptides (http://www.bicnirrh.res.in/antimicrobial/;
Thomas et al., Nucleic Acids Res. 2010; 38 (Database
issue):D774-80), U.S. Pat. Nos. 5,221,732, 5,447,914, 5,519,115,
5,607,914, 5,714,577, 5,734,015, 5,798,336, 5,821,224, 5,849,490,
5,856,127, 5,905,187, 5,994,308, 5,998,374, 6,107,460, 6,191,254,
6,211,148, 6,300,489, 6,329,504, 6,399,370, 6,476,189, 6,478,825,
6,492,328, 6,514,701, 6,573,361, 6,573,361, 6,576,755, 6,605,698,
6,624,140, 6,638,531, 6,642,203, 6,653,280, 6,696,238, 6,727,066,
6,730,659, 6,743,598, 6,743,769, 6,747,007, 6,790,833, 6,794,490,
6,818,407, 6,835,536, 6,835,713, 6,838,435, 6,872,705, 6,875,907,
6,884,776, 6,887,847, 6,906,035, 6,911,524, 6,936,432, 7,001,924,
7,071,293, 7,078,380, 7,091,185, 7,094,759, 7,166,769, 7,244,710,
7,314,858, and 7,582,301, the contents of which are incorporated by
references.
[0550] The antimicrobial peptides described herein may inhibit or
reduce the viability of one or more bacteria species, e.g., one or
more bacteria (e.g., Gram-negative bacteria) of different genera,
species, subspecies, and/or strains. In an embodiment, the
antimicrobial peptide is capable of inhibiting and/or reducing the
viability of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Gram-negative
bacterial strains. For instance, the antibody molecule may inhibit
and/or reduce the viability of one or more bacteria of different
species, subspecies, and/or strains from Enterobacteriaceae (e.g.,
Klebsiella, Enterobacter, Shigella, Escherichia, Salmonella,
Yersinia, or Citrobacter, e.g., pan-resistant Enterobacteriaceae),
one or more bacteria of different species, subspecies, and/or
strains from Pseudomonas, one or more bacteria of different
species, subspecies, and/or strains from Acinetobacter, or any
combination thereof. In an embodiment, the antimicrobial peptide is
capable of inhibiting or reducing the viability of Klebsiella
pneumonia (e.g., Klebsiella pneumoniae subsp. ozaenae, Klebsiella
pneumoniae subsp. pneumoniae, or Klebsiella pneumoniae subsp.
rhinoscleromatis), Enterobacter cancerogenous, Enterobacter
cloacae, Enterobacter hormaechei, Enterobacter asburiae, Shigella
boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Escherichia coli (e.g., Escherichia coli ATCC 11775, Escherichia
coli ATCC 25922, Escherichia coli ATCC 35401, or Escherichia coli
ATCC 43895), Escherichia fergusonii, Salmonella choleraesuis,
Salmonella choleraesuis subsp. indica, Salmonella enteritidis,
Salmonella virchow, Salmonella paratyphi B, Salmonella typhimurium,
Salmonella paratyphi A, Salmonella typhi, Salmonella choleraesuis
subsp. arizonae, Salmonella choleraesuis subsp. diarizonae,
Salmonella choleraesuis subsp. houtenae, Salmonella bongori,
Citrobacter sedlakii, Citrobacter braakii, Citrobacter werkmanii,
Citrobacter freundii, Citrobacter youngae, Citrobacter
amalonaticus, Yersinia enterocolitica, Yersinia frederiksenii,
Yersinia pestis, Yersinia pseudotuberculosis, or any combination
thereof.
[0551] In an embodiment, the antimicrobial peptide is capable of
inhibit and/or reduce the viability of one or more: Enterococcus
faecium (e.g., vancomycin-resistant (VRE) Enterococcus faecium),
Staphylococcus aureus (e.g., methicillin-resistant (MRSA)
Staphylococcus aureus), Clostridium difficile, Acinetobacter
baumannii (e.g., multidrug resistant (MDR) Acinetobacter),
Pseudomonas aeruginosa (e.g., multidrug resistant (MDR) P.
aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis). The antimicrobial peptides
described herein can have one or more (e.g., two, three or all) of
the following properties: strong bacterial inhibition activity,
strong bactericidal activity, low red blood cell (RBC) hemolytic
activity, or low cytotoxicity (e.g., off-target toxicity). In an
embodiment, the antimicrobial peptide has a minimum inhibitory
concentration (MIC) of less than 100 .mu.g/ml, e.g., less than 90,
80, 70, 60, 50, 40, 30, 20, 10, or 5 .mu.g/ml, against a bacterial
strain described herein, e.g., Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC27853, or both. In an embodiment, the
antimicrobial peptide has a minimum bactericidal concentration
(MBC) of less than 100 .mu.g/ml, e.g., less than 90, 80, 70, 60,
50, 40, 30, 20, 10, or 5 .mu.g/ml, against a bacterial strain
described herein, e.g., Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC27853, or both. In an embodiment, the antimicrobial
peptide has low hemolytic activity, e.g., has a PLC to MIC ratio
for a Gram-negative bacterium (e.g., a Gram-negative bacterium
described herein) which is equal to or greater than 1 (e.g.,
greater than 4:1, 8:1, 16:1, 24:1, or 32:1), e.g., as determined by
a red blood cell hemolysis assay and an MIC assay, respectively. In
an embodiment, the PLC is the concentration (e.g., minimum
concentration) required or needed to lyse 50% of the red blood
cells. In an embodiment, the antimicrobial peptide has low
hemolytic activity, e.g., has an MLC to MIC ratio for a
Gram-negative bacterium (e.g., a Gram-negative bacterium described
herein) which is greater than 4:1 (e.g., greater than 8:1, 16:1,
24:1, or 32:1), e.g., as determined by a red blood cell hemolysis
assay and an MIC assay, respectively. In an embodiment, the MLC is
the concentration (e.g., minimum concentration) required or needed
to lyse 100% of the red blood cells.
[0552] In an embodiment, the antimicrobial peptide is an
.alpha.-helical peptide. In an embodiment, the antimicrobial
peptide has two or more amino acid residues cross-linked. For
example, two cysteine residues in the antimicrobial peptide can be
cross-linked, e.g., chemically cross-linked. While not wishing to
be bound by theory, it is believed that in an embodiment,
cross-linking two or more amino acid residues in an antimicrobial
peptide may enhance .alpha.-helical conformation, enhance serum
stability, and/or increase antimicrobial potency. In an embodiment,
the antimicrobial peptide having two or more amino acid residues
cross-linked is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold more
stable, e.g., in serum, than an otherwise identical antimicrobial
peptide that does not have two or more amino acid residues
cross-linked. In an embodiment, the antimicrobial peptide having
two or more amino acid residues cross-linked has a MIC that is at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
lower than an otherwise identical antimicrobial peptide that does
not have two or more amino acid residues cross-linked.
[0553] In an embodiment, the antimicrobial peptide is coupled
(e.g., fused) to an antibody molecule, e.g., an antibody molecule
described herein, e.g., to form an antibody molecule-drug conjugate
(ADC).
[0554] The following criteria can be used to select a candidate
antimicrobial peptide: broad spectrum (e.g., activity against
multiple bacterial pathogens), alpha-helical secondary structure
(e.g., readily expressed in functional form), mechanism of action
by membrane disruption (e.g., bactericidal and/or synergy with
small molecules), in vivo stability (e.g., stable in human sera),
and adaptable to N-terminal modifications (e.g., to function as
C-terminal antibody conjugates).
[0555] The antimicrobial peptides described herein can contain a
sequence that does not have an antimicrobial activity by itself. In
an embodiment, the antimicrobial peptide comprises a linker
sequence. In an embodiment, the antimicrobial peptide comprises a
sortase donor sequence.
[0556] In an embodiment, the antimicrobial peptide alone does not
bind to LPS, e.g., a core region of LPS.
[0557] In an embodiment, a heavy chain of the antibody molecule
comprises a first sortase acceptor sequence and a light chain of
the antibody molecule comprises a second sortase acceptor sequence.
In an embodiment, the sortase acceptor sequence, e.g., the first
sortase recognition sequence, comprises the amino acid sequence of
(GS).sub.6LPETGGG (SEQ ID NO: 24). In another embodiment, the
sortase acceptor sequence, e.g., the second sortase acceptor
sequence, comprises the amino acid sequence of
P(G.sub.4S).sub.2LPETGGSG (SEQ ID NO: 26).
[0558] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, or 5 amino acid residues from, or has at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with, an amino acid
sequence described herein, e.g., any of SEQ ID NOS: 67-80, 94-102,
147-156, 158-159, or 163-164. In an embodiment, the antimicrobial
peptide comprises or consists of an amino acid sequence described
herein e.g., any of SEQ ID NOS: 67-80, 94-102, 147-156, 158-159, or
163-164, or a portion thereof, e.g., a functional fragment thereof
(e.g., one or more (e.g., two, three, four or more) of the
N-terminal G residues are omitted). For example, the first three
N-terminal G residues in any of SEQ ID NOS: 101, 147, 152-154, 156,
or 163-164 can be omitted. In an embodiment, the presence of one or
more (e.g., two, three, four or more) of the N-terminal G residues
does not reduce, or significantly reduce, the activity of the
antimicrobial peptide.
[0559] In an embodiment, the peptide comprises the amino acid
sequence of:
TABLE-US-00004 (SEQ ID NO: 68) RGLRRLGRKIAHGVKKYGPTVLRIIRIAG, (SEQ
ID NO: 80) GGGRGLRRLGRKIAHGVKKYGPTVLRIIRIAG, (SEQ ID NO: 101)
GGGGRFKRFRKKFKKLFKKLSPVIPLLHLG, or (SEQ ID NO: 102)
GRFKRFRKKFKKLFKKLSPVIPLLHLG.
[0560] In an embodiment, the antimicrobial peptide is a stapled
antimicrobial peptide. Without wishing to be bound by theory, it is
believed that in an embodiment, stapling can increase stability,
reduce non-specific binding, or both, e.g., in serum. Exemplary
stapling methods are described, e.g., in Alexander et al. J. Am.
Chem. Soc., 2013, 135 (16), 5946-5949; and Example 7.
[0561] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence described in Tables 3 or 6A-6B
or in FIG. 4 or 15A-15B. In another embodiment, the antimicrobial
peptide comprises or consists of an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
residues from, or has at least 80%, 85%, 90%, 95%, 97%, or 100%
homology with, the amino acid sequence described in Tables 3 or
6A-6B or in FIG. 4 or 15A-15B, or a portion thereof, e.g., a
functional fragment thereof. In an embodiment, the antimicrobial
peptide comprises or consists of an amino acid sequence that
differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
residues from, or has at least 80%, 85%, 90%, 95%, 97%, or 100%
homology with, the amino acid sequence of SEQ ID NOS: 67-80,
94-102, 147-156, 158-159, or 163-164. In an embodiment, the
antimicrobial peptide comprises a carboxamide group (e.g., a
C-terminal carboxamide functional group).
[0562] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 101.
[0563] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 147.
[0564] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 152.
[0565] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 153.
[0566] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 154.
[0567] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 156.
[0568] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 163.
[0569] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 164.
[0570] In an embodiment, the antimicrobial peptide comprises one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38. 39, 40, or more) D-amino acids. In an embodiment,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of
the amino acid residues in the antimicrobial peptide are D-amino
acids. In an embodiment, all of the amino acid residues in the
antimicrobial peptide are D-amino acids. Without wishing to be
bound by theory, it is believed that in an embodiment, the presence
of one or more (e.g., all) D-amino acids in the antimicrobial
peptide can increase the stability of the antimicrobial peptide or
ADC, e.g., in serum (e.g., human serum), e.g., compared to an
otherwise identical antimicrobial peptide that contains one or more
(e.g., all) L-amino acids.
[0571] In an embodiment, the antimicrobial peptide comprises one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38. 39, 40, or more) L-amino acids. In an embodiment,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of
the amino acid residues in the antimicrobial peptide are L-amino
acids. In an embodiment, all of the amino acid residues in the
antimicrobial peptide are L-amino acids.
[0572] In an embodiment, the antimicrobial peptide comprises one or
more D-amino acids and one or more L-amino acids. For example, any
of the amino acid residues in the antimicrobial peptides described
in Tables 3 or 6A-6B or in FIG. 4 or 15A-15B can be a D-amino acid
or an L-amino acid.
TABLE-US-00005 TABLE 3 Amino acid sequences of exemplary
antimicrobial peptides SEQ ID Peptide Amino Acid Sequences NO
Peptide 26 ALWKTLLKKVLKAAAK 67 Peptide 119
RGLRRLGRKIAHGVKKYGPTVLRIIRIAG 68 Peptide 109 GIGKFLKKAKKFGKAFVKILKK
69 Peptide 30 ALWKTLLKKVLKAAAKGGGGSGGGGS 70 Peptide 24
GIGKFLKKAKKFGKAFVKILKKGGGGSGGGGS 71 Peptide 126 KKLLKWLKKLL 72
Peptide 21 (MAL)-(EG3)-GIGKFLKKAKKFGKAFVKILKK 73 Peptide 128
RLGNFFRKAKKKIGRGLKKIGQKIKDFLGNLVPRTES 74 Peptide 23
GGGGSGGGGSGIGKFLKKAKKFGKAFVKILKK 75 Peptide 33
GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLA 76 Peptide 29
GGGGSGGGGSALWKTLLKKVLKAAAK 77 Peptide 85 GWKKWFNRAKKVGKTVGGLAVDHYLG
78 Peptide 70 GAFGNFLKGVAKKAGLKILSIAQCKLFGTC 79
GGGRGLRRLGRKIAHGVKKYGPTVLRIIRIAG 80 GIGKHVGKALKGLKGLLKGLGES 94
GRRKRKWLRRIGKGVKIIGGAALDHL 95 GGLRSLGRKILRAWKKYGPQATPATRQ 96
IKWKKLLRAAKRIL 97 IGKKWKRIVKRIKKFLRKL 98 ILGKIWKIKKLF 99
RLGDILQKAREKIEGGLKKLVQKIKDFFGKFAPRTES 100
GGGGRFKRFRKKFKKLFKKLSPVIPLLHLG 101 GRFKRFRKKFKKLFKKLSPVIPLLHLG 102
Peptide 265 GGGLLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 147 Peptide
266 GGGKWKSFIKKLTKAAKKVVTTAKKPLIV 148 Peptide 267
GGGGRFKRFRKKFKKLFKKLSPVIPLLHLG 101 Peptide 268 GGGVNWKKILGKIIKVVK
149 Peptide 269 GGGTLISWIKNKRKQRPRVSRRRRRRGGRRRR 150 Peptide 270
GGGGIGAILKVLATGLPTLISWIKNKRKQ 151 Peptide 271
GGGGLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLA 152 Peptide 293
GGGGLRRLGRKIAHGIKKYGPTILRIIRIAG 153 Peptide 294
GGGRGLRRLGRKIAHGVKKYGPTVLRIIKKYG 154 Peptide 295
GGGGRFKRFRKKFKKLFKKLSPVIPLLHLG 101 Peptide 296
GGGKRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPF 155 Peptide 297
GGGKFFRKLKKSVKKRAKEFFKKPRVIGVSIPF 156 Peptide 261
GGGGIGKFLKKAKKFGKAFVKILKK 163 GGG-Octapeptin
GGG-D-Dab-cyclic(L-Dab-L-Dab-D-Leu-L-Phe-L- 164 Dab-L-Dab-L-Leu)
MAL: maleimide; EG3: tri(ethylene glycol)
[0573] In an embodiment, the ADC comprises a peptide comprising the
amino acid sequence of
TABLE-US-00006 (SEQ ID NO: 68) RGLRRLGRKIAHGVKKYGPTVLRIIRIAG, (SEQ
ID NO: 80) GGGRGLRRLGRKIAHGVKKYGPTVLRIIRIAG, (SEQ ID NO: 101)
GGGGRFKRFRKKFKKLFKKLSPVIPLLHLG, or (SEQ ID NO: 102)
GRFKRFRKKFKKLFKKLSPVIPLLHLG.
[0574] In an embodiment, the antimicrobial peptide comprises or
consists of an amino acid sequence selected from:
RGLRRLGRKIAHGVKKYGPTVLRIIRIAG (SEQ ID NO: 68),
GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 69), KKLLKWLKKLL (SEQ ID NO:
72), RLGNFFRKAKKKIGRGLKKIGQKIKDFLGNLVPRTES (SEQ ID NO: 74),
GIGKHVGKALKGLKGLLKGLGES (SEQ ID NO: 94), GRRKRKWLRRIGKGVKIIGGAALDHL
(SEQ ID NO: 95), GGLRSLGRKILRAWKKYGPQATPATRQ (SEQ ID NO: 96),
IKWKKLLRAAKRIL (SEQ ID NO: 97), IGKKWKRIVKRIKKFLRKL (SEQ ID NO:
98), ILGKIWKIKKLF (SEQ ID NO: 99), or
RLGDILQKAREKIEGGLKKLVQKIKDFFGKFAPRTES (SEQ ID NO: 100).
[0575] Antibody Molecule-Drug Conjugates
[0576] As used herein, the term "antibody molecule-drug conjugate"
or ADC refers to an antibody molecule that is coupled to a
non-antibody moiety, e.g., a therapeutic agent or label, e.g., an
antimicrobial peptide. The antibody molecule can be coupled to the
non-antibody moiety directly, or indirectly, e.g., through a
linker.
[0577] In an embodiment, the antibody molecule is coupled to the
non-antibody moiety by a covalent bond. In an embodiment, the
antibody molecule is coupled to the non-antibody moiety by a
peptide bond. In an embodiment, the coupling of the antibody
molecule to the non-antibody moiety is mediated by a sortase. In an
embodiment, the coupling of the antibody molecule and the
non-antibody moiety forms a fusion protein. In an embodiment, the
antibody molecule and the non-antibody moiety forms a fusion
protein. In an embodiment, the fusion protein comprises a linker
between the antibody molecule (e.g., a heavy chain, a light chain,
or both) and the non-antibody moiety. In an embodiment, the
antibody molecule is coupled to the non-antibody moiety by a
non-peptide bond. In an embodiment, the antibody molecule is not
coupled to the non-antibody moiety by a non-peptide bond. In an
embodiment, a non-antibody moiety is also referred to as a
"payload."
[0578] In an embodiment, the non-antibody moiety is coupled to the
backbone of the antibody molecule. In another embodiment, the
non-antibody moiety is coupled to a side chain of the antibody
molecule. In an embodiment, the non-antibody moiety is a peptide
(e.g., an antimicrobial peptide) and the antibody molecule is
coupled to the backbone of the peptide (e.g., antimicrobial
peptide). In an embodiment, the non-antibody moiety is a peptide
(e.g., an antimicrobial peptide) and the antibody molecule is
coupled to a side-chain of the peptide (e.g., antimicrobial
peptide).
[0579] In an embodiment, two or more (e.g., three, four, five, six,
seven, eight, or more) non-antibody moieties (e.g., antimicrobial
peptides) are coupled to the antibody molecule. In an embodiment,
four non-antibody moieties (e.g., antimicrobial peptides) are
coupled to the antibody molecule. For example, the non-antibody
moieties can be the same, or at least some of the non-antibody
moieties are different from each other. In an embodiment, the
non-antibody moiety (e.g., antimicrobial peptide) is coupled to the
antibody molecule in a bivalent manner In another embodiment, the
non-antibody moiety (e.g., antimicrobial peptide) is coupled to the
antibody molecule in a tetravalent manner.
[0580] In an embodiment, the ADC comprises an antibody molecule
that binds to a bacterium (e.g., a Gram-negative bacterium). In an
embodiment, the ADC comprises an antibody molecule that binds to
LPS, e.g., on the outer membrane of a Gram-negative bacterium). In
an embodiment, the ADC comprises an antibody molecule described
herein.
[0581] In an embodiment, the ADC comprises one, two, or three CDRs
of the VH region of an antibody molecule described in Table 1 or 8
(e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any
of the humanized mAb001), using the Kabat or Chothia definitions of
CDRs. In an embodiment, the ADC comprises one, two, or three CDRs
of the VL region of an antibody molecule described in Table 1 or 8
(e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any
of the humanized mAb001), using the Kabat or Chothia definitions of
CDRs. In an embodiment, the ADC comprises one or more (e.g., two or
three) CDRs of the VH region and/or one or more (e.g., two or
three) CDRs of the VL region of an antibody molecule described in
Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7,
or 3D6, or any of the humanized mAb001), using the Kabat or Chothia
definitions of CDRs.
[0582] In an embodiment, the ADC comprises one, two, or three VH
CDRs described in Table 1 or 8. In an embodiment, the ADC comprises
one, two, or three VL CDRs described in Table 1 or 8. In an
embodiment, the ADC comprises one or more (e.g., two or three) VH
CDRs and/or one or more (e.g., two or three) VL CDRs described in
Table 1 or 8.
[0583] In an embodiment, the ADC comprises one, two, three, or four
frameworks of the VH region of an antibody molecule described in
Table 1 or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7,
or 3D6, or any of the humanized mAb001). In an embodiment, the ADC
comprises one, two, three, or four frameworks of the VL region of
an antibody molecule described in Table 1 or 8 (e.g., mAb001,
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the
humanized mAb001). In an embodiment, the ADC comprises one or more
(e.g., two, three, or four) frameworks of the VH region and/or one
or more (e.g., two, three, or four) frameworks of the VL region of
an antibody molecule described in Table 1 or 8 (e.g., mAb001,
A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the
humanized mAb001).
[0584] In an embodiment, the ADC comprises a heavy chain variable
region of an antibody molecule described in Table 1 or 8 (e.g.,
mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the
humanized mAb001). In an embodiment, the ADC comprises a light
chain variable region of an antibody molecule described in Table 1
or 8 (e.g., mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6,
or any of the humanized mAb001). In an embodiment, the ADC
comprises a heavy chain variable region and a light chain variable
region of an antibody molecule described in Table 1 or 8 (e.g.,
mAb001, A001-25, hWN01, hWNv1, 3E7, 3G1, 2C7, or 3D6, or any of the
humanized mAb001).
[0585] In an embodiment, the ADC comprises a heavy chain variable
region having an amino acid sequence described in Table 1 or 8. In
an embodiment, the ADC comprises a light chain variable region
having an amino acid sequence described in Table 1 or 8. In an
embodiment, the ADC comprises a heavy chain variable region having
an amino acid sequence described in Table 2 and a light chain
variable region having an amino acid sequence described in Table 1
or 8.
[0586] In an embodiment, the antibody molecule comprises a heavy
chain variable region encoded by a nucleotide sequence described in
Table 2. In an embodiment, the antibody molecule comprises a light
chain variable region encoded by a nucleotide sequence described in
Table 2. In an embodiment, the antibody molecule comprises a heavy
chain variable region encoded by a nucleotide sequence described in
Table 2 and a light chain variable region encoded by a nucleotide
sequence described in Table 2.
[0587] In an embodiment, the ADC comprises a heavy chain constant
region. In an embodiment, the ADC comprises a light chain constant
region. In an embodiment, the ADC comprises a heavy chain constant
region and a light chain constant region. In an embodiment, the ADC
comprises a heavy chain constant region, a light chain constant
region, and heavy and light chain variable regions of an antibody
molecule described in Table 1 or 8. In certain embodiments, the ADC
comprises a heavy chain constant region, a light chain constant
region, and variable regions that comprise one, two, three, four,
five, or six CDRs of antibody molecule described in Table 1 or
8.
[0588] In an embodiment, the ADC is capable of binding to two or
more Gram-negative bacteria of different genera, species,
subspecies, and/or strains. Antibody molecules or ADCs capable of
binding to two or more Gram-negative bacteria of different genera,
species, subspecies, and/or strains have several advantageous
properties. For example, one therapy can be used to treat, prevent,
or diagnose multiple bacterial infections. In addition, a physician
need not determine which bacterial genus, species, subspecies
and/or strain infected a patient in order to determine the
appropriate therapy. Accordingly, in an embodiment, the ADC is
capable of independently binding to 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more Gram-negative bacteria of different genera, species,
subspecies, and/or strains, with high affinity. For instance, the
antibody molecule may independently bind with high affinity to one
or more bacteria of different species, subspecies, and/or strains
from Enterobacteriaceae (e.g., Klebsiella, Enterobacter, Shigella,
Escherichia, Salmonella, Yersinia, or Citrobacter, e.g.,
pan-resistant Enterobacteriaceae), one or more bacteria of
different species, subspecies, and/or strains from Pseudomonas, one
or more bacteria of different species, subspecies, and/or strains
from Acinetobacter, or any combination thereof. In an embodiment,
the ADC is capable of binding to Klebsiella pneumonia (e.g.,
Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp.
pneumoniae, or Klebsiella pneumoniae subsp. rhinoscleromatis),
Enterobacter cancerogenous, Enterobacter cloacae, Enterobacter
hormaechei, Enterobacter asburiae, Shigella boydii, Shigella
dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli
(e.g., Escherichia coli ATCC 11775, Escherichia coli ATCC 25922,
Escherichia coli ATCC 35401, or Escherichia coli ATCC 43895),
Escherichia fergusonii, Salmonella choleraesuis, Salmonella
choleraesuis subsp. indica, Salmonella enteritidis, Salmonella
virchow, Salmonella paratyphi B, Salmonella typhimurium, Salmonella
paratyphi A, Salmonella typhi, Salmonella choleraesuis subsp.
arizonae, Salmonella choleraesuis subsp. diarizonae, Salmonella
choleraesuis subsp. houtenae, Salmonella bongori, Citrobacter
sedlakii, Citrobacter braakii, Citrobacter werkmanii, Citrobacter
freundii, Citrobacter youngae, Citrobacter amalonaticus, Yersinia
enterocolitica, Yersinia frederiksenii, Yersinia pestis, Yersinia
pseudotuberculosis, or any combination thereof.
[0589] In an embodiment, the ADC is capable of binding to one or
more of: Enterococcus faecium (e.g., vancomycin-resistant (VRE)
Enterococcus faecium), Staphylococcus aureus (e.g.,
methicillin-resistant (MRSA) Staphylococcus aureus), Clostridium
difficile, Acinetobacter baumannii (e.g., multidrug resistant (MDR)
Acinetobacter), Pseudomonas aeruginosa (e.g., multidrug resistant
(MDR) P. aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis).
[0590] In an embodiment, the ADC is capable of binding to one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) P. aeruginosa
strains in Table 7. In another embodiment, the ADC is capable of
binding to one or more (e.g., 2, 3, 4, 5, 6, or all)
multidrug-resistant P. aeruginosa strains in Table 7.
[0591] In an embodiment, the ADC binds to a linear or
conformational epitope on LPS. In another embodiment, the ADC binds
to a core pentasaccharide region on LPS. In an embodiment, the ADC
has an opsonophagocytic activity, e.g., as determined by an
opsonophagocytosis assay described herein.
[0592] In an embodiment, the ADC comprises an antimicrobial
peptide, e.g., an antimicrobial peptide described herein, e.g.,
having an amino acid sequence disclosed in Tables 3 or 6A-6B or in
FIG. 4 or 15A-15B. In another embodiment, the antimicrobial peptide
comprises or consists of an amino acid sequence that differs by no
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues
from, or has at least 80%, 85%, 90%, 95%, 97%, or 100% homology
with, the amino acid sequence described in Tables 3 or 6A-6B or in
FIG. 4 or 15A-15B, or a portion thereof, e.g., a functional
fragment thereof (e.g., one or more (e.g., two, three, four or
more) of the N-terminal G residues are omitted). In an embodiment,
the antimicrobial peptide comprises or consists of an amino acid
sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid residues from, or has at least 80%, 85%, 90%, 95%,
97%, or 100% homology with, the amino acid sequence of any of SEQ
ID NO: 67-80, 94-102, 147-156, 158-159, or 163-164. In an
embodiment, the ADC comprises one or more (e.g., two, three, four,
five, six, seven, eight, or more) antimicrobial peptides, each of
which has an amino acid sequence that differs by no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of any of SEQ ID NO: 67-80, 94-102, 147-156, 158-159,
or 163-164. In an embodiment, the ADC comprises four or more
antimicrobial peptides, each of which has an amino acid sequence
that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acid residues from, or has at least 80%, 85%, 90%, 95%, 97%, or
100% homology with, the amino acid sequence of any of SEQ ID NO:
67-80, 94-102, 147-156, 158-159, or 163-164. In an embodiment, the
ADC comprises four or more antimicrobial peptides, each of which
comprises or consists of the amino acid sequence of any of SEQ ID
NO: 67-80, 94-102, 147-156, 158-159, or 163-164. In an embodiment,
the antimicrobial peptide comprises a carboxamide group (e.g., a
C-terminal carboxamide functional group).
[0593] In an embodiment, the antimicrobial peptide comprises one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38. 39, 40, or more) D-amino acids. In an embodiment,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of
the amino acid residues in the antimicrobial peptide are D-amino
acids. In an embodiment, all of the amino acid residues in the
antimicrobial peptide are D-amino acids.
[0594] In an embodiment, the antimicrobial peptide comprises one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38. 39, 40, or more) L-amino acids. In an embodiment,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of
the amino acid residues in the antimicrobial peptide are L-amino
acids. In an embodiment, all of the amino acid residues in the
antimicrobial peptide are L-amino acids.
[0595] In an embodiment, the antimicrobial peptide comprises one or
more D-amino acids and one or more L-amino acids.
[0596] In an embodiment, the ADC is capable of inhibiting and/or
reducing the viability of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
bacteria (e.g., Gram-negative bacteria) of different genera,
species, subspecies, and/or strains. For instance, the antibody
molecule may inhibit and/or reduce the viability of one or more
bacteria of different species, subspecies, and/or strains from
Enterobacteriaceae (e.g., Klebsiella, Enterobacter, Shigella,
Escherichia, Salmonella, Yersinia, or Citrobacter, e.g.,
pan-resistant Enterobacteriaceae), one or more bacteria of
different species, subspecies, and/or strains from Pseudomonas, one
or more bacteria of different species, subspecies, and/or strains
from Acinetobacter, or any combination thereof. In an embodiment,
the ADC is capable of inhibiting or reducing the viability of
Klebsiella pneumonia (e.g., Klebsiella pneumoniae subsp. ozaenae,
Klebsiella pneumoniae subsp. pneumoniae, or Klebsiella pneumoniae
subsp. rhinoscleromatis), Enterobacter cancerogenous, Enterobacter
cloacae, Enterobacter hormaechei, Enterobacter asburiae, Shigella
boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Escherichia coli (e.g., Escherichia coli ATCC 11775, Escherichia
coli ATCC 25922, Escherichia coli ATCC 35401, or Escherichia coli
ATCC 43895), Escherichia fergusonii, Salmonella choleraesuis,
Salmonella choleraesuis subsp. indica, Salmonella enteritidis,
Salmonella virchow, Salmonella paratyphi B, Salmonella typhimurium,
Salmonella paratyphi A, Salmonella typhi, Salmonella choleraesuis
subsp. arizonae, Salmonella choleraesuis subsp. diarizonae,
Salmonella choleraesuis subsp. houtenae, Salmonella bongori,
Citrobacter sedlakii, Citrobacter braakii, Citrobacter werkmanii,
Citrobacter freundii, Citrobacter youngae, Citrobacter
amalonaticus, Yersinia enterocolitica, Yersinia frederiksenii,
Yersinia pestis, Yersinia pseudotuberculosis, or any combination
thereof.
[0597] In an embodiment, the ADC is capable of inhibiting or
reducing the viability of one or more of: Enterococcus faecium
(e.g., vancomycin-resistant (VRE) Enterococcus faecium),
Staphylococcus aureus (e.g., methicillin-resistant (MRSA)
Staphylococcus aureus), Clostridium difficile, Acinetobacter
baumannii (e.g., multidrug resistant (MDR) Acinetobacter),
Pseudomonas aeruginosa (e.g., multidrug resistant (MDR) P.
aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis).
[0598] In an embodiment, the ADC is capable of binding to one or
more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) P. aeruginosa
strains in Table 7. In another embodiment, the ADC is capable of
binding to one or more (e.g., 2, 3, 4, 5, 6, or all)
multidrug-resistant P. aeruginosa strains in Table 7.
[0599] While not wishing to be bound by theory, the antimicrobial
peptides suitable for the ADCs described herein can be selected, at
least in part, based on one or more (e.g., two, three or all) of
the following properties: strong bacterial inhibition activity,
strong bactericidal activity, low red blood cell (RBC) hemolytic
activity, or low cytotoxicity (e.g., off-target toxicity). In an
embodiment, the antimicrobial peptide has a minimum inhibitory
concentration (MIC) of less than 100 .mu.g/ml, e.g., less than 90,
80, 70, 60, 50, 40, 30, 20, 10, or 5 .mu.g/ml, against a bacterial
strain described herein, e.g., Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC27853, or both. In an embodiment, the
antimicrobial peptide has a minimum bactericidal concentration
(MBC) of less than 100 .mu.g/ml, e.g., less than 90, 80, 70, 60,
50, 40, 30, 20, 10, or 5 .mu.g/ml, against a bacterial strain
described herein, e.g., Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC27853, or both. In an embodiment, the antimicrobial
peptide has low hemolytic activity, e.g., has a PLC to MIC ratio
for a Gram-negative bacterium (e.g., a Gram-negative bacterium
described herein) which is equal to or greater than 1 (e.g.,
greater than 4:1, 8:1, 16:1, 24:1, or 32:1), e.g., as determined by
a red blood cell hemolysis assay and an MIC assay, respectively. In
an embodiment, the PLC is the concentration (e.g., minimum
concentration) required or needed to lyse 50% of the red blood
cells. In an embodiment, the antimicrobial peptide has low
hemolytic activity, e.g., has an MLC to MIC ratio for a
Gram-negative bacterium (e.g., a Gram-negative bacterium described
herein) which is greater than 4:1 (e.g., greater than 8:1, 16:1,
24:1, or 32:1), e.g., as determined by a red blood cell hemolysis
assay and an MIC assay, respectively. In an embodiment, the MLC is
the concentration (e.g., minimum concentration) required or needed
to lyse 100% of the red blood cells.
[0600] In an embodiment, the antimicrobial peptide is an
.alpha.-helical peptide. In an embodiment, the antimicrobial
peptide has two or more amino acid residues cross-linked. For
example, two cysteine residues in the antimicrobial peptide can be
cross-linked, e.g., chemically cross-linked. While not wishing to
be bound by theory, it is believed that in an embodiment,
cross-linking two or more amino acid residues in an antimicrobial
peptide may enhance .alpha.-helical conformation, enhance serum
stability, and/or increase antimicrobial potency. In an embodiment,
the antimicrobial peptide having two or more amino acid residues
cross-linked is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold more
stable, e.g., in serum, than an otherwise identical antimicrobial
peptide that does not have two or more amino acid residues
cross-linked. In an embodiment, the antimicrobial peptide having
two or more amino acid residues cross-linked has a MIC that is at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
lower than an otherwise identical antimicrobial peptide that does
not have two or more amino acid residues cross-linked.
[0601] In an embodiment, the ADC is produced by enzymatic
synthesis. For example, ADCs can be produced by expression of an
antibody molecule (e.g., a tagged antibody molecule), chemical
synthesis of a peptide (e.g., an antimicrobial peptide), and
enzymatic ligation of the peptide to the antibody molecule. In an
embodiment, 90% or more, e.g., 92% or more, 95% or more, 97% or
more, or 99% or more, reaction efficiency is achieved. In another
embodiment, the method further comprises purifying the ADC. In an
embodiment, the yield is 60% or more (e.g., 70% or more, 75% or
more, 80% or more, 90% or more, or 95% or more) after
purification.
[0602] In an embodiment, the ADC binds to a bacterial surface. In
another embodiment, the ADC binds to a secreted vesicle. In yet
another embodiment, the ADC binds to both a bacterial surface and a
secreted vesicle. In an embodiment, the binding is detected by
electron microscopy. In an embodiment, the ADC has enhanced binding
to bacteria from a first genus, species, or subspecies, compared to
binding to bacteria from a second genus, species, or subspecies. In
an embodiment, the ADC has enhanced binding to P. aeruginosa,
compared to binding to bacteria other than P. aeruginosa (e.g., E.
coli or Klebsiella spp.).
[0603] In an embodiment, the ADC comprising: a) an antibody
molecule that binds to lipopolysaccharide (LPS); and b) an
antimicrobial peptide,
[0604] wherein the antibody molecule comprises a VH and a VL,
wherein the VH comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the VL comprises
three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), and wherein the ADC or antibody molecule
comprises:
[0605] (a) an HCDR1 comprising the amino acid sequence of SEQ ID
NO: 108; an HCDR2 comprising the amino acid sequence of any of SEQ
ID NOS: 109, 145, or 146; an HCDR3 comprising the amino acid
sequence of SEQ ID NO: 107; an LCDR1 comprising the amino acid
sequence of any of SEQ ID NOS: 110, 138, 140, or 144; an LCDR2
comprising the amino acid sequence of any of SEQ ID NOS: 111, 139,
141, 142, or 143; and an LCDR3 comprising the amino acid sequence
of any of SEQ ID NO: 112, or
[0606] (b) an HCDR1 comprising the amino acid sequence of SEQ ID
NO: 105; an HCDR2 comprising the amino acid sequence of SEQ ID NO:
106; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 107;
an LCDR1 comprising the amino acid sequence of any of SEQ ID NOS:
110, 138, 140, or 144; an LCDR2 comprising the amino acid sequence
of any of SEQ ID NOS: 111, 139, 141, 142, or 143; and an LCDR3
comprising the amino acid sequence of any of SEQ ID NO: 112;
and
[0607] wherein the antimicrobial peptide comprises or consists
of:
[0608] (i) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 101;
[0609] (ii) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 147;
[0610] (iii) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 152;
[0611] (iv) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 153;
[0612] (v) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 154;
[0613] (vi) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 156;
[0614] (vii) an amino acid sequence that differs by no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has at
least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 163; or
[0615] (viii) an amino acid sequence that differs by no more than
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from, or has
at least 80%, 85%, 90%, 95%, 97%, or 100% homology with, the amino
acid sequence of SEQ ID NO: 164.
[0616] In an embodiment, the ADC comprises (a) and (i). In an
embodiment, the ADC comprises (a) and (ii). In an embodiment, the
ADC comprises (a) and (iii). In an embodiment, the ADC comprises
(a) and (iv). In an embodiment, the ADC comprises (a) and (v). In
an embodiment, the ADC comprises (a) and (vi). In an embodiment,
the ADC comprises (a) and (vii). In an embodiment, the ADC
comprises (a) and (viii). In an embodiment, the ADC comprises (b)
and (i). In an embodiment, the ADC comprises (b) and (ii). In an
embodiment, the ADC comprises (b) and (iii). In an embodiment, the
ADC comprises (b) and (iv). In an embodiment, the ADC comprises (b)
and (v). In an embodiment, the ADC comprises (b) and (vi). In an
embodiment, the ADC comprises (b) and (vii). In an embodiment, the
ADC comprises (b) and (viii).
Evaluation of Candidates: Opsonophagocytosis Assay (OPA)
[0617] The candidates of antibody molecules and antibody
molecule-drug conjugates (ADCs) described herein can be evaluated,
e.g., in vitro, for their opsonophagocytic activities, by
opsonophagocytosis assays.
[0618] Assays for antibody-mediated, complement-dependent
opsonization are described, e.g., in Hemachandra et al. Infect
Immun. 2001; 69(4):2223-2229. Briefly, the ability of the antibody
molecule or ADC candidates to opsonize bacteria for uptake by human
polymorphonuclear leukocytes (PMNs) can be measured by flow
cytometry. Bacteria are grown, heat killed, and FITC labeled.
Opsonization can be carried out by incubating the labeled bacteria
with antibody molecules or antibody molecule-drug conjugates with
or without 1% human serum from an agammaglobulinemic patient as the
complement source. Bacteria are washed in PBS containing 6% dextran
and 0.2% glucose and then are resuspended in Hanks balanced salt
solution with 0.1% gelatin. PMNs can be isolated from peripheral
human blood via venipuncture of healthy adult volunteers. PMNs are
resuspended to achieve a concentration of 10.sup.7 cells/ml and are
activated for 30 min with 10 .mu.l of a 10.sup.-6 dilution of
N-formyl-Met-Leu-Phe (FMLP; Peninsula Laboratories, San Carlos,
Calif.) per ml of cells. PMNs are added to each opsonized bacterial
opsonized bacterial sample, incubated at 37.degree. C., separated
from free bacteria by differential centrifugation, and resuspended
in PBS. Single-color flow cytometric analysis of PMN can be
performed utilizing a FACScan and CellQuest software (Becton
Dickinson, Mountain View, Calif.), and phagocytosis is expressed in
relative units of mean fluorescence of 10,000 PMN for each sample.
To demonstrate that the observed opsonophagocytosis is associated
with bacterial killing, an alternative assay can be used in which
25,000 CFU of live bacteria are mixed with agammaglobulinemic human
serum, various concentrations of antibody molecules or antibody
molecule-drug conjugates, and 10.sup.6 fresh human PMN obtained as
described above in RPMI medium (400-ml final volume). Samples are
obtained at the beginning and end of a 90-min 37.degree. C.
incubation, after which bacteria are diluted and then plated for
bacterial enumeration.
[0619] An exemplary opsonophagocytosis assay is also described in
Example 1.
Evaluation of Candidates: Minimal Inhibitory Concentration
Determination
[0620] The candidates of antimicrobial peptides and antibody
molecule-drug conjugates (ADCs) described herein can be evaluated,
e.g., in vitro, for their microbial inhibitory activities, by
determining the minimal inhibitory concentration (MIC).
[0621] In an embodiment, the MIC is determined in the presence of
human serum (e.g., 50% human serum), and sometimes referred to
herein as hsMIC. In an embodiment, the MIC is determined in the
presence of phosphate-buffered saline (PBS). In an embodiment, the
MIC or hsMIC is determined on per ADC basis. In an embodiment, the
MIC or hsMIC is determined on per payload or antimicrobial peptide
basis. In an embodiment, the ratio of hsMIC to MIC is equal to or
less than 2, e.g., equal to or less than 1.5 or 1.
[0622] Methods for determining MIC are also described, e.g., in
Clinical and Laboratory Standards Institute. 2012. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow
aerobically; approved standard, 9th ed. M07-A8, vol 29, no. 2
Clinical and Laboratory Standards Institute, Wayne, Pa. For
example, MIC can be determined according to CLSI guidelines, using
2-fold serial compound dilutions, in 96-well microtiter plates.
Briefly, compounds are diluted in water across a mother plate then
2 .mu.l is stamped to assay plates, one plate for each strain to be
tested. Bacterial strains are sub-cultured overnight on agar plates
at 37.degree. C. Overnight plates are used to prepare 0.5 McFarland
cultures in 0.85% saline. These concentrated cultures are diluted
1:200 in growth media to approximately 5.times.10.sup.5 cells/ml.
All assay plates receive 100 .mu.l diluted culture per well. All
plates are placed at 37.degree. C. overnight. After 18 hours the
plates are read using a mirrored plate reader and reflected
incandescent light. In some embodiments, the MIC can be defined as
the lowest concentration of compound that inhibits growth by at
least 80%. Wells at and above the MIC should appear void of growth
when read by eye.
[0623] An exemplary method for determination of MIC is also
described in Example 2.
Animal Models
[0624] The antibody molecules, antibody molecule-drug conjugates
(ADCs), and antimicrobial peptides described herein can be
evaluated in vivo, e.g., using various animal models. For example,
an animal model can be used to test the efficacy of an antibody
molecule, anti-bacterial peptide, or antibody molecule-drug
conjugate described herein in reducing or inhibiting bacterial
infection Animal models can also be used, e.g., to investigate for
side effects, measure concentrations of antibody molecules,
anti-bacterial peptides or antibody molecule-drug conjugates in
situ, demonstrate correlations between bacterial infection and
bacteria under controlled conditions.
[0625] Exemplary animal models that can be used for evaluating an
antibody molecule, anti-bacterial peptide, or antibody
molecule-drug conjugate described herein include, but are not
limited to, basic antimicrobial screening models (e.g., as
described in Zak and O'Reilly, Antimicrob. Agents Chemother. 1991;
35(8): 1527-1531); primary rodent infection models (e.g., as
described in Marra and Girard Curr. Protoc. Pharmacol. 2006;
Chapter 13: Unit 13A.4); ex vivo models (e.g., as described in Zak
and O'Reilly, Antimicrob. Agents Chemother. 1991; 35(8):
1527-1531); monoparametric or discriminative models (e.g., as
described in Zak and O'Reilly, Antimicrob. Agents Chemother. 1991;
35(8): 1527-1531); mice models for wound healing (e.g., as
described in Samy et al. Methods Mol. Biol. 2011; 716: 245-265);
rabbit models for evaluation of bacterial migration and
colonization (e.g., as described in Allan et al. J. Biomed.
Biotechnol. 2012; 2012: 921617). Pharmacokinetic-pharmacodynamic
modeling of antimicrobial drugs are described, e.g., in Nielsen and
Friberg Pharmacol. Rev. 2013; 65(3):1053-1090.
[0626] Exemplary types of animals that can be used to evaluate
antibody molecules, anti-bacterial peptides, or antibody
molecule-drug conjugates described herein include, but are not
limited to, mice, rats, rabbits, guinea pigs, and monkeys. Various
methods of immunosuppression can be used for enhancement of
virulence of bacteria for inoculation. These methods include, e.g.,
targeting bone marrow (e.g., by irradiation), targeting neutrophils
(e.g., using cytostatics), targeting macrophages (e.g., using mucin
or baker's yeast), targeting complement (e.g., using cobra venom
factor), targeting tuftsin (e.g., by splenectomy), targeting
immunoglobulins (e.g., using anti-Ig), targeting T-lymphocytes
(e.g., by thymectomy), or targeting interleukins (e.g., using
antibodies or chemical compounds). Other considerations that may
influence anti-bacterial activity in in vivo tests include, e.g.,
inoculum size, virulence, growth or generation time in vivo, timing
of treatment, method of administration,
pharmacokinetics/pharmacodynamics, and development of resistance in
vivo.
Pharmaceutical Compositions and Kits
[0627] In some aspects, this disclosure provides compositions,
e.g., pharmaceutically acceptable compositions, which include an
antibody molecule, an antibody molecule-drug conjugate (ADC), or an
antimicrobial peptide, as described herein, formulated together
with a pharmaceutically acceptable carrier.
[0628] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. The carrier can be suitable for intravenous,
intramuscular, subcutaneous, parenteral, rectal, spinal,
transdermal or epidermal administration (e.g., by injection or
infusion). In certain embodiments, less than about 5%, e.g., less
than about 4%, 3%, 2%, or 1% of the antibody molecules, ADCs, or
antimicrobial peptides in the pharmaceutical composition are
present as aggregates. In other embodiments, at least about 95%,
e.g., at least about 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, or
more of the antibody molecules, ADCs, or antimicrobial peptides in
the pharmaceutical composition are present as monomers. In some
embodiments, the level of aggregates or monomers is determined by
chromatography, e.g., high performance size exclusion
chromatography (HP-SEC).
[0629] The compositions set out herein may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, liposomes, and
suppositories. A suitable form depends on the intended mode of
administration and therapeutic application. Typical suitable
compositions are in the form of injectable or infusible solutions.
One suitable mode of administration is parenteral (e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In some
embodiments, the antibody molecule, ADC, or antimicrobial peptide
is administered by intravenous infusion or injection. In certain
embodiments, the antibody, ADC, or antimicrobial peptide is
administered by intramuscular or subcutaneous injection.
[0630] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0631] Therapeutic compositions typically should be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
antibody concentration. Sterile injectable solutions can be
prepared by incorporating the active compound (i.e., antibody or
antibody portion) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying that yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0632] The antibody molecules ADC, or antimicrobial peptide can be
administered by a variety of methods. Several are known in the art,
and for many therapeutic, prophylactic, or diagnostic applications,
an appropriate route/mode of administration is intravenous
injection or infusion. For example, the antibody molecules, ADC, or
antimicrobial peptide can be administered by intravenous infusion
at a rate of less than 10 mg/min; preferably less than or equal to
5 mg/min to reach a dose of about 1 to 100 mg/m.sup.2, preferably
about 5 to 50 mg/m.sup.2, about 7 to 25 mg/m.sup.2 and more
preferably, about 10 mg/m.sup.2. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0633] In certain embodiments, an antibody molecule, ADC, or
antimicrobial peptide can be orally administered, for example, with
an inert diluent or an assimilable edible carrier. The antibody
molecule, ADC, or antimicrobial peptide (and other ingredients, if
desired) may also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the antibody
molecule, ADC, or antimicrobial peptide may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. To administer an antibody molecule, ADC, or
antimicrobial peptide by other than parenteral administration, it
may be necessary to coat the compound with, or co-administer the
compound with, a material to prevent its inactivation. Therapeutic,
prophylactic, or diagnostic compositions can also be administered
with medical devices, and several are known in the art.
[0634] Dosage regimens are adjusted to provide the desired response
(e.g., a therapeutic, prophylactic, or diagnostic response). For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation. It is especially advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit contains a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
are dictated by and directly dependent on (a) the unique
characteristics of the antibody molecule, ADC, or antimicrobial
peptide, and the particular therapeutic, prophylactic, or
diagnostic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an antibody molecule, ADC, or
antimicrobial peptide for the treatment of sensitivity in
individuals.
[0635] An exemplary, non-limiting range for a therapeutically,
prophylactically, or diagnostically effective amount of an antibody
molecule, ADC, or antimicrobial peptide is 0.1-100 mg/kg, e.g.,
0.1-50 mg/kg or 0.1-20 mg/kg, e.g., about 1-10, 1-5, 5-10, or 1-3
mg/kg, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. The
antibody molecule, ADC, or antimicrobial peptide can be
administered by intravenous infusion at a rate of less than 10
mg/min, e.g., less than or equal to 5 mg/min to reach a dose of
about 1 to 100 mg/m.sup.2, e.g., about 5 to 50 mg/m.sup.2, about 7
to 25 mg/m.sup.2, e.g., about 10 mg/m.sup.2. It is to be noted that
dosage values may vary with the type and severity of the condition
to be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed compositions.
[0636] The pharmaceutical compositions herein may include a
"therapeutically effective amount," "prophylactically effective
amount," or "diagnostically effectively amount" of an antibody
molecule, ADC, or antimicrobial peptide.
[0637] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody molecule, ADC, or antimicrobial peptide may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the antibody or
antibody portion to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the antibody molecule, ADC, or antimicrobial
peptide is outweighed by the therapeutically beneficial effects. A
"therapeutically effective dosage" typically inhibits a measurable
parameter by at least about 20%, e.g., by at least about 40%, by at
least about 60%, or by at least about 80% relative to untreated
subjects. The measurable parameter may be, e.g., bacterial load,
fever, headache, muscle or joint pains, skin rash, bleeding,
reduced platelet levels, and reduced blood pressure. The ability of
an antibody molecule, ADC, or antimicrobial peptide to inhibit a
measurable parameter can be evaluated in an animal model system
predictive of efficacy in reducing, inhibiting, or preventing a
bacterial infection. Alternatively, this property of a composition
can be evaluated by examining the ability of the antibody molecule,
ADC, or antimicrobial peptide to inhibit or reduce the viability of
bacteria, e.g., by an in vitro assay described herein.
[0638] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0639] A "diagnostically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired diagnostic result. Typically, a diagnostically
effective amount is one in which a bacterial infection or a related
disorder can be diagnosed in vitro, ex vivo, or in vivo.
[0640] Also within this disclosure is a kit that comprises an
antibody molecule, ADC, or antimicrobial peptide described herein.
The kit can include one or more other elements including:
instructions for use; other reagents, e.g., a label, a therapeutic
agent, or an agent useful for chelating, or otherwise coupling, an
antibody molecule, ADC, or antimicrobial peptide to a label or
therapeutic agent, or a radioprotective composition; devices or
other materials for preparing the antibody molecule, ADC, or
antimicrobial peptide for administration; pharmaceutically
acceptable carriers; and devices or other materials for
administration to a subject.
Nucleic Acids
[0641] The present disclosure also features nucleic acids
comprising nucleotide sequences that encode the antibody molecules
(e.g., heavy and light chain variable regions and CDRs of the
antibody molecules), antibody molecule-drug conjugates (e.g., heavy
and light chain variable regions and CDRs of the antibody
molecule-drug conjugates), or antimicrobial peptide, as described
herein.
[0642] For example, the present disclosure features a first and
second nucleic acid encoding heavy and light chain variable
regions, respectively, of an antibody molecule chosen from one or
more of the antibody molecules disclosed herein, e.g., an antibody
molecule of Table 1 or 8, or a portion of an antibody molecule,
e.g., the variable regions of Table 1 or 8. The nucleic acid can
comprise a nucleotide sequence encoding any one of the amino acid
sequences in the tables herein, or a sequence substantially
identical thereto (e.g., a sequence at least about 85%, 90%, 95%,
99% or more identical thereto, or which differs by no more than 3,
6, 15, 30, or 45 nucleotides from the sequences shown in the tables
herein).
[0643] In certain embodiments, the nucleic acid can comprise a
nucleotide sequence encoding at least one, two, or three CDRs from
a heavy chain variable region having an amino acid sequence as set
forth in the tables herein, or a sequence substantially homologous
thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more
identical thereto, and/or having one or more substitutions, e.g.,
conserved substitutions). In some embodiments, the nucleic acid can
comprise a nucleotide sequence encoding at least one, two, or three
CDRs from a light chain variable region having an amino acid
sequence as set forth in the tables herein, or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or having one or
more substitutions, e.g., conserved substitutions). In some
embodiments, the nucleic acid can comprise a nucleotide sequence
encoding at least one, two, three, four, five, or six CDRs from
heavy and light chain variable regions having an amino acid
sequence as set forth in the tables herein, or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or having one or
more substitutions, e.g., conserved substitutions).
[0644] In certain embodiments, the nucleic acid can comprise a
nucleotide sequence encoding at least one, two, or three CDRs from
a heavy chain variable region having the nucleotide sequence as set
forth in Table 2, a sequence substantially homologous thereto
(e.g., a sequence at least about 85%, 90%, 95%, 99% or more
identical thereto, and/or capable of hybridizing under the
stringency conditions described herein). In some embodiments, the
nucleic acid can comprise a nucleotide sequence encoding at least
one, two, or three CDRs from a light chain variable region having
the nucleotide sequence as set forth in Table 2, or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or capable of
hybridizing under the stringency conditions described herein). In
certain embodiments, the nucleic acid can comprise a nucleotide
sequence encoding at least one, two, three, four, five, or six CDRs
from heavy and light chain variable regions having the nucleotide
sequence as set forth in Table 2, or a sequence substantially
homologous thereto (e.g., a sequence at least about 85%, 90%, 95%,
99% or more identical thereto, and/or capable of hybridizing under
the stringency conditions described herein).
[0645] In certain embodiments, the nucleic acid comprises a
nucleotide sequence as set forth in Table 2 or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or capable of
hybridizing under the stringency conditions described herein). In
some embodiments, the nucleic acid comprises a portion of a
nucleotide sequence as set forth in Table 2 or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or capable of
hybridizing under the stringency conditions described herein). The
portion may encode, for example, a variable region (e.g., VH or
VL); one, two, or three or more CDRs; or one, two, three, or four
or more framework regions.
[0646] In certain embodiments, the nucleic acid comprises a
nucleotide sequence encoding an amino acid sequence as set forth in
Tables 3 or 6A-6B or in FIG. 4 or 15A-15B, or a sequence
substantially homologous thereto (e.g., a sequence at least about
80%, 85%, 90%, 95% or more identical thereto, and/or capable of
hybridizing under the stringency conditions described herein). In
some embodiments, the nucleic acid comprises a portion of a
nucleotide sequence encoding an amino acid sequence as set forth in
Tables 3 or 6A-6B or in FIG. 4 or 15A-15B, or a sequence
substantially homologous thereto (e.g., a sequence at least about
80%, 85%, 90%, 95%, or more identical thereto, and/or capable of
hybridizing under the stringency conditions described herein).
[0647] The nucleic acids disclosed herein include
deoxyribonucleotides or ribonucleotides, or analogs thereof. The
polynucleotide may be either single-stranded or double-stranded,
and if single-stranded may be the coding strand or non-coding
(antisense) strand. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
The sequence of nucleotides may be interrupted by non-nucleotide
components. A polynucleotide may be further modified after
polymerization, such as by conjugation with a labeling component.
The nucleic acid may be a recombinant polynucleotide, or a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin
which either does not occur in nature or is linked to another
polynucleotide in a non-natural arrangement.
[0648] In some aspects, the application features host cells and
vectors containing the nucleic acids described herein. The nucleic
acids may be present in a single vector or separate vectors present
in the same host cell or separate host cell, as described in more
detail below.
Vectors
[0649] Further provided herein are vectors that comprise nucleotide
sequences encoding an antibody molecule, antibody molecule-drug
conjugate (ADC), or antimicrobial peptide, described herein. In
some embodiments, the vectors comprise nucleotides encoding an
antibody molecule described herein. In some embodiments, the
vectors comprise the nucleotide sequences described herein. The
vectors include, but are not limited to, a virus, plasmid, cosmid,
lambda phage or a yeast artificial chromosome (YAC).
[0650] Numerous vector systems can be employed. For example, one
class of vectors utilizes DNA elements which are derived from
animal viruses such as, for example, bovine papilloma virus,
polyoma virus, adenovirus, vaccinia virus, baculovirus,
retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus.
Another class of vectors utilizes RNA elements derived from RNA
viruses such as Semliki Forest virus, Eastern Equine Encephalitis
virus and Flaviviruses.
[0651] Additionally, cells which have stably integrated the DNA
into their chromosomes may be selected by introducing one or more
markers which allow for the selection of transfected host cells.
The marker may provide, for example, prototropy to an auxotrophic
host, biocide resistance (e.g., antibiotics), or resistance to
heavy metals such as copper, or the like. The selectable marker
gene can be either directly linked to the DNA sequences to be
expressed, or introduced into the same cell by cotransformation.
Additional elements may also be needed for optimal synthesis of
mRNA. These elements may include splice signals, as well as
transcriptional promoters, enhancers, and termination signals.
[0652] Once the expression vector or DNA sequence containing the
constructs has been prepared for expression, the expression vectors
may be transfected or introduced into an appropriate host cell.
Various techniques may be employed to achieve this, such as, for
example, protoplast fusion, calcium phosphate precipitation,
electroporation, retroviral transduction, viral transfection, gene
gun, lipid based transfection or other conventional techniques. In
the case of protoplast fusion, the cells are grown in media and
screened for the appropriate activity.
[0653] Methods and conditions for culturing the resulting
transfected cells and for recovering the antibody molecule,
anti-bacterial peptide, or antibody molecule-drug conjugate
produced are known to those skilled in the art, and may be varied
or optimized depending upon the specific expression vector and
mammalian host cell employed, based upon the present
description.
Cells
[0654] The present disclosure also provides host cells comprising a
nucleic acid encoding an antibody molecule, anti-bacterial peptide,
or antibody molecule-drug conjugate as described herein. For
example, the host cells may comprise a nucleic acid of Table 2, a
sequence substantially homologous thereto (e.g., a sequence at
least about 85%, 90%, 95%, 99% or more identical thereto, and/or
capable of hybridizing under the stringency conditions described
herein), or a portion of one of said nucleic acids. Additionally,
the host cells may comprise a nucleic acid encoding an amino acid
sequence described in Tables 1, 3, 6A-6B, or 8, a sequence
substantially homologous thereto (e.g., a sequence at least about
80%, 85%, 90%, 95%, 99% or more identical thereto), or a portion of
one of said sequences.
[0655] In some embodiments, the host cells are genetically
engineered to comprise nucleic acids encoding the antibody
molecule, ADC, or antimicrobial peptide.
[0656] In certain embodiments, the host cells are genetically
engineered by using an expression cassette. The phrase "expression
cassette," refers to nucleotide sequences, which are capable of
affecting expression of a gene in hosts compatible with such
sequences. Such cassettes may include a promoter, an open reading
frame with or without introns, and a termination signal. Additional
factors necessary or helpful in effecting expression may also be
used, such as, for example, an inducible promoter.
[0657] The disclosure also provides host cells comprising the
vectors described herein.
[0658] The cell can be, but is not limited to, a eukaryotic cell, a
bacterial cell, an insect cell, or a human cell. Suitable
eukaryotic cells include, but are not limited to, Vero cells, HeLa
cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII
cells. Suitable insect cells include, but are not limited to, Sf9
cells.
Uses of Antibody Molecules, Antibody Molecule-Drug Conjugates, and
Antimicrobial Peptides
[0659] The antibody molecules, antibody molecule-drug conjugates
(ADCs), or antimicrobial peptides disclosed herein, as well as the
pharmaceutical compositions disclosed herein, have in vitro, ex
vivo, and in vivo therapeutic, prophylactic, and/or diagnostic
utilities.
[0660] In an embodiment, the antibody molecule, ADC, or
antimicrobial peptide, inhibits or reduces the viability of
bacteria, e.g., Gram-negative bacteria. For example, these
molecules can be administered to cells in culture, in vitro or ex
vivo, or to a subject, e.g., a human subject, e.g., in vivo, to
inhibit or reduce the viability of bacteria, e.g., Gram-negative
bacteria. Accordingly, in an aspect, the disclosure provides a
method of treating or preventing a bacterial infection in a
subject, comprising administering to the subject an antibody
molecule, ADC, or antimicrobial peptide, described herein, such
that the bacterial infection is treated or prevented. For example,
these antibody molecules can be administered to cells in culture,
e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat,
prevent, and/or diagnose a bacterial infection, or to inhibit or
reduce a bacterial infection.
[0661] As used herein, the term "subject" is intended to include
human and non-human animals In some embodiments, the subject is a
human subject, e.g., a human patient infected with bacteria, e.g.,
disease-causing bacteria, e.g., Gram-negative bacteria, or at risk
of being infected with bacteria, e.g., disease-causing bacteria,
e.g., Gram-negative bacteria. The term "non-human animals" includes
mammals and non-mammals, such as non-human primates. In some
embodiments, the subject is a human. The methods and compositions
described herein are suitable for treating human patients infected
with bacteria, e.g., disease-causing bacteria, e.g., Gram-negative
bacteria. Patients infected with bacteria, e.g., disease-causing
bacteria, e.g., Gram-negative bacteria include those who have been
exposed to the bacteria but are (at least temporarily)
asymptomatic, patients having a bacterial infection, or patients
having a disorder related to a bacterial infection.
Methods of Treating or Preventing Bacterial Infection
[0662] Gram-negative bacteria display lipopolysaccharides (LPS) on
the outer membrane of the bacteria. While not wishing to be bound
by theory, in an embodiment, the antibody molecules, antibody
molecule-drug conjugates (ADCs), or antimicrobial peptides
described herein can inhibit or reduce the viability of
Gram-negative bacteria, at least in part, by binding to LPS.
[0663] The antibody molecules, ADCs, and antimicrobial peptides
described herein, can be used to treat or prevent bacterial
infections, as well as disorders, conditions or symptoms associated
with bacterial infections.
[0664] In an embodiment, the bacterial infection is caused by one
or more of the following bacteria: Klebsiella pneumonia (e.g.,
Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp.
pneumoniae, or Klebsiella pneumoniae subsp. rhinoscleromatis),
Enterobacter cancerogenous, Enterobacter cloacae, Enterobacter
hormaechei, Enterobacter asburiae, Shigella boydii, Shigella
dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli
(e.g., Escherichia coli ATCC 11775, Escherichia coli ATCC 25922,
Escherichia coli ATCC 35401, or Escherichia coli ATCC 43895),
Escherichia fergusonii, Salmonella choleraesuis, Salmonella
choleraesuis subsp. indica, Salmonella enteritidis, Salmonella
virchow, Salmonella paratyphi B, Salmonella typhimurium, Salmonella
paratyphi A, Salmonella typhi, Salmonella choleraesuis subsp.
arizonae, Salmonella choleraesuis subsp. diarizonae, Salmonella
choleraesuis subsp. houtenae, Salmonella bongori, Citrobacter
sedlakii, Citrobacter braakii, Citrobacter werkmanii, Citrobacter
freundii, Citrobacter youngae, Citrobacter amalonaticus, Yersinia
enterocolitica, Yersinia frederiksenii, Yersinia pestis, Yersinia
pseudotuberculosis, or any combination thereof.
[0665] In an embodiment, the bacterial infection is caused by one
or more of: Enterococcus faecium (e.g., vancomycin-resistant (VRE)
Enterococcus faecium), Staphylococcus aureus (e.g.,
methicillin-resistant (MRSA) Staphylococcus aureus), Clostridium
difficile, Acinetobacter baumannii (e.g., multidrug resistant (MDR)
Acinetobacter), Pseudomonas aeruginosa (e.g., multidrug resistant
(MDR) P. aeruginosa, e.g., carbapenem-resistant P. aeruginosa),
Enterobacteriaceae (e.g., E. coli, K. pneumoniae, or Enterobacter
spp., e.g., carbapenem-resistant Enterobacteriaceae (CRE)), N.
gonorrhoaeae (e.g., drug-resistant N. gonorrhoaeae), Salmonella
(e.g., drug resistant Salmonella), Shigella (e.g., drug-resistant
Shigella), a bacterium producing an extended spectrum
.beta.-lactamase (ESBL), or Mycobacterium tuberculosis (e.g.,
drug-resistant M. tuberculosis).
[0666] Exemplary disorders or conditions that can be associated
with bacterial infections include, but are not limited to pneumonia
(e.g., community-acquired pneumonia and hospital-acquired
pneumonia), a urinary tract infection (UTI), septicemia,
meningitis, diarrhea (e.g., traveler's diarrhea), a soft tissue
infection, a skin infection, bacteremia, a respiratory system
infection (e.g., a lower respiratory tract infection),
endocarditis, an intra-abdominal infection, septic arthritis,
osteomyelitis, a CNS infection, an ophthalmic infection,
cholecystitis, cholangitis, meningitis (e.g., neonatal meningitis),
typhoid fever, food poisoning, gastroenteritis, enteric fever,
shigellosis, a blood stream infection, intra-abdominal sepsis, a
brain abscess, meningitis, sepsis (e.g., neonatal sepsis), a joint
infection, a bone infection, a gastrointestinal infection, or a
wound infection.
[0667] Certain antibody molecules, ADCs, and antimicrobial peptides
described herein are capable of treating at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 25, 30, 35, 40, 50, 100, 200, or more bacteria (e.g.,
Gram-negative bacteria) of different genera, species, subspecies,
and/or strains. Accordingly, in an embodiment, the antibody
molecule, ADC, or antimicrobial peptide is administered to a
patient infected with or with a risk of being infected with
bacterial infection, when no test has been performed to determine
the genus, species. subspecies, and/or strain of the bacteria,
e.g., the type of infected or disease-causing bacteria may be
unknown.
[0668] The antibody molecules, ADCs, or antimicrobial peptides are
typically administered at a frequency that keeps a therapeutically
effective level of antibody molecules, ADCs, or antimicrobial
peptides in the patient's system until the patient recovers. For
example, the antibody molecules, ADCs, or antimicrobial peptides
may be administered at a frequency that achieves a serum
concentration sufficient for at least about 1, 2, 5, 10, 20, 30, or
40 antibody molecules, ADCs, or antimicrobial peptides to bind each
bacterium. In an embodiment, the antibody molecules, ADCs, or
antimicrobial peptides are administered every 1, 2, 3, 4, 5, 6, or
7 days.
[0669] Methods of administering various antibody molecules, ADCs,
or antimicrobial peptides are known in the art and are described
below. Suitable dosages of the antibody molecules, ADCs, or
antimicrobial peptides used will depend on the age and weight of
the subject and the particular drug used.
[0670] The antibody molecules or antimicrobial peptides can be used
by themselves or conjugated to a second agent, e.g., an
antibacterial agent, toxin, or protein, e.g., a second
anti-bacterial (e.g., anti-LPS) antibody molecule or antimicrobial
peptide. This method includes: administering the antibody molecule
or antimicrobial peptide, alone or conjugated to a second agent, to
a subject requiring such treatment. The antibody molecules can be
used to deliver a variety of therapeutic agents, e.g., a toxin or
anti-viral agent, or mixtures thereof.
Combination Therapies
[0671] The antibody molecules, antibody molecule-drug conjugates
(ADCs), and antimicrobial peptides can be used in combination with
other therapies. For example, the combination therapy can include
an antibody molecule, ADC, or antimicrobial peptide co-formulated
with, and/or co-administered with, one or more additional
therapeutic agents, e.g., anti-bacterial agents (including
antibiotics or other anti-bacterial antibodies), vaccines, or
agents that enhance an immune response. In other embodiments, the
antibody molecules, anti-bacterial peptides, or antibody
molecule-drug conjugates are administered in combination with other
therapeutic treatment modalities, such as intravenous hydration,
fever-reducing agents (such as acetaminophen), or blood
transfusion. Such combination therapies may advantageously utilize
lower dosages of the administered therapeutic agents, thus avoiding
possible toxicities or complications associated with the various
monotherapies.
[0672] Administered "in combination", as used herein, means that
two (or more) different treatments are delivered to the subject
before, or during the course of the subject's affliction with a
bacterial infection or disease. In one embodiment, two or more
treatments are delivered prophylactically, e.g., before the subject
is infected or diagnosed with bacteria, e.g., Gram-negative
bacteria. In another embodiment, the two or more treatments are
delivered after the subject has been infected or diagnosed with
bacteria, e.g., Gram-negative bacteria. In some embodiments, the
delivery of one treatment is still occurring when the delivery of
the second begins, so that there is overlap. This is sometimes
referred to herein as "simultaneous" or "concurrent delivery." In
other embodiments, the delivery of one treatment ends before the
delivery of the other treatment begins. In some embodiments of
either case, the treatment is more effective because of combined
administration. For example, the second treatment is more
effective, e.g., an equivalent effect is seen with less of the
second treatment, or the second treatment reduces symptoms to a
greater extent, than would be seen if the second treatment were
administered in the absence of the first treatment, or the
analogous situation is seen with the first treatment. In some
embodiments, delivery is such that the reduction in a symptom, or
other parameter related to the bacterial infection or disorder is
greater than what would be observed with one treatment delivered in
the absence of the other. The effect of the two treatments can be
partially additive, wholly additive, or greater than additive. The
delivery can be such that an effect of the first treatment
delivered is still detectable when the second is delivered.
[0673] In some embodiment, the additional antimicrobial agent is an
antibiotic. For example, the antibiotic can be a beta-lactam
antibiotic (e.g., a penicillin, a cephalosporin, a monobactam, or a
carbapenem), a monobactam, a carbapenem, a macrolide, a
lincosamide, a streptogramin, an aminoglycoside, a quinolone, a
sulfonamide, a tetracycline, a glycopeptide, a lipoglycopeptide, an
oxazolidinone, a rifamycin, a polypeptide, or a tuberactinomycin.
Exemplary antibiotics include, but are not limited to, amikacin,
amoxicillin, ampicillin, azithromycin, aztreonam, bacampicillin,
bacitracin, balofloxacin, besifloxacin, capreomycin, carbenicillin,
cefacetrile (cephacetrile), cefaclomezine, cefaclor, cefadroxil
(cefadroxyl), cefalexin (cephalexin), cefaloglycin (cephaloglycin),
cefalonium (cephalonium), cefaloram, cefaloridine (cephaloradine),
cefalotin (cephalothin), cefamandole, cefaparole, cefapirin
(cephapirin), cefatrizine, cefazaflur, cefazedone, cefazolin
(cephazolin), cefcanel, cefcapene, cefclidine, cefdaloxime,
cefdinir, cefditoren, cefedrolor, cefempidone, cefepime, cefetamet,
cefetrizole, cefivitril, cefixime, cefluprenam, cefmatilen,
cefmenoxime, cefmepidium, cefmetazole, cefodizime, cefonicid,
cefoperazone, cefoselis, cefotaxime, cefotetan, cefovecin,
cefoxazole, cefoxitin, cefozopran, cefpimizole, cefpirome,
cefpodoxime, cefprozil (cefproxil), cefquinome, cefradine
(cephradine), cefrotil, cefroxadine, cefsumide, ceftaroline,
ceftaroline (teflaro), ceftazidime, cefteram, ceftezole,
ceftibuten, ceftiofur, ceftiolene, ceftioxide, ceftizoxime,
ceftobiprole, ceftriaxone, cefuracetime, cefuroxime, cefuzonam,
chloramphenicol, ciprofloxacin, clarithromycin, clinafloxacin,
clindamycin, cloxacillin, cycloserine, daptomycin (cubicin),
demeclocycline, dicloxacillin, dirithromycin, doripenem,
doxycycline, enoxacin, ertapenem, erythromycin, flucloxacillin,
flumequine, gatifloxacin, gemifloxacin, gemifloxacin (factive),
gentamicin, grepafloxacin, imipenem, imipenem/cilastatin,
kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin,
macrocyclics, meropenem, metronidazole, mezlocillin, minocycline,
moxifloxacin, nadifloxacin, nafcillin, nalidixic acid, neomycin,
netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin,
oxolinic acid, oxytetracycline, paromomycin, pazufloxacin,
pefloxacin, penicillin g, penicillin v, pipemidic acid,
piperacillin, piromidic acid, pivampicillin, pivmecillinam,
polymyxin b, pristinamycin, prulifloxacin,
quinupristin/dalfopristin, rifabutin, rifampin, rifapentine,
rosoxacin, roxithromycin, rufloxacin, sitafloxacin, sparfloxacin,
streptomycin, sulfamethizole, sulfamethoxazole, sulfisoxazole,
teicoplanin, telavancin, telavancin (vibativ), telithromycin,
temafloxacin, tetracycline, ticarcillin, tigecycline, tinidazole,
tobramycin, tosufloxacin, trimethoprim-sulfamethoxazole,
trovafloxacin, vancomycin, viomycin, or zeocin.
[0674] In some embodiments, the additional anti-bacterial agent is
a vaccine. The vaccine may be, e.g., live, attenuated, or
inactivated bacteria, e.g., anthrax vaccine (e.g., BIOTHRAX.RTM.),
DTaP vaccine (e.g., DAPTACEL.RTM. or INFANRIX.RTM.), DT vaccine,
Haemophilus influenzae type b (Hib) vaccine (e.g., ACTHIB.RTM.,
HIBERIX.RTM., or PEDVAXHIB.RTM.), meningococcal vaccine (e.g.,
MENOMUNE.RTM., MENACTRA.RTM., MENVEO.RTM., TRUMENBA.RTM., or
BEXSERO.RTM.), pneumococcal vaccine (e.g., PNEUMOVAX.RTM. 23 or
PREVNAR.RTM. 13), tetanus/diphtheria vaccine (e.g., DECAVAC.RTM. or
TENIVAC.RTM.), tetanus/diphtheria/pertussis vaccine (e.g.,
BOOSTRIX.RTM. or ADACEL.RTM.), typhoid vaccine (e.g., TYPHIM
VI.RTM. or VIVOTIF.RTM.), DTaP/polio vaccine (e.g., KINRIX.RTM.),
DTaP/hepatitis B/polio vaccine (e.g., PEDIARIX.RTM.),
DTaP/polio/Haemophilus influenza type b vaccine (e.g.,
PENTACEL.RTM.), Haemophilus influenza type b/hepatitis B vaccine
(e.g., COMVAX.RTM.), and Haemophilus influenza type b/meningococcal
vaccine (e.g., MENHIBRIX.RTM.).
[0675] In certain embodiments, the additional antiviral agent is a
second antibody molecule, ADC, or antimicrobial peptide, e.g., an
antibody molecule, ADC, or antimicrobial peptide different from a
first antibody molecule, ADC, or antimicrobial peptide. Exemplary
antibody molecules that can be used in combination include, but are
not limited to, any combination of the antibody molecules listed in
Table 1 or 8.
[0676] In some embodiments, the additional anti-bacterial agent is
an antimicrobial (e.g., antibacterial) peptide. Exemplary
antimicrobial peptides include, but are not limited to, pexiganan
acetate (MSI 78), omiganan (MX-226/MBI-226 or CLS001), iseganan
(IB-367), hLF1-11, XOMA 629, PAC-113, CZEN-002, IMX942, OP-145,
Ghrelin, PMX-30063, delmitide (RDP58), plectasin, and HB1345.
[0677] In some embodiments, the additional anti-bacterial agent is
a resistance-modifying agent. Exemplary resistance-modifying agents
include, but are not limited to, an efflux inhibitor (e.g.,
Phe-Arg-.beta.-naphthylamide) and beta-lactamase inhibitor (e.g.,
clavulanic acid or sulbactam).
Methods of Diagnosis
[0678] In some aspects, the present disclosure provides a
diagnostic method for detecting the presence of a bacterium in
vitro (e.g., in a biological sample, such as a blood sample) or in
vivo (e.g., in vivo imaging in a subject). The method includes: (i)
contacting the sample with an antibody molecule or antibody
molecule-drug conjugate (ADC) described herein, or administering to
the subject, the antibody molecule or ADC; (optionally) (ii)
contacting a reference sample, e.g., a control sample (e.g., a
control biological sample, such as plasma or blood) or a control
subject with an antibody molecule or ADC described herein; and
(iii) detecting formation of a complex between the antibody
molecule or ADC, and the sample or subject, or the control sample
or subject, wherein a change, e.g., a statistically significant
change, in the formation of the complex in the sample or subject
relative to the control sample or subject is indicative of the
presence of the bacterium in the sample. The antibody molecule or
ADC can be directly or indirectly labeled with a detectable
substance to facilitate detection of the bound or unbound antibody.
Suitable detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials and
radioactive materials, as described above and described in more
detail below.
[0679] The term "sample," as it refers to samples used for
detecting bacteria includes, but is not limited to, cells, cell
lysates, proteins or membrane extracts of cells, body fluids such
as blood, or tissue samples.
[0680] Complex formation between the antibody molecule or ADC, and
a bacterium or lipopolysaccharide, can be detected by measuring or
visualizing either the antibody molecule or antibody molecule-drug
conjugate bound to the bacterium or lipopolysaccharide or unbound
antibody molecule or ADC. Any suitable detection assays can be
used, and conventional detection assays include an enzyme-linked
immunosorbent assays (ELISA), a fluorescence-activated cell sorting
(FACS) assay, a radioimmunoassay (RIA) or tissue
immunohistochemistry. Alternative to labeling the antibody molecule
or ADC, the presence of a bacterium or lipopolysaccharide can be
assayed in a sample by a competition immunoassay utilizing
standards labeled with a detectable substance and an unlabeled
antibody molecule or ADC. In this assay, the biological sample, the
labeled standards and the antibody molecule or ADC are combined and
the amount of labeled standard bound to the unlabeled binding
molecule is determined. The amount of bacteria or
lipopolysaccharides in the sample is inversely proportional to the
amount of labeled standard bound to the antibody molecule or
ADC.
EXAMPLES
Example 1: In Vitro Evaluation of Candidate Antibody Molecules by
Opsonophagocytic Assay (OPA)
[0681] Candidate antibody molecules were evaluated in vitro for
opsonophagocytic killing activity against multiple gram-negative
bacteria. The opsonophagocytic assay (OPA) evaluates the ability of
an antibody molecule to opsonize bacteria in the presence of
complement and neutrophils. Opsonization of bacteria is a major
pathway by which antibodies have been shown to kill bacteria in
vivo. Activity in this assay is a pre-requisite for selection and
further evaluation of antibody molecules in vivo.
[0682] Briefly, the following assay components were utilized:
[0683] Complement (C'): normal human serum adsorbed with specific
bacterial test strain.
[0684] Neutrophils: fresh human blood from healthy adult
donors.
[0685] Bacteria: E. coli ATCC strain 25922.
[0686] Positive controls: opsonic polyclonal IgG (pAb) to each
bacterium tested.
[0687] Screening: antibody molecules were screened for OPA from 0.1
.mu.g/ml to 25 .mu.g/ml.
[0688] The complement reagent was prepared by adsorption of
bacterial specific antibody molecules from normal human serum.
Briefly, .about.10.sup.9 CFU bacteria were suspended in serum (10
mL) and incubated on ice for 30 min with mixing. The sample was
centrifuged and the serum was transferred and resuspended in
10.sup.9 CFU bacteria. This was incubated on ice with mixing for an
additional 30 min. The serum/complement was recovered by
centrifugation and sterilized by filtration through a 0.22 .mu.M
filter.
[0689] Polymorphonuclear cells (PMNs) were prepared from a single
donor. Fresh human blood was mixed with an equal volume of
HISTOPAQUE.RTM. and incubated for 1 hour at 37.degree. C. The upper
layer was collected and cells pelleted by centrifugation at 250 g
for 5 min. The remaining erythrocytes were lysed with 1% NH.sub.4Cl
by incubation at room temperature for 10 min. The cells were washed
and re-suspended in MEM. The cell viability was determined by
trypan blue and suspended to a final concentration of
5.times.10.sup.6 cells/ml.
[0690] Bacteria were prepared by seeding a 6 ml tube of Mueller
Hinton Broth, cation adjusted (MHB), to a 600 nM absorbance of 0.1
from and overnight growth of E. coli 25922 on a blood agar plate.
The cells were grown to mid-log phase (A.sub.600 nm=0.6-1.0) and
then diluted in 0.9% saline to an A.sub.600 nM=0.2. This provided a
culture at approximately 1.times.10.sup.8 cfu/ml. The culture was
diluted 1:100 in Minimal Essential Medium (MEM) to obtain a culture
at 1.times.10.sup.6 cfu/ml. This was the bacterial suspension us in
the OPA. The culture absorbance required varied amongst bacterial
strains.
[0691] Each run of the assay contained multiple controls and test
articles. In addition to bacteria, the assay groups were stratified
by: C' alone, PMNs+C', PMNs+heat inactivated C',
PMNs+C'+non-specific mAb; PMNs+C'+positive control pAb, C'+positive
control pAb, PMNs+C'+test article mAbs (dilution series). The OPA
was performed as follows. 100 .mu.l each of PMN suspension, the
bacterial suspension, the antisera or antibody, and the complement,
were mixed for a total volume of 400 .mu.l. A 25 .mu.l sample was
taken from this mixture immediately at T=0. After incubation in a
rotating rack at 37.degree. C. for 90 minutes, a sample was taken
again at T=90 minutes. The samples were diluted 1:10 into 225 .mu.l
TSB+0.05% Tween to lyse the white blood cells (alternative lysis
saponin). Samples were plated onto duplicate TSA plates (100 .mu.l
each) and incubated overnight at 37 C. Since the preparation was
1.times.10.sup.6 CFU/ml and there was 0.1 ml/tube in a final volume
of 0.4 ml, there would be 250,000 CFU/ml. Plating a 1:100 of that
should yield a readily countable 250 CFUs per plate in duplicate. A
one log drop would be 25 CFUs per plate. Temperature was adjusted
for each bacterial species to obtain better resolution. The plates
were counted. The reduction was calculated in the number of CFUs at
T=90 minutes as compared to T=0 and reported as the percentage of
killing. At least a one log drop would be desirable.
[0692] Antibody molecules were ranked based on their OPA activity
against E. coli. The antibody molecules having the highest OPA
activity against E. coli. would be selected for in vivo
evaluation.
[0693] Methods for performing an OPA assay is also described, e.g.,
in Hemachandra et al. Infect Immun. 2001; 69(4): 2223-2229, which
is incorporated by reference in its entirety.
[0694] The methods described herein can also be used to evaluate
the in vitro opsonophagocytic killing activity of the antibody
molecule-drug conjugates against multiple Gram negative
bacteria.
Example 2: Minimal Inhibitory Concentration Assay
[0695] The minimal inhibitory concentration (MIC) assay was
performed based on standards and practices published by the CLSI
(documents M07-A9, M100-24). It was used to determine minimum
inhibitory concentrations for test compounds against several
microbial species using broth microdilution. The assay mixes
compounds with bacteria in the presence of rich broth and measures
the minimum concentration of compound at which bacterial growth is
decreased by at least 80%. The assay can use a 96-well plate/high
throughput format such that compounds can be tested against a large
panel of bacterial strains simultaneously. For each test strain
there is a published inhibitory standard and results for standards,
published in CSLI documents, should fall within two-fold of
published values and should not vary more than two-fold in
subsequent tests.
[0696] The procedures are described as follows:
[0697] On Day 1, all strains to be used in the assay were taken
from -80.degree. C. storage, thawed on ice, and subcultured to an
appropriate agar plate using a 10 .mu.l inoculating loop. All
plates were placed at 37.degree. C. overnight. Alternatively,
strains can be subcultured from a fresh overnight agar plate or a
refrigerated agar plate less than one week old.
[0698] On Day 2, all overnight plates were examined for homology
and appropriate colony morphology. All strains were subcultured to
an appropriate agar plate using a 10 .mu.l inoculating loop. All
plates were placed at 37.degree. C. overnight.
[0699] On Day 3, all overnight plates were examined for homology
and appropriate colony morphology. Mother plates were prepared for
test compounds and standards. In a 96 well polypropylene plate, 40
.mu.l of a 50.times. concentration of compound (50 times the
desired top final assay concentration) was placed in column 1 (8
compounds per plate). 20 .mu.l diluent was placed in columns 2-12.
20 .mu.l was carried into 20 .mu.l (doubling dilutions) across the
plate, in columns 2-11. Column 12 was used as a growth control. The
diluent can vary depending on the solubility of the compound
(common diluents are water, DMSO, and 0.1 N HCl).
[0700] Concentration ranges can also be varied depending on the
efficacy of the compounds. Typically, compounds were resuspended at
3.2 mg/ml for a top final assay concentration of 64 .mu.g/ml,
however, less efficacious compounds were resuspended at 25.6 mg/ml
for a top final assay concentration of 512 .mu.g/ml. Alternatively,
compounds with even higher MICs or compounds whose stock is at a
low starting concentration can be run using an alternate protocol
for high concentration MIC determinations in which the compound is
diluted directly in the assay plate using 40 .mu.l undiluted stock
in column 1 and 20 .mu.l is carried into 20 .mu.l across the plate
columns 2-11. The assay then adds 80 .mu.l (as opposed to 100
.mu.l) of cultured media so that the compound dilution is only 1:5
(instead of 1:50).
[0701] Daughter plates were prepared as follows. Daughter plates
were stamped from the mother plate by carrying 2 .mu.l from each
row of the mother plate to a corresponding row in the daughter
plate, making one daughter plate for each strain to be tested.
[0702] Cultured media were prepared as follows. For each strain to
be tested, a culture equivalent to McFarland 0.5 was prepared. A 10
.mu.l inoculating loop was used to seed cells from overnight agar
plates into 10 ml Pyrex tubes containing 5 ml of 0.85% Saline.
Using a densitometer, each tube was adjusted to 0.5 McFarland units
by adding more cells or saline. These cultures should contain
approximately 1.times.10.sup.8 cells/ml. These cultures were
diluted to 1:200 in appropriate media to reduce the cell
concentration to approximately 5.times.10.sup.5 cells/ml. The
exception to this is strains of yeast including C. albicans, which
should be diluted 1:2000. Each plate was seeded with each diluted
culture with 100 .mu.l per well (approximately 10 ml per plate).
All daughter plates were seeded with 100 .mu.l/well of appropriate
culture. All plates were set at 37.degree. C. overnight.
[0703] On Day 4, all plates were read 18 hours after seeding using
a mirrored plate reader and reflected incandescent light. In
certain experiments, the MIC was considered as the lowest
concentration of compound that inhibits growth by at least 80%. The
well should appear void of growth when read by eye and even a
partial button would constitute observable growth. For example, as
shown in FIG. 5, the duplicate MICs would be read as 3, 3, 2, 2, 6,
6, 2, 2.
[0704] For each test strain the corresponding control compound
should have an MIC within 2 fold of the expected value. If controls
were verified, values for all test compounds were reported.
[0705] Exemplary MIC control compound values for various strains
are shown in Table 4.
TABLE-US-00007 TABLE 4 Exemplary MIC control compound values Strain
# Name Strain MIC Control Compound MIC (ug/ml) 1 P. aeruginosa ATCC
27853 Ciprofloxacin 0.25 2 E. coli ATCC 25922 Ciprofloxacin 0.25 3
S. aureus ATCC 29213 Ciprofloxacin 0.5 4 E. coli ATCC 43745
Ciprofloxacin 0.25 5 K. pneumoniae ATCC 700603 Ciprofloxacin 0.5 6
C. albicans ATCC 90028 Amphotericin B 4.0 7 P. aeruginosa ATCC
39324 Ciprofloxacin 0.125 8 P. aeruginosa ATCC 27313 Ciprofloxacin
0.125 9 P. aeruginosa ATCC 15692 Ciprofloxacin 0.25 10 P.
aeruginosa ATCC 33350 Ciprofloxacin 0.125 11 P. aeruginosa ATCC
25102 Ciprofloxacin 0.125 12 P. aeruginosa 12-4-4 Ciprofloxacin
0.125 13 P. aeruginosa PA01 Ciprofloxacin 0.25 14 P. aeruginosa PAK
Ciprofloxacin 0.125 15 S. aureus MN8 Ciprofloxacin 0.25 16 A.
baumannii ATCC 17978 Ciprofloxacin 0.125 17 A. baumannii ATCC 19606
Ciprofloxacin 0.5
Example 3: Targeted In Vitro Activity of Antibody Drug
Conjugates
[0706] The effect of an exemplary ADC (Anti-Pseudomonas antibody
with Peptide 2 fused at the C-terminus of the Heavy Chain) on
binding and inhibiting bacteria was investigated. In this example,
the bacterial strain ATCC 27853 was employed. The exemplary ADC
displayed similar binding to bacterial surface as the antibody
alone as determined by FACS (data not shown). As shown in Table 5,
the ADC showed about 10-fold enhancement in activity relative to
the peptide alone. The ADC also retained opsonophagocytic activity.
Further, the ADC demonstrated specificity for Gram-negative
pathogens as no killing of Gram-positive bacteria was observed in
this experiment.
TABLE-US-00008 TABLE 5 Targeted In Vitro Activity Sample MIC
(.mu.g/ml) MW (g/mol) MIC (.mu.M) MIC Per Payload (.mu.M) Peptide
Alone 16 3400 4.7 4.7 Antibody Alone -- 149000 -- N/A ADC 77 162000
0.48 0.95
Example 4: Generation and Testing of Exemplary Antibodies
[0707] Exemplary antibodies hWN01 and hWNv1 were designed by
structure guided engineering of WN1 222-5 (Di Padova et al., Infect
Immun. 1993; 61(9):3863-3872). Antibodies hWN01 and hWNv1 target
the conserved core glycan of LPS and were engineered to bring them
proximal to human germline and improve binding to K. pneumoniae. As
shown in FIG. 2, hWN01 showed picomolar (pM) binding to E. coli and
nanomolar (nM) binding to K. pneumoniae strains. In addition, hWN01
and hWNv1 are proximal to human germline sequence and have improved
expression.
[0708] Exemplary antibodies 2C7, 3D6, 3E7 and 3G1 were also
generated. The binding of antibodies 2C7, 3D6, 3E7 and 3G1 to
representative E. coli, K. pneumoniae and S. typhimurium strains
was determined by ELISA. The results are shown in FIGS. 7-10.
Example 5: Novel Scaffold for Engineering Mouse Immunization mAb
A001-25
[0709] Towards identification of a broadly reactive antibody
targeting the core of LPS, CD-1 mice were immunized with E. coli
J5-OMP vaccine (10 .mu.g) and Hiltonol adjuvant (10 .mu.g). The
mice were dosed intraperitoneally with the immunogen-adjuvant mix
weekly for 3 weeks, at which point the sera from the immunized mice
were assessed for their titers against multiple pathogens by whole
cell ELISA. Mice with the highest titers against E. coli and K.
pneumoniae were selected for fusion. Splenic fusions were performed
with myeloma partner cells and seeded into 96-well for clonal
development. Two weeks later, the hybridomas were screened for
their ability to recognize E. coli J5 LPS. One such clone with
binding to E. coli, K. pneumoniae and S. typhimurium by whole cell
ELISA is mAb A001-25. It targets the conserved core glycan of LPS.
As shown in FIG. 3, A001-25 has strong picomolar (pM) binding to E.
coli and K. pneumoniae. Structural assessment based of the modeled
structure indicated pathways for affinity enhancement.
Example 6: Ranking Exemplary Antimicrobial Peptides by Cascade
Testing
[0710] Candidate antimicrobial peptides were tested for their
inhibitory activity on bacteria and their hemolytic activity.
Peptides with high killing activity against both E. coli and
Pseudomonas in broth, mouse and humans serum along with low
cytotoxicity or hemolytic activity were prioritized as lead
candidates. The results are shown in Table 6A. Antimicrobial
peptides with strong inhibitory or bactericidal activity, low red
blood cell hemolysis, and low off-target toxicity, were selected
for further analysis.
TABLE-US-00009 TABLE 6A Inhibitory Activity of Exemplary
Antimicrobial Peptide on Bacteria and Hemolytic Activity SEQ ID MIC
.mu.g/ml PLC Sample Sequence NO Eco 25922 Pae 27853 .mu.g/ml
Peptide 26 ALWKTLLKKVLKAAAK 67 4 8 64 peptide 119
RGLRRLGRKIAHGVKKYGPTVLRIIRIAG 68 4 2 128 peptide 109
GIGKFLKKAKKFGKAFVKILKK 69 4 8 128 peptide 30
ALWKTLLKKVLKAAAKGGGGSGGGGS 70 64 128 512 peptide 24
GIGKFLKKAKKFGKAFVKILKKGGGGSGGGGS 71 32 8 512 peptide 126
KKLLKWLKKLL 72 64 32 640 peptide 21
(MAL)-(EG3)-GIGKFLKKAKKFGKAFVKILKK 73 8 64 64 peptide 128
RLONFFRKAKKKIGRGLKKIGQKIKDFLONLVPRTES 74 16 8 128 peptide 23
GGGGSGGGGSGIGKFLKKAKKFGKAFVKILKK 75 8 32 32 Peptide 33
GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLA 76 8 8 32 peptide 29
GGGGSGGGGSALWKTLLKKVLKAAAK 77 32 128 128 peptide 85
GWKKWFNRAKKVGKTVGGLAVDHYLG 78 64 256 256 peptide 70
GAFGNFLKGVAKKAGLKILSIAQCKLFGTC 79 4 8 8
[0711] Structure-activity relationships for exemplary AMPs were
examined for ADC construction. Candidate AMPs were selected based,
e.g., on killing activity, level of hemolysis, and activity in
human serum. The results are shown in Table 6B. Exemplary AMPs,
P265, P271, GGG-Octapeptin, P293, P294, P295, P261, and P297 were
selected for evaluation as ADCs.
TABLE-US-00010 TABLE 6B Structure-Activity Relationships for
Exemplary AMPs 50% Hemo- Hemo- hsMIC MIC (mg/ml) lysis lysis
(.mu.g/ml) Sample Pae Pae Eco Eco Sau Kpn MLC MLC/ PLC PLC/ Pae
hsMIC/ (P = Peptide) 27853 39324 25922 43745 29213 700603
(.mu.g/ml) MIC (.mu.g/ml) MIC 27853 MIC P265 32 16 32 8 >128
>128 128 >4 32 1 >128 >4 P271 16 8 4 4 16 32 >128
>8 64 4 32 2 GGG- 2 2 32 16 >128 >128 >128 >64
>128 >64 2 1 Octapeptin P289 >128 32 128 64 64 >128
>128 ND >128 ND >128 ND P291 >128 >128 >128
>128 >128 >128 >128 ND 32 <0.25 >128 ND P292
>128 >128 >128 >128 >128 >128 128 ND 128 <1
>128 ND P293 8 2 4 4 4 4 128 16 32 4 32 4 P294 64 2 4 2 4 8
>128 >2 >128 >2 64 1 P295 4 4 4 4 16 16 >128 >32
>128 >32 2 0.5 P296 4 2 4 4 16 32 >128 >32 128 32 1
0.25 P261 16 8 4 4 32 128 >128 >8 >128 >8 32 2 P297 4 2
4 2 32 128 >128 >32 >128 >32 8 2 Ceftazadime 1 1 0.25
0.25 4 32 16 16 8 8 1 1 Ciprofloxacin 0.25 0.03 0.015 0.03 0.5 0.25
>1 >4 >1 >4 0.25 1
[0712] In Table 6B, MIC was determined in the presence of PBS,
hsMIC was determined in the presence of 50% human serum, MLC was
determined as the concentration (e.g., minimum concentration) that
led to 100% red blood cell lysis; PLC was determined as the
concentration (e.g., minimum concentration) that led to 50% red
blood cell lysis.
[0713] Pae: P. aeruginosa; Eco: E. coli; Sau: S. aureus; Kpn: K.
pneumoniae
Example 7: Peptide Stapling to Improve Potency and Stability of
Antimicrobial Peptides
[0714] This example shows that peptide stapling improved potency
and stability of an exemplary .alpha.-helical antimicrobial
peptide. The process of peptide stapling by chemical crosslinking
was illustrated in FIG. 4. The inhibitory activity of the
antimicrobial peptide before and after stapling was determined. The
MIC for the exemplary peptide is 160 .mu.g/ml, whereas the MIC for
the stapled peptide is 10 .mu.g/ml. Exemplary stapling methods are
described, e.g., in Alexander et al. J. Am. Chem. Soc., 2013, 135
(16), 5946-5949.
Example 8: Production of Antibody Drug Conjugates Using
Sortase-Based Ligation
[0715] The sortase ligation forms a native peptide bond between a
sortase recognition sequence and a sortase donor sequence.
Quantitative addition was achieved of the peptide
GGGRGLRRLGRKIAHGVKKYGPTVLRIIRIAG (SEQ ID NO: 80) onto the C-termini
of antibody heavy chains containing (GS).sub.6LPETGGG (SEQ ID NO:
24), and of light chains containing P(G.sub.4S).sub.2LPETGGSG (SEQ
ID NO: 26) using sortase A pentamutant (Chen et al. Proc. Natl.
Acad. Sci. USA, 2011, 108 (28): 11399-11404). The sequence of GGG
is the sortase donor domain and the sequence of
RGLRRLGRKIAHGVKKYGPTVLRIIRIAG (SEQ ID NO: 68) is the antimicrobial
peptide domain.
[0716] A representative method is described as follows. 1.5 mg/mL
antibody in 150 mM NaCl, 50 mM Tris (pH 7.5), 10 mM CaCl.sub.2, 20
mol equivalents of sortase donor peptide per sortase acceptor
sequence, 1 mol equivalent of sortase per 75 mol equivalents of
sortase acceptor sequence were combined. The reaction was incubated
at 25.degree. C. for 20 hours, and extent of conversion was
monitored by Q-TOF mass spectrometry. Upon complete conversion of
the reaction, as determine by Q-TOF, the reaction mixture was
diluted 10-fold in PBS and purified on a Protein A column. The FPLC
purified constructs were further characterized by Q-TOF and by gel
electrophoresis. The results are shown in FIG. 6.
[0717] Sortase ligation was also performed as follows. Peptides
containing an N-terminal GGG sortase donor sequence were ligated to
the C-termini of the antibody heavy chain containing the sortase A
recognition sequence LPETGGG. Antibodies had been buffer exchanged
into 150 mM NaCl/50 mM Tris (pH 7.5) prior to ligation. Optimized
sortase ligation conditions were performed in 150 mM NaCl/50 mM
Tris (pH 7.5) using 20 mol peptide per mol mAb at 1.5 mg/mL mAb
(150 kDa), 10 mM CaCl2, 5.8 .mu.g/mL Sortase A (from BPS
Bioscience, (21.7 kDa)). After incubation at ambient temperature in
the dark for 18 hours, samples were diluted to 10 ml total volume
in PBS and purified by FPLC. Conjugation efficiency was determined
by Q-TOF mass spectrometry using a reduced antibody prepared by
heating a 5 .mu.g sample at 65.degree. C. for 15 min in 10 mM
DTT.
Example 9: Evaluation of Selectivity of Antibody Drug
Conjugates
[0718] An exemplary ADC was evaluated for selectivity for target
bacteria using a mixed microbial killing assay. As shown in FIGS.
11A-11D, killing activity of ADC targeting Pseudomonas was
preferential for Pseudomonas over E. coli and Klebsiella spp.,
compared to antibody alone, peptide alone, or a combination of
antibody and peptide.
Example 10: Binding to P. aeruginosa Including Multi-Drug Resistant
Strains
[0719] The binding of an exemplary ADC (comprising mAb001) to P.
aeruginosa, including multi-drug resistant strains, was tested. As
shown in FIG. 12 and Table 7, the exemplary ADC showed strong
binding to P. aeruginosa. The exemplary ADC is highly selected for
P. aeruginosa (data not shown). The binding is LPS core
specific.
TABLE-US-00011 TABLE 7 Binding Avidity to P. aeruginosa (EC50) P.
aeruginosa Strain Binding (pM) BAA-2108* 53 BAA-2109* 73 BAA-2110*
68 BAA-2111* 53 BAA-2112* 104 BAA-2113* 58 BAA-2114* 56 33348 73
27316 72 33358 72 27853 113 *Strains are multi-drug resistant
Example 11: Engagement to Bacterial Surface
[0720] The binding of an exemplary ADC (comprising mAb001) to
bacterial surface was examined. The binding was visualized by
electron microscopy using immunogold secondary labeling. As shown
in FIG. 13, the exemplary ADC binds to bacterial surface. Enhanced
surface binding was observed compared to other targets.
Example 12: Antibody In Vivo Activity: Murine Acute Pneumonia
[0721] The in vivo activity of an exemplary anti-LPS antibody
molecule (mAb001) was tested in a murine acute pneumonia model.
[0722] Mice were supplied by Charles River (Margate UK) and were
specific pathogen free. The strain of mice used was ICR (also known
as CD1 mice) which is a well characterized outbred murine strain.
Mice (male) were 11-15 g on receipt and were allowed to acclimatise
for at least 7 days. Mice were rendered neutropenic by
immunosuppression with cyclophosphamide at 200 mg/kg 4 days before
infection and 150 mg/kg 1 day before infection by intraperitoneal
injection. The immunosuppression regime leads to neutropenia
starting 24 hours post administration of the first injection which
continues throughout the study. Pseudomonas aeruginosa strain ATCC
27853 was used for in vivo studies.
[0723] Mice were infected approximately 24 hours after the second
dose of immunosuppressive agent by intranasal instillation with P.
aeruginosa ATCC 27853 prepared from fresh broth and diluted to an
optimal concentration with PBS. For infection, animals were
anaesthetized with Ketamine/Xylazine (90 mg/kg Ketamine/9 mg/kg
Xylazine) via IP injection delivered at .about.15 mL/kg.
Anaesthetized mice were infected with 0.04 mL inoculum by
intranasal instillation (20 .mu.L per nostril, 5 min between
nostrils) and were kept in an upright position on a string rack for
.about.10 minutes post-infection. The inoculum concentration was
6.67.times.10.sup.5 CFU/mL (.about.2.67.times.10.sup.4 CFU/mouse
lung). Stock solutions of test articles were prepared in PBS
(Dulbecco's Phosphate Buffered Saline). Following reconstitution,
all test dosing solutions remained translucent and non-particulate
for the duration of the dosing period.
[0724] Test articles were dosed once by the intravenous (IV) route
at 12 hours before the planned infection time. The comparators
tobramycin and polymyxin B were dosed three times a day (TID) by
the IV and subcutaneous (SC) routes, respectively, starting at 2
hours post-infection. The dosing volume was 10 mL/kg for all test
article doses and comparators.
[0725] Each group included 8 neutropenic animals Animals received
intranasal inoculation of P. aeruginosa ATCC 27853. The antibody
molecule was administered at 5 mg/kg or 45 mg/kg intravenously.
Polymyxin B was used as a positive control. The 24-hour bacterial
burden in lung was measured. As shown in FIG. 14, the exemplary
antibody molecule showed 2-log reduction (CFU/g). The results
demonstrated the in vivo efficacy of an LPS core-targeting antibody
in a murine acute pneumonia model.
Example 13: Effect of AMP Stapling on Stability and Non-Specific
Binding
[0726] The effect of stapling on stability and non-specific binding
(NSB) was examined using an exemplary antimicrobial peptide. As
shown in FIGS. 15A-15B, both T=0 and T=60 min serum measurements
show increased amounts of the stapled AMP relative to the unstapled
version. This data is quantitative and was generated on an LS/MS.
The increase was attributed to reduced NSB with the stapled
compound. FIG. 15C illustrates the difference between unoptimized
payload and optimized payload. Payloads can be selected based on
serum stability. Payloads having reduced non-specific binding
and/or enhanced protease stability can be selected.
[0727] Serum stability sample generation was performed as follows.
Normal Human Serum (NHS) (Sigma S-7023) was thawed, diluted in
water, centrifuged at 13000 rpm for 10 minutes and the supernatant
was warmed to 37.degree. C. in a water bath. Twenty .mu.l of each
test article was placed in a 2.0 ml round bottom microfuge tube.
Two ml of diluted NHS was added to each tube and immediately the
tubes were vortexed and 200 .mu.l was transferred to a fresh
microfuge tube with 40 .mu.l of 15% Trichloroacetic acid (TCA).
Assay tubes were placed at 37.degree. C. in a rotating rack between
time-points. TCA tubes were placed on ice for 15 minutes and then
centrifuged at 13000 rpm for 10 minutes. Supernatant from each tube
was collected and frozen at -20.degree. C. for analysis. Samples
were harvested and processed at various time-points up to 6 hours.
Exemplary methods are also disclosed in Nguyen et al. (2010) PLoS
ONE 5(9): e12684.
Example 14: Effect of Anti-LPS Antibody on Endotoxin Signal
[0728] A cell-based colorimetric assay was used for the detection
and quantification of endotoxin signal LPS. This assay is based on
the activation of Toll-like receptor (TLR) 4, the mammalian
endotoxin sensor (Beutler et al. Curr Top Microbiol Immunol. 2002;
270:109-20). TLR4 recognizes LPS from Gram-negative bacteria and
activates NF-.kappa.B. Cells engineered to become sensitive to LPS,
such as HEK-Blue.TM.-4 cells (InvivoGen), stably express human TLR4
and an NF-.kappa.B-inducible secreted embryonic alkaline
phosphatase (SEAP) reporter gene. The presence of LPS can be
detected by the HEKBlue.TM.-4 cells leading to the activation of
NF-.kappa.B. Using QUANTI-Blue.TM. (InvivoGen), a SEAP detection
medium that produces a color signal, NF-kB activation can be
detected at 620-655 nm. Since the absorbance is in direct
proportion to the amount of endotoxin present, the concentration of
endotoxin can be measured from a standard curve obtained using
serial dilutions of the HEK-Blue.TM. Endotoxin Standard
(InvivoGen).
[0729] As shown in FIG. 16, the endotoxin signal (Pseudomonas-LPS)
was completely abolished in the presence of an exemplary anti-LPS
antibody molecule, mAb001. Negative control (a C. difficile
anti-toxin antibody, CDA1) did not show any effect on the endotoxin
signal. 0.5 EU/ml of Pseudomonas-LPS was used in this study.
Example 15: Microbial Killing Activity with Antibody Drug
Conjugates
[0730] Exemplary ADCs were tested for their microbial killing
activity.
[0731] The microbial killing assay was performed as follows.
Bacterial cells were grown aerobically overnight on agar plates at
37.degree. C. Overnight plates were used to seed 30 ml cultures of
growth media in 250 ml vented flasks. Cultures were grown
aerobically at 37.degree. C., shaking at 150 rpm. Growth was
monitored at A600 nM and bacterial cells were harvested at mid-log
growth. Ten ml of culture was pelleted at 4000.times.G for 10
minutes and washed one time with PBS plus 1% BSA before
re-suspending in 2 ml PBS+BSA. The concentrated culture was used to
seed 6 ml tubes of PBS plus BSA to an OD giving a concentration of
1.times.10.sup.8 cells/ml. Cultures were diluted to
1.times.10.sup.4 cells/ml in PBS plus BSA. Test articles were
diluted in PBS+1% BSA and 50 .mu.l per concentration tested was
loaded into a 96 well polypropylene microtiter plate. Fifty .mu.l
of diluted culture was added to all test wells as well as no
compound control wells. Plates were shaken and incubated at
37.degree. C., 90 minutes, static. Ten .mu.l from each assay well
was plated onto agar plates and incubated at 37.degree. C.
overnight. Percent killing was determined by the CFU for test wells
compared to the CFU for no compound control wells.
[0732] As shown in Table 9, multiple exemplary ADCs showed 50%
killing activity against P. aeruginosa stains at a concentration
less than 10 .mu.g/ml.
TABLE-US-00012 TABLE 9 Microbial Killing Activity of Exemplary ADCs
(SEQ (SEQ 50% Killing ID NO: ID NO: Activity 157) 157) (.mu.g/ml)
Exemplary Linker- Linker- Val- Pay- Pae Pae Conjugates mAb HC LC
ency load 27853 39324 1 mAb001 (GS)15 (GS)15 Tetra- P271 25 6.3 2
mAb001 (GS)15 (GS)15 Tetra- P293 25 12.5 3 mAb001 (GS)15 (GS)15
Tetra- P294 6.3 3.1 4 mAb001 (GS)15 (GS)15 Tetra- P295 6.3 0.8 5
mAb001 (GS)15 (GS)15 Tetra- P297 1.6 0.4
Example 16: Microbial Killing Activity Against Multiple Drug
Resistant Strains
[0733] Exemplary compounds were tested for their microbial killing
activity against multiple drug resistant P. aeruginosa strain.
[0734] The microbial killing assay was performed as described in
Example 15. As shown in Table 10, exemplary ADCs showed microbial
killing activity against multiple drug resistant (MDR) strains of
P. aeruginosa.
TABLE-US-00013 TABLE 10 Microbial Killing Activity of Exemplary
ADCs against MDR Strains Linker- Linker- HC LC (SEQ (SEQ 50%
Killing ID ID Activity (.mu.g/ml) Com- NO: NO: Val- Pay- Pae BAA-
BAA- BAA- pounds mAb 157) 157) ency load 27853 2110 2114 2108
mAb001 mAb001 >100 >100 >100 >100 P295 -- P295 0.3 0.6
0.14 0.6 P297 -- P297 0.04 0.28 0.07 0.28 mAb001- mAb001 (GS)15
(GS)15 Tetra- P295 12.5 12.5 6.3 6.3 Conjugate1 mAb001- mAb001
(GS)15 (GS)15 Tetra- P297 3.1 6.3 3.1 3.1 Conjugate2
Example 17: Selective Killing of Bacteria by Antibody Drug
Conjugates
[0735] Exemplary compounds were tested for their killing
selectivity against different bacteria. The Pseudomonas strains
used in the assay were P. aeruginosa 27853 (ATCC), P. aeruginosa
39324 (ATCC), P. aeruginosa PA01 (UMD). The E. coli strains used in
the assay were E. coli 25922 (ATCC) and E. coli 43745 (J5) (ATCC).
The Klebsiella strain used in the assay was K. pneumoniae 700603
(ATCC). Bacterial cells were grown on agar plates from frozen
stocks. All bacterial strains were grown on blood agar plates
(TSA+5% Sheep blood). All plates were grown at 37.degree. C.
overnight.
[0736] Overnight plates were used to establish 0.5 McFarland
Cultures in 10 ml Pyrex tubes in 6 ml 1.times.PBS. The concentrated
cultures (approximately 1.times.10.sup.8 cells/ml) were diluted
2.times.1:100 in PBS (0.1 mls culture in 9.9 ml PBS) to a
concentration of approximately 1.times.10.sup.4 cells/ml. Ten .mu.l
of each of the diluted cultures were plated onto blood agar plates
(BAPs) using sterile "hockey stick" spreaders to determine the
initial concentration, check for purity, and establish strain
morphology. One ml of each diluted culture was then mixed with
enough PBS to bring the volume to 10 ml. This mixed culture had
approximately 1.times.10.sup.3 cells of each bacterial strain per
ml. 25 .mu.l of this mixed culture was plated onto a BAP to
establish the t=0 CFUs/ml for each strain.
[0737] Antibodies, anti-microbial peptides (AMPs), and antibody
drug conjugates (ADCs) to be tested were diluted in 1.times.PBS.
Dilutions were either 2 fold or four-fold and 3 or 4 concentrations
of each compound were tested. For antibodies and conjugates,
typical final assay concentrations for the assay were 100, 25,
6.25, and 1.56 .mu.g/ml although higher, lower, and broader
dilution ranges had been used. Antimicrobial peptides were tested
at molar equivalents to the amount of peptide that was found in a
corresponding conjugate (most commonly 4.4, 1.1, 0.28, and 0.07
.mu.g/ml). All compounds were at a final volume of 200 .mu.l in a
2.0 ml round bottom Eppendorf tube. A no compound control tube was
also included. 200 .mu.l of the mixed bacterial culture above was
added to all assay tubes. The tubes were votexed and 50 .mu.l of
each assay tube were plated on separate BAPs. The assay tubes were
then placed at 37.degree. C. in a rotating rack. The plating
procedure was repeated at one or two hour intervals over four hours
with the tubes rotating at 37.degree. C. between timepoints. All
plates were put at 37.degree. C. overnight.
Total plates required=(number of assay tubes.times.number of
time-points)+t=0 controls (3-4)
[0738] The following day, all plates were counted for CFU of each
bacterial strain.
[0739] For each compound, percent killing (% CFU reduction compared
to a no compound control) was calculated for each strain at each
timepoint. Data was tabled and graphed as % killing vs. time.
[0740] As shown in FIG. 17, the exemplary ADC selectively killed
Pseudomonas and rapid killing (within an hour) was achieved.
Example 18: Antibody Drug Conjugate In Vivo Activity: Murine Acute
Pneumonia
[0741] The in vivo activity of an exemplary ADC (mAb001-conj2) was
tested in a murine acute pneumonia model.
[0742] Mice were supplied by Charles River (Margate UK) and were
specific pathogen free. The strain of mice used was ICR (also known
as CD1 mice) which is a well characterized outbred murine strain.
Mice (male) were 11-15 g on receipt and were allowed to acclimatise
for at least 7 days. Mice were rendered neutropenic by
immunosuppression with cyclophosphamide at 200 mg/kg 4 days before
infection and 150 mg/kg 1 day before infection by intraperitoneal
injection. The immunosuppression regime leads to neutropenia
starting 24 hours post administration of the first injection which
continues throughout the study. Pseudomonas aeruginosa strain ATCC
27853 was used for in vivo studies.
[0743] Mice were infected approximately 24 hours after the second
dose of immunosuppressive agent by intranasal instillation with P.
aeruginosa ATCC 27853 prepared from fresh broth and diluted to an
optimal concentration with PBS. For infection, animals were
anaesthetized with Ketamine/Xylazine (90 mg/kg Ketamine/9 mg/kg
Xylazine) via IP injection delivered at .about.15 mL/kg.
Anaesthetized mice were infected with 0.04 mL inoculum by
intranasal instillation (20 .mu.L per nostril, 5 min between
nostrils) and were kept in an upright position on a string rack for
.about.10 minutes post-infection. The inoculum concentration was
6.67.times.10.sup.5 CFU/mL (.about.2.67.times.10.sup.4 CFU/mouse
lung). Stock solutions of test articles were prepared in PBS
(Dulbecco's Phosphate Buffered Saline). Following reconstitution,
all test dosing solutions remained translucent and non-particulate
for the duration of the dosing period.
[0744] For intravenous dosing, test articles were dosed once by the
IV route at 12 hours before the planned infection time. The
comparators tobramycin and polymyxin B were dosed TID by the IV and
SC route respectively starting at 2 hours post-infection. The
dosing volume was 10 mL/kg for all test article doses and
comparators.
[0745] For intranasal (IN) dosing, test articles and comparator
Tobramycin were dosed once intranasally (IN) post infection (15 min
for Co-administration; 2 h for therapeutic). Animals were
anaesthetized with isoflurane. Anaesthetized mice were dosed with
0.04 mL inoculum by intranasal instillation (20 .mu.L per nostril,
5 min between nostrils) and were kept in an upright position on a
string rack for .about.10 minutes post-dosing
[0746] 2 Hours post infection, pre-treatment control animals were
humanely euthanized using a pentobarbitone overdose. Clinical
condition of the remaining animals was monitored and animals that
succumbed to the disease were humanely euthanized. The study was
terminated .about.23 h post infection when most of the vehicle mice
were at the ethically agreed endpoint.
[0747] At 23 hours post infection, the clinical condition of all
remaining animals was assessed and they were humanely euthanized by
pentobarbitone overdose Animal weights were determined before the
lungs were removed and weighed. Lung samples were homogenized in
ice cold sterile phosphate buffered saline using a Precellys bead
beater; the homogenates were quantitatively cultured onto
Pseudomonas selective agar and incubated at 37.degree. C. for 16 to
24 hours before colonies were counted. Bacterial burden in the lung
was reported on a CFU/g basis. The murine model is also described
in Secher et al. PLoS One. 2013; 8(9):e73396.
[0748] In the Co-administration study arm, each group included 6
neutropenic animals Animals received co-administration of ADC or
antibody molecule with bacteria. The ADC (mAb001-Conj2) was
administered at 10 .mu.g or 200 .mu.g (mAb001-Conj2=mAb001-P297
conjugate), and mAb001 was administered at 200 .mu.g. The 24-hour
bacterial burden in lung was measured. As shown in FIG. 18A, the
exemplary ADC showed rapid activity when co-administered with
bacteria. The activity was comparable to Tobramycin.
[0749] In the intranasal (IN) study arm, each group included 6
neutropenic animals Animals received intranasal inoculation of P.
aeruginosa ATCC 27853. The ADC (mAb001-Conj2) was administered at
10 .mu.g or 200 .mu.g 2 hours post inoculation. The 24-hour
bacterial burden in lung was measured. As shown in FIG. 18B, the
exemplary ADC showed about 2 log.sub.10 reduction in CFU/g.
Example 19: Bioavailability of Antibody Drug Conjugate
[0750] The bioavailability of an exemplary ADC was studied in a
mouse model. C57/BL6 mice, 6 week old, male, were used. Each group
included four mice. mAb001 and mAb001-P297 were dosed at 5 mg/kg by
intravenous injection. Data were collected 24 hours and 120 hours
post-administration. Human IgG was quantified by ELISA.
[0751] As shown in FIG. 19, the bioavailability of the mAb001
conjugate was comparable to mAb001.
Example 20: Study of Phosphorylated Glycans in Core LPS
[0752] Phosphorylated glycans represent a key, conserved motif in
all P. aeruginosa strains. LPS was prepared from PAC557 strain. NMR
analysis revealed multiple glycoforms, variable 0-acetylation in
outer core, and hyper-phosphorylated L, D-mannoheptose units.
Example 21: Effect of D-Amino Acids on Peptide Serum Stability
[0753] The effect of D-amino acids on the stability of
antimicrobial peptide in human serum was studied. An exemplary
antimicrobial peptide, P297, was used in this study. (D)-P297
contains all D-amino acids and (L)-P297 contains all L-amino acids.
The percentages of remaining intact peptides were measured either
in the absence of human serum, or in the presence of 2%, 5%, or 10%
human serum, over a period of 60 minutes. As shown in FIGS. 20-21,
(L)-P297 degraded rapidly (and completely under certain testing
conditions), and (D)-P297 was considerably more stable than
(L)-P297.
INCORPORATION BY REFERENCE
[0754] All publications, patents, and accession numbers mentioned
herein are hereby incorporated by reference in their entirety as if
each individual publication or patent was specifically and
individually indicated to be incorporated by reference.
EQUIVALENTS
[0755] While specific embodiments of the compositions and methods
herein have been discussed, the above specification is illustrative
and not restrictive. Many variations of the invention will become
apparent to those skilled in the art upon review of this
specification and the claims below. The full scope of the invention
should be determined by reference to the claims, along with their
full scope of equivalents, and the specification, along with such
variations.
Sequence CWU 1
1
1671118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp His 20 25 30Tyr Ile Asn Trp Val Lys Gln Ser His
Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Tyr His Gly
Ile Thr Lys Tyr Asn Arg Asn Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Asn Ser
Leu Thr Ser Glu Leu Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly
Ser Arg Arg Tyr Phe Asp Val Trp Gly Thr Gly Thr 100 105 110Thr Val
Thr Val Ser Ser 1152120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Thr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Leu Ile
Arg Asn Lys Arg Asn Gly Asp Thr Ala Glu Tyr Ser Ala 50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Ala Arg Gln Gly Arg Gly Tyr Thr Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
1203120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25 30Trp Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Leu Ile Arg Ala Lys Ala Asn
Gly Asp Thr Ala Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg
Gln Gly Arg Gly Tyr Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 1204116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5
10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Asp Ile Tyr Pro Gly Ser Gly Ser Thr Asn Tyr Asn
Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Ser Leu Asp Tyr
Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser
1155117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Glu Val Gln Leu Gln Gln Ser Val Ala Glu Leu
Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asn Thr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Pro Ser Asn
Tyr His Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Thr
Val Ser Ser 1156117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Asp Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Ser Val
Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30Tyr Tyr Trp Asn Trp Ile Arg
Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Ser Tyr
Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60Lys Asn Arg Ile Ser
Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Lys Leu
Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg
Trp Asn Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105
110Leu Thr Val Ser Ser 1157120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Glu Val Gln Leu Gln Gln
Ser Gly Pro Val Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp
Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Val Ile
Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Arg Gln Leu Gly Leu Arg Trp Phe Ala Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala 115
1208112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Arg Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
1109107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asn Ile Asn Ile Trp 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu His Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln Gly Gln Ser Tyr Pro Arg 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 10510112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly1
5 10 15Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly
Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Arg Gly Ser Gly Thr Asp Phe
Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ser 85 90 95Leu Glu Tyr Pro Leu Thr Phe Gly Ala
Gly Thr Lys Leu Glu Leu Lys 100 105 11011112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly1
5 10 15Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln His 85 90 95Leu Glu Tyr Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 11012112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1
5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn
Ser 20 25 30Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
Val Tyr Tyr Cys Lys Gln 85 90 95Ser Tyr Asn Leu Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 11013107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly1
5 10 15Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu
Leu Ile 35 40 45Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser
Ser Leu Gln Ala65 70 75 80Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln
Leu Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105147PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Gly Tyr Thr Phe Thr Asp His1
5156PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Tyr Pro Tyr His Gly Ile1 5169PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Gly
Gly Ser Arg Arg Tyr Phe Asp Val1 5175PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Asp
His Tyr Ile Asn1 51817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 18Gly Ile Tyr Pro Tyr His Gly
Ile Thr Lys Tyr Asn Arg Asn Phe Lys1 5 10 15Asp197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gly
Phe Thr Phe Ser Asp Tyr1 5208PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 20Arg Asn Lys Arg Asn Gly Asp
Thr1 5219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gln Gly Arg Gly Tyr Thr Leu Asp Tyr1
5225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Asp Tyr Tyr Met Thr1 52319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Leu
Ile Arg Asn Lys Arg Asn Gly Asp Thr Ala Glu Tyr Ser Ala Ser1 5 10
15Val Lys Gly2419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Leu Pro Glu Thr1 5 10 15Gly Gly Gly258PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Arg
Ala Lys Ala Asn Gly Asp Thr1 52619PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 26Pro Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly1 5 10 15Gly Ser
Gly275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Asp Tyr Trp Met Thr1 52819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Leu
Ile Arg Ala Lys Ala Asn Gly Asp Thr Ala Glu Tyr Ser Ala Ser1 5 10
15Val Lys Gly297PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Gly Tyr Thr Phe Thr Ser Tyr1
5306PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Tyr Pro Gly Ser Gly Ser1 5317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Gly
Ser Tyr Ser Leu Asp Tyr1 5325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Ser Tyr Trp Ile Thr1
53317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Asp Ile Tyr Pro Gly Ser Gly Ser Thr Asn Tyr Asn
Glu Lys Phe Lys1 5 10 15Ser347PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 34Gly Phe Asn Ile Lys Asn
Thr1 5356PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Asp Pro Ala Asn Gly Asn1 5368PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Ser
Asn Tyr His Ala Met Asp Tyr1 5375PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 37Asn Thr Tyr Met His1
53817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Ala
Pro Lys Phe Gln1 5 10 15Gly398PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Gly Tyr Ser Ile Thr Ser Gly
Tyr1 5405PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Ser Tyr Asp Gly Ser1 5418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Trp
Asn Gly Asn Tyr Phe Asp Tyr1 5426PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 42Ser Gly Tyr Tyr Trp Asn1
54316PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro
Ser Leu Lys Asn1 5 10 15447PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 44Gly Tyr Thr Phe Thr Asp
Tyr1 5456PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Asn Pro Tyr Asn Gly Gly1 54611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Thr
Arg Gln Leu Gly Leu Arg Trp Phe Ala Tyr1 5 10475PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Asp
Tyr Tyr Met Asn1 54817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Val Ile Asn Pro Tyr Asn Gly
Gly Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10 15Gly4916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Arg
Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu His1 5 10
15507PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Lys Val Ser Asn Arg Phe Ser1 5519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 51Ser
Gln Ser Thr His Val Pro Tyr Thr1 55211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Arg
Ala Ser Gln Asn Ile Asn Ile Trp Leu Ser1 5 10537PRTArtificial
SequenceDescription of Artificial
Sequence Synthetic peptide 53Lys Ala Ser Asn Leu His Thr1
5549PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Leu Gln Gly Gln Ser Tyr Pro Arg Thr1
55516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn
Thr Tyr Leu Tyr1 5 10 15567PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 56Tyr Met Ser Asn Leu Ala
Ser1 5579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Met Gln Ser Leu Glu Tyr Pro Leu Thr1
55816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn
Thr Tyr Leu Tyr1 5 10 15597PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 59Arg Met Ser Asn Leu Ala
Ser1 5609PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Met Gln His Leu Glu Tyr Pro Tyr Thr1
56117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg
Lys Asn Tyr Leu1 5 10 15Ala627PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 62Trp Ala Ser Thr Arg Glu
Ser1 5638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Lys Gln Ser Tyr Asn Leu Trp Thr1
56411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala1 5
10657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Ala Ala Thr Ser Leu Ala Asp1 5669PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Gln
Gln Leu Tyr Ser Thr Pro Trp Thr1 56716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Ala
Leu Trp Lys Thr Leu Leu Lys Lys Val Leu Lys Ala Ala Ala Lys1 5 10
156829PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His
Gly Val Lys Lys1 5 10 15Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile
Ala Gly 20 256922PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 69Gly Ile Gly Lys Phe Leu Lys Lys Ala
Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Lys Ile Leu Lys Lys
207026PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Ala Leu Trp Lys Thr Leu Leu Lys Lys Val Leu Lys
Ala Ala Ala Lys1 5 10 15Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
257132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys
Phe Gly Lys Ala Phe1 5 10 15Val Lys Ile Leu Lys Lys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 20 25 307211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Lys
Lys Leu Leu Lys Trp Leu Lys Lys Leu Leu1 5 107322PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Gly
Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe1 5 10
15Val Lys Ile Leu Lys Lys 207437PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 74Arg Leu Gly Asn Phe
Phe Arg Lys Ala Lys Lys Lys Ile Gly Arg Gly1 5 10 15Leu Lys Lys Ile
Gly Gln Lys Ile Lys Asp Phe Leu Gly Asn Leu Val 20 25 30Pro Arg Thr
Glu Ser 357532PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 75Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Ile Gly Lys Phe Leu1 5 10 15Lys Lys Ala Lys Lys Phe Gly
Lys Ala Phe Val Lys Ile Leu Lys Lys 20 25 307632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
76Gly Leu Arg Lys Arg Leu Arg Lys Phe Arg Asn Lys Ile Lys Glu Lys1
5 10 15Leu Lys Lys Ile Gly Gln Lys Ile Gln Gly Leu Leu Pro Lys Leu
Ala 20 25 307726PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 77Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Leu Trp Lys Thr Leu1 5 10 15Leu Lys Lys Val Leu Lys Ala Ala
Ala Lys 20 257826PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 78Gly Trp Lys Lys Trp Phe Asn Arg Ala
Lys Lys Val Gly Lys Thr Val1 5 10 15Gly Gly Leu Ala Val Asp His Tyr
Leu Gly 20 257930PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 79Gly Ala Phe Gly Asn Phe Leu Lys
Gly Val Ala Lys Lys Ala Gly Leu1 5 10 15Lys Ile Leu Ser Ile Ala Gln
Cys Lys Leu Phe Gly Thr Cys 20 25 308032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
80Gly Gly Gly Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly1
5 10 15Val Lys Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala
Gly 20 25 3081354DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 81gaggtccagc tgcagcagtc
tggacctgtg ctggtgaagc ctggggcttc agtgaagatg 60tcctgtaagg cttctggata
cacattcact gaccactata taaactgggt gaagcagagc 120catggaaaga
gccttgagtg gattggaggt atttatcctt accacggtat tactaagtac
180aaccggaatt tcaaggacaa ggccacattg actgttgaca agtcctccag
cacagcctac 240atggagctca acagcctgac atctgaactc tctgcagtct
attactgtgc aagcggggga 300agtcgccggt acttcgatgt ctggggcaca
gggaccacgg tcaccgtctc ctca 35482360DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
82gaagtgcagc tcgtggaatc tggaggagga cttgtgcaac ctggaggttc cctgcgactg
60tcgtgtgccg catccggttt caccttttcc gactactaca tgacctgggt cagacaggcg
120ccggggaagg gactggagtg ggtcggcttg atccgcaaca agaggaacgg
cgatactgct 180gaatactcgg ccagcgtgaa ggggcggttc accatctcga
gagatgacag caagaactcc 240ctgtacctcc aaatgaactc cctgaaaacc
gaggacactg cggtgtacta ctgcgcccgc 300cagggtcgcg gctacacgct
ggactattgg ggccagggca ccctggtcac tgtgtcaagc 36083360DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
83gaagtgcagc tcgtggaatc tggaggagga cttgtgcaac ctggaggttc cctgcgactg
60tcgtgtgccg catccggttt caccttttcc gactactgga tgacctgggt cagacaggcg
120ccggggaagg gactggagtg ggtcggcttg atccgcgcca aggcgaacgg
cgatactgct 180gaatactcgg ccagcgtgaa ggggcggttc accatctcga
gagatgacag caagaactcc 240ctgtacctcc aaatgaactc cctgaaaacc
gaggacactg cggtgtacta ctgcgcccgc 300cagggtcgcg gctacacgct
ggactattgg ggccagggca ccctggtcac tgtgtcaagc 36084348DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
84caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagatg
60tcctgcaagg cttctggcta caccttcacc agctactgga taacctgggt gaagcagagg
120cctggacaag gccttgagtg gattggagat atttatcctg gtagtggtag
tactaactac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca
catcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac
tctgcggtct attactgtgc aagaggtagc 300tactcccttg actactgggg
ccaaggcacc actctcacag tctcctca 34885351DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
85gaggttcagc tgcagcagtc tgtggcagag cttgtgaggc caggggcctc agtcaagttg
60tcctgcacag cttctggctt caacattaaa aacacctata tgcactgggt gaagcagagg
120cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa
tactaaatat 180gccccgaagt tccagggcaa ggccactata actgcagaca
catcctccaa cacagcctac 240ctgcagctca gcagcctgac atctgaggac
actgccatct attactgtgc tcctagtaac 300taccatgcta tggactactg
gggtcaagga acctcagtca ccgtctcctc a 35186351DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
86gatgtacagc ttcaggagtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc
60acctgctctg tcactggcta ctccatcacc agtggttatt actggaactg gatccggcag
120tttccaggaa acaaactgga atggatgggc tacataagct acgatggtag
caataactac 180aacccatctc tcaaaaatcg aatctccatc actcgtgaca
catctaagaa ccagtttttc 240ctgaagttga attctgtgac tactgaggac
acagccacat attactgtgc aagatggaat 300ggtaactact ttgactactg
gggccaaggc accactctca cagtctcctc a 35187360DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
87gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtgaagatg
60tcctgtaagg cttctggata cacattcact gactactata tgaactgggt gaagcagagc
120catggaaaga gccttgagtg gattggagtt attaatcctt acaacggtgg
tactagctac 180aaccagaagt tcaagggcaa ggccacattg actgttgaca
agtcctccag cacagcctac 240atggagctca acagcctgac atctgaggac
tctgcagtct attactgtgc aagaaccaga 300cagctcgggc tacgttggtt
tgcttactgg ggccaaggga ctctggtcac tgtctctgca 36088336DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
88gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc
60atctcttgca gatctagtca gagacttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc
caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga
cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt
tatttctgct ctcaaagtac acatgttccg 300tacacgttcg gaggggggac
caagctggaa ataaaa 33689321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 89gacatccaga
tgactcagtc cccgtcctca gtctccgcat ccgtgggaga tcgcgtgacg 60attacttgcc
gggcgtcgca gaacatcaac atctggctgt cgtggtacca gcagaagccc
120gggaaggctc cgaagctgct gatctacaag gcctcaaact tgcacaccgg
cgtgccttcc 180cgcttttctg gttcgggctc cgggactgac ttcaccctga
ccatcagcag cctgcaaccc 240gaggacttcg ccacctatta ctgcctccaa
ggacagtcct acccaagaac cttcggcgga 300ggaaccaagg tcgaaatcaa a
32190336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 90gatattgtga tgactcaggc tgcaccctct
gtacctgtca ctcctggaga gtcagtatcc 60atctcctgca ggtctagtaa gagtcttctg
catagtaatg gcaacactta cttgtattgg 120ttcctgcaga ggccaggcca
gtctcctcag cgcctgatat attatatgtc caaccttgcc 180tcaggagtcc
cagacaggtt cagtggcaga gggtcaggaa ctgatttcac actgagaatc
240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaaagtct
agaatatcct 300ctcacgttcg gtgctgggac caagctggag ctgaaa
33691336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 91gatattgtga tgactcaggc tgcaccctct
gtacctgtca ctcctggaga gtcagtatcc 60atctcctgca ggtctagtaa gagtctcctg
catagtaatg gcaacactta cttgtattgg 120ttcctgcaga ggccaggcca
gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180tcaggagtcc
cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc
240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct
agaatatccg 300tacacgttcg gaggggggac caagctggaa ataaaa
33692336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 92gacattgtga tgtcacagtc tccatcctcc
ctggctgtgt cagcaggaga gaaggtcact 60atgagctgca aatccagtca gagtctgctc
aacagtagaa cccgaaagaa ctacttggct 120tggtaccagc agaaaccagg
gcagtctcct aaactgctga tctactgggc atccactagg 180gaatctgggg
tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc
240atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc
ttataatctg 300tggacgttcg gtggaggcac caagctggaa atcaaa
33693321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 93gacattcaga tgacccagtc tcctgcctcc
cagtctgcat ctctgggaga aagtgtcacc 60atcacatgcc tggcaagtca gaccattggt
acatggttag catggtatca gcagaaacca 120gggaaatctc ctcagctcct
gatttatgct gcaaccagct tggcagatgg ggtcccatca 180aggttcagtg
gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct
240gaagattttg taagttatta ctgtcaacaa ctttacagta ctccgtggac
gttcggtgga 300ggcaccaagc tggaaatcaa a 3219423PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Gly
Ile Gly Lys His Val Gly Lys Ala Leu Lys Gly Leu Lys Gly Leu1 5 10
15Leu Lys Gly Leu Gly Glu Ser 209526PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Gly
Arg Arg Lys Arg Lys Trp Leu Arg Arg Ile Gly Lys Gly Val Lys1 5 10
15Ile Ile Gly Gly Ala Ala Leu Asp His Leu 20 259627PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 96Gly
Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys1 5 10
15Tyr Gly Pro Gln Ala Thr Pro Ala Thr Arg Gln 20
259714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Ile Lys Trp Lys Lys Leu Leu Arg Ala Ala Lys Arg
Ile Leu1 5 109819PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 98Ile Gly Lys Lys Trp Lys Arg Ile Val
Lys Arg Ile Lys Lys Phe Leu1 5 10 15Arg Lys Leu9912PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 99Ile
Leu Gly Lys Ile Trp Lys Ile Lys Lys Leu Phe1 5 1010037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
100Arg Leu Gly Asp Ile Leu Gln Lys Ala Arg Glu Lys Ile Glu Gly Gly1
5 10 15Leu Lys Lys Leu Val Gln Lys Ile Lys Asp Phe Phe Gly Lys Phe
Ala 20 25 30Pro Arg Thr Glu Ser 3510130PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
101Gly Gly Gly Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu1
5 10 15Phe Lys Lys Leu Ser Pro Val Ile Pro Leu Leu His Leu Gly 20
25 3010227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 102Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys
Lys Leu Phe Lys Lys1 5 10 15Leu Ser Pro Val Ile Pro Leu Leu His Leu
Gly 20 25103119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 103Glu Val Lys Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Glu Phe Thr Phe Ser Asp Tyr 20 25 30Ala Met Ser Trp Val Arg
Gln Thr Pro Ala Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Ser
Asp Gly Asp Ser Thr Tyr Tyr Pro Asp Asn Ile 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys 85 90 95Ala
Arg Glu Ile Arg Leu Arg Gly Tyr Phe Asp Val Trp Gly Ala Gly 100 105
110Thr Thr Val Thr Val Ser Ser 115104111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
104Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1
5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Phe Gly
His 20 25 30Gly Ile Ser Pro Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Lys Phe Gly
Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr
Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro Arg Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 1101057PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 105Glu
Phe Thr Phe Ser Asp Tyr1 51066PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 106Ser Ser Asp Gly Asp Ser1
510710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Glu Ile Arg Leu Arg Gly Tyr Phe Asp Val1 5
101085PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Asp Tyr Ala Met Ser1 510917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 109Tyr
Ile Ser Ser Asp Gly Asp Ser Thr Tyr Tyr Pro Asp Asn
Ile Lys1 5 10 15Gly11015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 110Arg Ala Ser Glu Ser Val
Phe Gly His Gly Ile Ser Pro Met His1 5 10 151117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Arg
Ala Ser Asn Leu Lys Phe1 51129PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 112Gln Gln Ser Asn Glu Tyr
Pro Arg Thr1 5113357DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 113gaagtgaagt tggtggagtc
tgggggagac ttggtgaaac ctggagggtc cctgagactc 60tcctgtgcag cctctgaatt
cactttcagt gattatgcca tgtcttgggt tcgccagact 120ccggcgaaga
ggctggagtg ggtcgcatac attagtagtg atggtgatag tacctactat
180ccggacaata ttaagggccg attcaccatc tccagagaca atgccaagaa
caccctatac 240ctgcaaatga acagtctgag gtctgaggac acggccatgt
atttttgtgc aagagaaata 300cggctaaggg ggtacttcga tgtctggggc
gcagggacca cggtcaccgt ctcctca 357114333DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
114gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc 60atatcctgca gagccagtga aagtgttttt ggtcatggca ttagtcctat
gcactggtac 120cagcagaaac caggacagcc acccaaactc ctcatctatc
gtgcatccaa cctaaaattt 180gggatccctg ccaggttcag tggcagtggg
tctaggacag acttcaccct caccattaat 240cctgtggagg ctgatgatgt
tgcaacctat tactgtcagc aaagtaatga atatcctcgg 300acgttcggtg
gaggcaccaa gctggagatc aaa 333115119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
115Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Glu Phe Thr Phe Ser Asp
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Tyr Ile Ser Ser Asp Gly Asp Ser Thr Tyr Tyr Pro
Asp Asn Ile 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Val Gln Met Ser Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Glu Ile Arg Leu Arg Gly Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115116119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 116Glu Val Lys Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Glu Phe Thr Phe Ser Asp Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Ser Asp Gly
Asp Ser Ile Tyr Tyr Pro Asp Asn Ile 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Phe Cys 85 90 95Ala Arg Glu
Ile Arg Leu Arg Gly Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110Thr
Thr Val Thr Val Ser Ser 115117119PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 117Glu Val Lys Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Glu Phe Thr Phe Ser Asp Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr
Ile Ser Ser Asp Gly Asp Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Phe Cys
85 90 95Ala Arg Glu Ile Arg Leu Arg Gly Tyr Phe Asp Val Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 115118119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
118Glu Val Lys Leu Val Glu Ser Gly Glu Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Ser Asp
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu
Trp Val 35 40 45Ala Tyr Ile Ser Ser Asp Gly Asp Ser Thr Tyr Tyr Pro
Asp Asn Ile 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Gly Ser Leu Arg Ala Glu Asp
Met Ala Met Tyr Phe Cys 85 90 95Ala Arg Glu Ile Arg Leu Arg Gly Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115119111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 119Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Arg Lys Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ser
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110120111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 120Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110121111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 121Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala
Ser Asn Arg Lys Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ser
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110122111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 122Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110123111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 123Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Ile Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110124111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 124Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Ile Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110125111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 125Asp Ile Val Leu Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Met His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110126111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 126Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110127111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 127Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Phe Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala
Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110128111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 128Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Ser Leu Lys Phe Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala
Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110129111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 129Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala
Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110130111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 130Asp Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ser
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110131111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 131Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Arg Lys Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Pro Val Gln Ser
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110132111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 132Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Ile Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95Glu Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110133111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 133Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Lys Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly
Ser Gly Ser Arg Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu
Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110134111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 134Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110135111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 135Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Glu Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110136111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 136Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110137111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Ile Phe Gly His 20 25 30Gly Ile Ser Pro Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Lys Ser Gly Ile Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Tyr Pro
Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11013815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Arg Ala Ser Glu Ser Val Phe Gly His Gly Ile
Ser Pro Leu His1 5 10 151397PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 139Arg Ala Ser Asn Arg Lys
Thr1 514015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Arg Ala Ser Glu Ser Ile Phe Gly His Gly Ile
Ser Pro Met His1 5 10 151417PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 141Arg Ala Ser Ser Leu Lys
Phe1 51427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Arg Ala Ser Asn Leu Lys Ser1
51437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Arg Ala Ser Asn Leu Lys Thr1
514415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Arg Ala Ser Glu Ser Ile Phe Gly His Gly Ile
Ser Pro Leu His1 5 10 1514517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 145Tyr Ile Ser Ser Asp Gly
Asp Ser Ile Tyr Tyr Pro Asp Asn Ile Lys1 5 10
15Gly14617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Tyr Ile Ser Ser Asp Gly Asp Ser Thr Tyr Tyr
Pro Asp Ser Val Lys1 5 10 15Gly14740PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
147Gly Gly Gly Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile1
5 10 15Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu
Arg 20 25 30Asn Leu Val Pro Arg Thr Glu Ser 35 4014829PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 148Gly
Gly Gly Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala1 5 10
15Lys Lys Val Val Thr Thr Ala Lys Lys Pro Leu Ile Val 20
2514918PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gly Gly Gly Val Asn Trp Lys Lys Ile Leu Gly
Lys Ile Ile Lys Val1 5 10 15Val Lys15032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
150Gly Gly Gly Thr Leu Ile Ser Trp Ile Lys Asn Lys Arg Lys Gln Arg1
5 10 15Pro Arg Val Ser Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg
Arg 20 25 3015129PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 151Gly Gly Gly Gly Ile Gly Ala Ile Leu
Lys Val Leu Ala Thr Gly Leu1 5 10 15Pro Thr Leu Ile Ser Trp Ile Lys
Asn Lys Arg Lys Gln 20 2515235PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 152Gly Gly Gly Gly Leu
Arg Lys Arg Leu Arg Lys Phe Arg Asn Lys Ile1 5 10 15Lys Glu Lys Leu
Lys Lys Ile Gly Gln Lys Ile Gln Gly Leu Leu Pro 20 25 30Lys Leu Ala
3515331PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 153Gly Gly Gly Gly Leu Arg Arg Leu Gly Arg
Lys Ile Ala His Gly Ile1 5 10 15Lys Lys Tyr Gly Pro Thr Ile Leu Arg
Ile Ile Arg Ile Ala Gly 20 25 3015432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
154Gly Gly Gly Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly1
5 10 15Val Lys Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Lys Lys Tyr
Gly 20 25 3015537PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 155Gly Gly Gly Lys Arg Phe Lys Lys
Phe Phe Lys Lys Leu Lys Asn Ser1 5 10 15Val Lys Lys Arg Ala Lys Lys
Phe Phe Lys Lys Pro Arg Val Ile Gly 20 25 30Val Ser Ile Pro Phe
3515633PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 156Gly Gly Gly Lys Phe Phe Arg Lys Leu Lys
Lys Ser Val Lys Lys Arg1 5 10 15Ala Lys Glu Phe Phe Lys Lys Pro Arg
Val Ile Gly Val Ser Ile Pro 20 25 30Phe15730PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
157Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1
5 10 15Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 20
25 3015822PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 158Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys
Cys Gly Lys Ala Cys1 5 10 15Val Lys Ile Leu Lys Lys
2015930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 159Gly Gly Gly Gly Arg Cys Lys Arg Phe Arg
Lys Lys Cys Lys Lys Leu1 5 10 15Phe Lys Lys Leu Ser Pro Val Ile Pro
Leu Leu His Leu Gly 20 25 301605PRTStaphylococcus
aureusMOD_RES(3)..(3)Any amino acid 160Leu Pro Xaa Thr Gly1
51615PRTStaphylococcus pyogenesMOD_RES(3)..(3)Any amino acid 161Leu
Pro Xaa Thr Ala1 51625PRTStaphylococcus sp. 162Leu Pro Glu Thr Gly1
516325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 163Gly Gly Gly Gly Ile Gly Lys Phe Leu Lys Lys
Ala Lys Lys Phe Gly1 5 10 15Lys Ala Phe Val Lys Ile Leu Lys Lys 20
2516411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(4)..(4)D-DabMOD_RES(5)..(6)DabMISC_FEATURE(5)..(11)Cy-
clicMOD_RES(7)..(7)D-LeuMOD_RES(9)..(10)Dab 164Gly Gly Gly Xaa Xaa
Xaa Leu Phe Xaa Xaa Leu1 5 1016517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(9)..(9)Ile or
ThrMOD_RES(14)..(14)Asn or SerMOD_RES(15)..(15)Ile or Val 165Tyr
Ile Ser Ser Asp Gly Asp Ser Xaa Tyr Tyr Pro Asp Xaa Xaa Lys1 5 10
15Gly16615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(6)..(6)Val or IleMOD_RES(14)..(14)Met or
Leu 166Arg Ala Ser Glu Ser Xaa Phe Gly His Gly Ile Ser Pro Xaa His1
5 10 151677PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Asn or SerMOD_RES(5)..(5)Leu or
ArgMOD_RES(7)..(7)Phe, Thr or Ser 167Arg Ala Ser Xaa Xaa Lys Xaa1
5
* * * * *
References