FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patent (Amendment) Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION:
FC VARIANTS AND PREPARATION THEREOF
APPLICANT:
NAME : ZYDUS LIFESCIENCES LIMITED
NATIONALITY : Indian
ADDRESS : Zydus Lifesciences Limited, Zydus Corporate Park, Scheme No. 63,
Survey No. 536, Khoraj (Gandhinagar), Nr. Vaishnodevi Circle, Ahmedabad,
Gandhinagar, India 382481
PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which
it has to be performed.
Field of the invention
The present invention relates to Fc variants protein and preparation thereof.
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Background of the invention
Neonatal Fc receptor (FcRn) is structurally homologous to the Major Histocompatibility
Complex (MHC) Class I heterodimer molecule consisting of a type I transmembrane heavy
chain that non-covalently associates with the soluble light chain, β2-microglobulin (β2m).
Monoclonal antibody based antagonists have been developed to inhibit the endogenous
IgG–FcRn interaction. One approach is the use of monoclonal antibodies directed against
FcRn working via the classical antibody: antigen binding mechanism. Examples of such
antibodies are M281 and UCB7665 which bind human FcRn to inhibit IgG binding to FcRn
and thereby accelerate the clearance of endogenous IgG. A mean reduction of endogenous
IgG ranging between 25-80 % was observed with M281 in a dose dependent manner.
Moreover, single doses of M281 at 30 mg / kg or 60 mg / kg maintained serum IgG at 50 %
of baseline or below for 18 and 27 days, respectively 1. In healthy subjects, IgG
concentrations in the serum were reduced by up to 50 % at a single IV dose of 7 mg / kg of
UCB7665. The observed reductions in serum IgG concentrations with UCB7665 persisted
for weeks, with maximal reductions achieved by days 7 to 10 and thereafter gradually
returning to baseline by day 57 2.
The present invention provides an improved FcRn binder that solves various unmet needs
such as, minimal toxicity, downregulation of the FcRn activity, increasing the circulating
half-life of a variety of drugs, targeting of drugs and / or vaccines to specific cell types etc.
The FcRn binder of the present invention can be used in treating a wide variety of diseases.
WO2021234655 discloses various FcRn binding molecules. The present invention provides
therapeutically effective FcRn binding molecule(s) that may be used in the treatment of
autoimmune diseases.
Summary of the invention
The present invention provides novel Fc variants with altered binding affinity towards
FcRn, preferably higher binding affinity towards FcRn. In one aspect, the Fc variant(s)
according to the present invention comprises L234A, L235A, T307N, V308P, L309Y,
P329G, H433R, N434W amino acid substitutions. In another aspect, the Fc variant(s)
according to the present invention comprises T307N, V308P, L309Y, H433R, N434W,
M252Y, S254T, T256E amino acid substitutions. In one more aspect, the Fc variant(s) of
the present invention comprises T307N, V308P, L309Y, H433R, N434W, M252Y, S254T,
T256E, L234A, L235A, P329G amino acid substitutions. The amino acid substitutions as
mentioned herein the present invention are as per EU numbering system (EU position). The
said Fc variants can be used to develop drug compositions with FcRn antagonist function
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or increased circulating half-life, or for targeting to specific cells and / or tissues. The present
invention also provides methods of making the novel Fc variants of the present invention.
The Fc variants according to the present invention are further used in the preparation of a
drug either for treating diseases where activity of FcRn is detrimental or for increasing the
circulating half-life of a drug or for targeting the drug to certain cells or tissues.
Brief description of figures
Figure 1: It depicts a map of the vector used for the generation of Fc monomers and Fc
dimers of the present invention.
Figure 2: It depicts a map of the vector used for the generation of monoclonal antibodies
containing Fc monomers of the present invention.
Figure 3: It depicts the relative percentage IgG concentration with respect to pre-dose levels
across the various treatment groups - A3H11A4.0, A3H11A4.1, B14H11A4.0 and
B14H11A4.1.
Figure-4: It depicts the relative percentage IgG concentration with respect to pre-dose levels
across the antibody molecules of the various treatment groups - A3H11A4.0, A3H11A4.2,
A3H11A4.3.
Figure-5: It depicts the relative percentage IgG concentration with respect to pre-dose levels
across the candidates of the various treatment groups - H11A4.1, H11A4.0, H11A4.2,
H11A4.3 Fc.
Figure-6: It depicts the relative percentage IgG concentration across study time points with
respect to pre-dose levels for the H11A4.1 Fc candidate.
Figure-7: It depicts the relative percentage IgG concentration with respect to pre-dose levels
across study time points for the A3H11A4.1 antibody candidate.
Figure-8: It depicts the relative percentage IgG concentration with respect to pre-dose levels
across study time points for the B14H11A4.1 antibody candidate.
Definitions
Unless otherwise defined herein, scientific and technical terms used in connection with the
present invention shall have the meanings that are commonly understood by those of
ordinary skill in the art. The meaning and scope of the terms should be clear, however, in
the event of any latent ambiguity, definitions provided herein take precedent over any
dictionary or extrinsic definition. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the singular. Generally,
nomenclature used in connection with, and techniques of, cell and tissue culture, molecular
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biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and
hybridization described herein are those well known and commonly used in the art.
As used herein, the term “Fc domain” refers to the portion of a single immunoglobulin heavy
chain beginning in the hinge region just upstream of the papain cleavage site and ending at
the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a
portion of a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain,
and a CH3 domain.
The term “amino acid modification” as used herein is an amino acid substitution, insertion,
and / or deletion in a polypeptide sequence.
The term “amino acid substitution” or “substitution” as used herein is the replacement of an
amino acid at a particular position in a parent polypeptide sequence with another amino
acid. For example, the substitution T307N refers to a variant(s) polypeptide, in this case an
Fc variant(s), in which the threonine at position 307 is replaced with asparagine, where ‘T’
& ‘N’ represent the one letter code for threonine and asparagine respectively.
The term “antigen-binding molecule” according to the present invention refers to a protein
that can bind to target antigen and comprising an “FcRn binding domain”. FcRn Binding
domain according to the present invention is present in Fc protein, preferably, present in Fc
variant(s) of the present invention. Preferred “antigen-binding molecule” according to the
present invention may include antibody or peptibody or fusion proteins or monomer or
dimer comprising Fc variant(s) of the present invention.
The term "FcRn antagonist" refers to any agent comprising an Fc region that binds
specifically to FcRn through the Fc region and inhibits the binding of immunoglobulin to
FcRn. The terms, “FcRn antagonist”, “Fc variant(s) protein” or “Fc protein variant(s)” or
“Fc variant(s), “Fc variant(s) containing molecule”, “Fc variant(s) containing protein” etc.
can be used interchangeably herein the present invention.
The term “antibody” as used herein includes whole antibodies and any antigen- binding
fragments (i.e., “antigen-binding portion”) that include an Fc portion. An “antibody” refers
to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-
connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain
constant region (Fc). The heavy chain constant region is comprised of three domains, CH1,
CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated
herein as VL) and a light chain constant region. The light chain constant region is comprised
of one domain, CL. The VH and VL regions can be further subdivided into regions of hyper
variability, termed complementarity determining regions (CDR), interspersed with regions
that are more conserved, termed framework regions (FR). Each VH and VL is composed of
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three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the
heavy and light chains contain a binding domain that interacts with an antigen. The constant
regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g., effector cells such as, NK cells,
T cells, macrophages and dendritic cells etc.) and the first component (C1q) of the classical
complement system.
The term “operatively linked” is intended to mean that a gene is ligated into a vector such
that transcriptional and translational control sequences within the vector serve their intended
function of regulating the transcription and translation of such gene.
The term “ka” is the association rate of interaction between two molecules, whereas the term
“kd” is the dissociation rate of the interaction between two molecules. The term “KD” is an
affinity rate constant, which is obtained from the ratio of kd to ka. It can be measured by
using surface plasma resonance method which is well known in the art.
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.
The term “bispecific antibody” refers to a homogeneous antibody population involved in
the highly specific recognition and binding of two different antigenic determinants, or
epitopes.
The term “recombinant antibody”, as used herein, includes all antibodies that are prepared,
expressed, created or isolated by recombinant means. In certain embodiments, however,
such recombinant antibodies can be obtained by in vitro mutagenesis and thus the amino
acid sequences of the VH and VL regions of the recombinant antibodies as described herein
are sequences that may not naturally exist within the human antibody germline repertoire in
vivo.
The term “Fc” fragment, whose name reflects its ability to crystallize readily. The crystal
structure of the human IgG Fc region has been determined.3 In human IgG molecules, the
Fc region can be separated from the rest of the molecule by digestion with papain which
cleaves N-terminus to Cys226. The Fc region is central to the effector functions of
antibodies.
The term “Fc protein” as used herein refers to the portion of a single immunoglobulin heavy
chain beginning in the hinge region just upstream of the papain cleavage site and ending at
the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a
portion of a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain,
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and a CH3 domain. The Fc protein comprises of two Fc chains attached to each other via
two or more disulphide bridges. The Fc protein includes but not limited to “Fc protein
variant(s) known in the art.”
The term “Fc variant(s) containing protein” as used herein refers to any molecule that
comprises an Fc variant(s) of the present invention. Preferably, Fc variant(s) containing
protein includes antibody, fusion protein and peptibody. Antibody, fusion protein and
peptibody as referred herein include any approved or under clinical trial or under pre-clinical
trial antibody, fusion protein and peptibody.
The term “Fc variant(s) containing molecule” as used herein refers to any molecule that
comprises an Fc variant(s) of the present invention. It can be fusion product where Fc
variant(s) of the present invention is linked or conjugated to a drug, wherein drug can be
small peptide or receptor or toxin or any chemical molecule.
The terms, “Fc variant(s) protein” or “Fc protein variant(s)” or “Fc variant(s)” as used herein
refer to an Fc protein that differs from that of a wild type Fc protein by virtue of at least one
amino acid modification in wild type Fc. Fc variant(s) may refer to the Fc variant(s) itself,
a composition comprising the Fc variant(s), or the amino acid sequence that encodes it.
Preferably, Fc variant(s) includes monomer, dimer or multimer. Preferably, the Fc variant(s)
has at least one amino acid modification compared to the wild type Fc protein e.g. from
about one to about eleven amino acid modifications, and preferably from about one to about
eight amino acid modifications compared to the wild type Fc protein . The term “Fc
variant(s) protein known in the art” or “Fc protein variant(s) known in the art” or “Fc
variant(s) known in the art” as used herein the present invention refers to “Fc variant(s)
protein” or “Fc protein variant(s)s” or “Fc variant(s)” disclosed in the public domain before
the priority date of this patent specification, preferably one or more “Fc variant(s) protein”
or “Fc protein variant(s)” or “Fc variant(s)” disclosed in the PCT/IB2021/054423
(WO2021234655).
The term “EU position” as used herein refers to the amino acid position in the EU numbering
convention for the Fc region as described in reference.4
The term “CH1 domain” as used herein refers to the first (amino terminal) constant region
domain of an immunoglobulin heavy chain that extends from about EU positions 118-215.
The CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an
immunoglobulin heavy chain molecule, and does not form a part of the Fc region of an
immunoglobulin heavy chain.
The term “hinge region” as used herein refers to the portion of a heavy chain molecule that
joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25
residues and is flexible, thus allowing the two N-terminal antigen binding regions to move
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independently. Hinge regions can be subdivided into three distinct domains: upper, middle,
and lower hinge domains.5 The Fc variant(s) of the present invention can include all or a
portion of a hinge region.
The term “CH2 domain” as used herein refers to the portion of a heavy chain
immunoglobulin molecule that extends from about EU positions 231-340.
The term “CH3 domain” as used herein refers to the portion of a heavy chain
immunoglobulin molecule that extends approximately 110 residues from N-terminus of the
CH2 domain, e.g., from about position 341-447 (EU numbering system).
The term “about” as used herein refers to variation in the numerical quantity that can occur,
for example, through typical measuring and handling procedures used for making
compounds, compositions, concentrates or formulations; through inadvertent error in these
procedures; through differences in the manufacture, source, or purity of starting materials
or ingredients used to carry out the methods, and other similar considerations.
The term “FcRn” or “neonatal Fc Receptor” as used herein is a protein that binds the IgG
antibody Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from
any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is
known in the art, the functional FcRn protein comprises two polypeptides, often referred to
as the heavy chain and light chain. The light chain is beta-2-microglobulin and the heavy
chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn protein
refers to the complex of FcRn heavy chain with beta-2-microglobulin.
The term “wild-type Fc” as used herein is an unmodified Fc polypeptide that is subsequently
modified to generate a variant(s). The wild-type Fc may be a naturally occurring
polypeptide, or recombinant version of a naturally occurring polypeptide. The wild-type Fc
may refer to the unmodified Fc polypeptide itself, compositions that comprise the
unmodified Fc polypeptide, or the amino acid sequence that encodes it.
The term “position” as used herein is a location in the sequence of a protein. Positions may
be numbered sequentially or according to an established format, for example the EU
numbering system or EU numbering or EU index or Kabat numbering. For example,
position 105 is the location of the 105th amino acid residue from the N terminus of the heavy
or light chain in the human antibody IgG1.
The terms “patient” and “subject” are used interchangeably and are used in their
conventional sense to refer to a living organism suffering from or prone to a condition that
can be prevented or treated by administration of a composition of the present invention, and
includes both humans and non-human animals. Examples of subjects include, but are not
limited to, humans, chimpanzees and other apes and monkey species; farm animals such as
cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory
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animals including rodents such as mice, rats and guinea pigs; birds, including domestic,
wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese,
and the like. The term does not denote a particular age. Thus, adult, juvenile and new born
individuals are of interest.
The term “residue” as used herein is a position in a protein and its associated amino acid
identity. For example, Threonine 307 (also referred to as Thr307, also referred to as T307)
is a residue in the human antibody IgG1.
The term “immunoconjugate” or “conjugate” as used herein refers to a compound or a
derivative thereof that is linked to a cell binding agent (i.e., antibody or Fc variant(s) as
described herein) and is defined by a generic formula: A-L-D, wherein A = cell binding
agent or antibody or Fc variant(s) of the present invention, L = linker and D = Drug (toxin
or pharmaceutical drug). Immunoconjugates can also be defined by the generic formula in
reverse order: D-L-A.
The term “linker” is any chemical moiety that is capable of linking a compound, usually a
drug, such as a maytansinoid or auristatin, to a cell-binding agent (i.e., antibody or Fc
variant(s) as described herein) in a stable, covalent manner. Linkers can be susceptible to or
be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-
induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions
under which the compound and/or the antibody remains active. Suitable linkers are well
known in the art and include, for example, disulfide groups, thioether groups, acid labile
groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also
include charged linkers, and hydrophilic forms thereof as described herein and known in the
art.
The term, “antagonist” is one which inhibits or reduces biological activity of the antigen
such as FcRn, that it binds. In a certain embodiment antagonist substantially or completely
inhibits the biological activity of the antigen, such as FcRn. Desirably, the biological activity
is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.
The term “treatment” or “therapeutics” as used herein, refers to any treatment of a disease
in a mammal, particularly in a human. It includes: (a) preventing the disease from occurring
in a subject which may be predisposed to the disease or at risk of acquiring the disease but
has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its
development; and (c) relieving the disease, i.e., causing regression of the disease.
The term “wild type” or “WT” herein is an amino acid sequence or a nucleotide sequence
that is found in nature, including allelic variations. A WT protein has an amino acid
sequence or a nucleotide sequence that has not been intentionally modified.
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The term “non-naturally encoded amino acid” refers to an amino acid that is not one of the
common amino acids or natural amino acids or pyrolysine, pyrroline-carboxy-lysine, or
selenocysteine. Other terms that may be used synonymously with the term “non-naturally
encoded amino acid” are “non-natural amino acid”, “unnatural amino acid”, “non-naturally-
occurring amino acid” and variously hyphenated and non-hyphenated versions thereof. The
term “non-naturally encoded amino acid” also includes, but is not limited to, amino acids
that occur by modification (e.g. post-translational modifications) of a naturally encoded
amino acid (including but not limited to, the 20 common amino acids or pyrrolysine,
pyrroline-carboxy-lysine, and selenocysteine) but are not themselves naturally incorporated
into a growing polypeptide chain by the translation complex. Examples of such non-
naturally-occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-
serine, N-acetylglucosaminyl-L-threonine and Ophosphotyrosine.
Table 1: Abbreviations of amino acid as used in the present application
Full Name Abbreviation
(3 Letter)
Abbreviation
(1 Letter)
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartate Asp D
Cysteine Cys C
Glutamate Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
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Tyrosine Tyr Y
Valine Val V
Other abbreviations used in the present patent application:
%: percentage
°C: degree Celsius
μg: microgram
μL: microliter
A: Adenine
ANCA: Anti-Neutrophilic Cytoplasmic Autoantibody.
APC: Antigen presenting cell
ADC: Antibody-drug conjugate
C: Cytosine
CFU: Colony forming unit
CHO: Chinese Hamster Ovary
DNA: Deoxyribonucleic acid
EC50: 50% of maximal effective concentration of any drug
EC75: 75% of maximal effective concentration of any drug
ELISA: Enzyme linked immunosorbent assay
Fc: Fragment crystallizable
FcGRT: Fc fragment of IgG receptor and transporter
FcRn: Neonatal Fc receptor
G: Guanine
h: Hour
H2SO4: Sulfuric acid
HRP: Horseradish peroxidase
IC: Immune-complex
IgG: Immunoglobulin G
IPTG: Isopropyl β-D-1-thiogalactopyranoside
ITP: Acute immune thrombocytopenia
IVIg: Intravenous immunoglobulin
ka / kassoc: Association constant
kd / kdissoc: Dissociation constant
KD: Equilibrium dissociation constant
M: molar
mg: milligram
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MgCl2: Magnesium chloride
min: Minute
mL: milliliter
mM: millimolar
mAb: Antibody
MOI: Multiplicity of infection
NaCl: Sodium chloride
NaHCO3: Sodium bicarbonate
ng: nanogram
nm: nanometer
OD: Optical density
OPD: o-phenylenediamine dihydrochloride
PBS: Phosphate buffered saline
PBST: Phosphate buffered saline with tween 0.05%
PEG: Polyethylene glycol
PFU: Plaque-forming unit
rFcRn: Recombinant neonatal Fc receptor
rhFcRn: Recombinant human neonatal Fc receptor
rpm: Revolution per minute
s: Second
SCIg: Subcutaneous immunoglobulin
SEQ / seq: Sequence
SPR: Surface plasmon resonance
T: Thymine
YT media: Yeast extract tryptone media
Embodiments of the invention
The disclosure of the present invention provides novel FcRn antagonist(s) comprising Fc
variant(s) as disclose herein the description.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprises L234A, L235A, T307N,
V308P, L309Y, P329G, H433R, N434W amino acid substitutions.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprises T307N, V308P, L309Y,
H433R, N434W, M252Y, S254T, T256E amino acid substitutions.
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In one of the preferred embodiment, the Fc variant(s) of the present invention comprises
T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G
amino acid substitutions.
In one embodiment, the Fc variant(s) of the present invention comprises single chain of Fc
domain or double chain of Fc domain, preferably two identical chain of Fc domain or
multiple chain of Fc domain.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention binds with high affinity to human
FcRn.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention reduces serum IgG level in subjects
undertaking the therapy.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention binds with higher affinity to human
FcRn relative to the wild-type Fc protein.
In one of the embodiments, the Fc variant(s) of the present invention has a KD of 10-8 M or
less, preferably 10-9 M or less, more preferably 10-10 M or less for FcRn. KD value is a
measurement of the binding affinity of the drug candidate to FcRn. In one of the
embodiments, the Fc variant(s) of the present invention has a KD in the range between 10-11
M and 10-8 M for FcRn.
In one embodiment, the amino acid sequence of Fc variant(s) is of the IgGl, IgG2, IgG3,
IgG4 or IgG2/G4 isotype, preferably the IgGl isotype.
In one of the embodiments, the Fc variant(s) of the present invention does not significantly
interfere with the binding property of FcRn to albumin.
In one of the embodiments, the Fc variant(s) of the present invention cross-reacts with FcRn
of mouse, monkey and human.
In one of the preferred embodiments, the Fc variant(s) of the present invention has higher
binding affinity towards human FcRn relative to the wild-type Fc protein or one or more
protein variant(s) known in the art.
In one of the preferred embodiments, the Fc variant(s) of the present invention has higher
binding affinity towards mouse FcRn relative to the wild-type Fc protein or one or more
protein variant(s) known in the art.
In one of the preferred embodiments, the Fc variant(s) of the present invention has higher
binding affinity towards monkey FcRn relative to the wild-type Fc protein or one or more
protein variant(s) known in the art.
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In one of the embodiments, Fc variant(s) or Fc variant(s) containing protein or Fc variant(s)
containing molecule of the present invention has a higher half-life in subject relative one or
more protein variant(s) known in the art. In certain embodiments, polyethylene glycol or
human serum albumin may be linked to the Fc variant(s) of the present invention to further
increase the half-life of the said Fc variant(s). In an alternate embodiment, the Fc variant(s)
of the present invention may include mutations to increase its half-life in the subject. In
another alternate embodiment, the Fc variant(s) of the present invention can be expressed
in monomer, dimer or multimer form to increase half-life by increasing the molecular size
and affinity through higher avidity of the Fc variant(s). The term “multimer form” as used
herein refers to a form of protein, which includes more than one unit of the protein,
preferably the Fc proteins of the present invention. For example, it can be a monomer, dimer,
trimer, tetramer, pentamer, hexamer, etc. For example, monomer Fc protein has two binding
sites for FcRn. In a similar manner, Fc dimer according to the present invention has four
binding sites for FcRn.
In another embodiment, the Fc variant(s) of the present invention is able to bind to the mouse
and monkey FcRn enabling ease of drug development by providing relevant animal models
for pharmacology and toxicology studies.
In one of the embodiments, the present invention provides a composition comprising Fc
variant(s) protein or Fc variant(s) containing protein or Fc variant(s) containing molecule
and an acceptable carrier.
In another embodiment, the Fc variant(s) protein or Fc variant(s) containing protein of the
present invention can be used for the treatment of disease such as infections, various
cancers, autoimmune disorders and the like.
In certain embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc variant(s)
containing molecule of the present invention comprises amino acid sequences selected from
SEQ ID No. 1, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 20 and SEQ ID No. 38.
In another embodiment, the Fc variant(s) of the present invention can be used to make full-
length antibody, where antibody has any of the Fc variant(s) of the present invention in
place of conventional Fc region. Said antibody can be modified form of an already approved
or a newly discovered antibody, where the modified form of the existing antibody has Fc
variant(s) of the present invention. Such antibodies may be selected and/or designed in such
a way that it does not bind to any target in the treated subject via its paratopes.
In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 3 and SEQ ID No. 5.
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In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 7 and SEQ ID No. 9.
In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 22 and SEQ ID No. 24.
In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 26 and SEQ ID No. 28.
In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 30 and SEQ ID No. 32.
In one of the embodiments, the Fc variant(s) or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention comprise an amino acid sequences
set forth in SEQ ID No. 34 and SEQ ID No. 36.
In one embodiment, the Fc variant(s) of the present invention can be used to make a drug
with higher half-life, where Fc variant(s) of the present invention is either fused or linked
or conjugated to drug of which original half-life in circulation is low.
In another embodiment, the Fc variant(s) of the present invention can be used to increase
the half-life of an ADC by incorporating the novel variant(s) into the antibody of the ADC,
thereby reducing its off-target toxicity by increasing its re-circulation through FcRn
receptor.
In one of the embodiments, Fc variant(s) of the present invention can be fused with any drug
to improve its half-life and in-vivo stability.
In one of the embodiments, Fc variant(s) of the present invention can be used to replace
albumin in an albumin fusion drug, to improve the pharmacokinetics of the drug.
In one of the embodiments, mutations in the Fc variant(s) of the present invention can be
used to improve the affinity of a full antibody towards FcRn at about pH 6, to increase its
circulating half-life by reducing catabolism.
In one of the embodiments, the Fc variant(s) containing antibody molecule can be used as a
scavenger to remove pathogenic proteins or toxins from the circulation such as TNF alpha,
VEGF etc.
In one of the embodiments, the Fc variant(s) containing antibody molecule or protein can
be used as a trapper to catch desired proteins, peptides, carbohydrates or drugs from the
environment and bring inside the FcRn expressing target cell.
15
In one of the embodiments, Fc variant(s) of the present invention can be used to develop
monomeric Fc fusion protein in fusion with any therapeutic peptide to improve its
penetration in a tissue along with retaining its FcRn binding activity.
In one of the embodiments, Fc variant(s) of the present invention when present in a
functional antibody molecule can help in the transcytosis of target antigens bound by the
said antibody thereby increasing their tissue penetration. Such target antigens being
peptides, proteins, carbohydrates, lipids or such molecules that need to be delivered into
tissues to mediate their effect.
In one of the embodiments, Fc variant(s) of the present invention blocks IgG binding site
on FcRn and compete with endogenous IgG for FcRn leading to higher clearance of
endogenous IgG.
In one embodiment, the improved Fc variant(s) of the present invention produces lesser
morbidity as compared to the one or more Fc variant(s) disclosed in WO2021234655
(PCT/IB2021/054423). In a preferred embodiment, the improved Fc variant(s) of the
present invention causes no morbidity when given in the same dose.
In one of the embodiments, the Fc variant(s) of the present invention binds human FcRn,
mouse FcRn and monkey FcRn in-vitro. The advantageous characteristics of cross-
reactivity with monkey and mouse FcRn shows that the Fc variant(s) of the present
invention can be tested in non-human primates and mice and the resulting data can be very
useful in predicting their pharmacokinetics, pharmacodynamics and toxicity profiles in
humans.
In one of the embodiments, the Fc variant(s) of the present invention in fusion with suitable
immunogens / antigens can improve the delivery of the said antigen to the FcRn expressing
APC s such as dendritic cells etc., leading to improved immune response.
Detailed description of the invention
The present invention relates to improved Fc variant(s) or Fc variant(s) containing protein
that comprises L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino
acid substitutions. In one of the embodiments, the Fc variant(s) protein or Fc variant(s)
containing protein or Fc variant(s) containing molecule of the present invention comprises
T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions.
In further embodiments, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention further comprises T307N, V308P,
L309Y, H433R, N434W, M252Y, S254T, T256E , L234A, L235A, P329G amino acid
substitutions. The amino acid substitutions in Fc variant(s) of the present invention are as
per EU numbering system. The amino acid substitutions of the present invention are in Fc
16
region. The Fc domain comprises at least a portion of a hinge (e.g., upper, middle, and/or
lower hinge region) domain, a CH2 domain, and a CH3 domain).
In one embodiment, the improved Fc variant(s) of the present invention produces lesser
morbidity as compared to the one or more Fc variant(s) disclosed in WO2021234655
(PCT/IB2021/054423). At some doses, intravenous administration of the Fc variant(s) of
WO2021234655 (PCT/IB2021/054423) led to severe morbidity in animals. The inventors
of the present invention surprisingly found that introducing additional mutations (amino
acid substitutions) in the Fc region as mentioned herein the present invention help resolves
this problem. The improved Fc variant(s) of the present invention causes significantly lesser
morbidity as compared to the one or more Fc variant(s) disclosed in WO2021234655
(PCT/IB2021/054423) when administered at the same dose. Preferably, the improved Fc
variant(s) of the present invention causes no morbidity. In some embodiments, the improved
Fc variant(s) of the present invention has higher endogenous IgG reduction as compared to
the one or more Fc variant(s) as disclosed in WO2021234655 (PCT/IB2021/054423). Thus,
the improved Fc variant(s) of the present invention has lesser morbidity, preferably no
morbidity as compared to the one or more Fc variant(s) as disclosed in WO2021234655
(PCT/IB2021/054423). In addition, the Fc variant(s) of the present invention reduces serum
IgG and thus retains its efficacy. In one of the embodiment, the Fc variant(s) of the present
invention reduces serum IgG.
In one more embodiment, the improved Fc variant(s) of the present invention has significant
binding to the FcRn receptors of human, mouse and other non-human primates such as
monkey. The advantageous characteristics of cross-reactivity with monkey and mouse FcRn
and its resulting data may be useful in predicting drug pharmacokinetics,
pharmacodynamics and toxicology profiles in humans.
In one embodiment, the Fc variant(s) protein or Fc variant(s) containing protein or Fc
variant(s) containing molecule of the present invention binds with high affinity to human
FcRn. The Fc variant(s) of the present invention has lower KD value (i.e. higher binding)
than wild type Fc to FcRn at about pH 6.0. The Fc variant(s) of the present invention has a
KD of 10-8 M or less, preferably 10-9 M or less, more preferably 10-10 M or less for FcRn at
about pH 6.0. In one of the embodiments, the Fc variant(s) of the present invention has KD
in the range between 10-11 M to 10-8 M for FcRn. KD value is a measurement of the binding
affinity of a binder (in this case Fc variant(s)) towards its target antigen (in this case FcRn).
Amino acid sequences of the Fc variant(s)
The amino acid sequences and nucleotide sequences of Fc variant(s) of the present invention
have been mentioned herein in table 2.
17
Table 2: List of amino acid and nucleotide sequences
SEQ ID No. Sequence Listing
1. Amino acid sequence of Fc variant(s) H11A4.1
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLNPYHQDW
LNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLSLSPGK
2. Light Chain Nucleotide Sequence of A3 H11A4.1 (mAb)
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAATCTGCTTTGACTCAGCCTGCCTCCGTGTCTGG
CTCTCCTGGCCAGTCTATCACCATCTCTTGTACCGGCACCTCTAGC
GACGTCGGCGGCTACAATTACGTGTCCTGGTATCAGCAGCACCCC
GGCAAGGCTCCCAAGCTGATGATCTACGATGTGTCCAAGCGGCCC
TCCGGCGTGTCCAACAGATTCTCTGGCTCTAAGTCTGGCAACACC
GCCAGCCTGACAATCTCTGGCCTGCAGTCTGAGGACGAGGCCGAC
TACTACTGCAACTCCCTGACCTCCATCTCCACCTGGGTTTTCGGCG
GAGGCACAAAGCTGACAGTGCTGGGCCAGCCTAAGGCCAATCCT
ACCGTGACACTGTTCCCTCCATCCTCCGAGGAACTGCAGGCTAAC
AAGGCTACCCTCGTGTGCCTGATCTCCGATTTTTACCCTGGCGCTG
TGACCGTGGCTTGGAAGGCTGATGGATCTCCTGTGAAGGCCGGCG
TGGAAACCACCAAGCCTAGCAAGCAGTCCAACAACAAATACGCC
GCCTCCTCCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCAC
CGGTCCTACTCTTGCCAAGTGACCCATGAGGGCTCCACCGTGGAA
AAGACAGTGGCCCCTACCGAGTGCTCTTAATGA
18
3. Light Chain Amino Acid Sequence of A3 H11A4.1 (mAb)
MGWSCIILFLVATATGVHSQSALTQPASVSGSPGQSITISCTGTSSDVG
GYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLT
ISGLQSEDEADYYCNSLTSISTWVFGGGTKLTVLGQPKANPTVTLFPP
SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK
QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
4. Heavy Chain Nucleotide Sequence of A3 H11A4.1 (mAb):
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGG
TGCAGCCTGGAAGAAGTCTGAGACTGTCTTGTGCCGCCTCCGGCT
TCACCTTCTCCAACTACGCTATGTACTGGGTCCGACAGGCCCCTG
GCAAAGGACTGGAATGGGTCGCCGTGATCTCCTACGACGGCTCCA
ACAAGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTC
GGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGA
GAACCGAGGACACCGCCGTGTACTACTGTGCCTCTGGCTCTGACT
ACGGCGACTACCTGCTGGTGTATTGGGGACAGGGAACCCTGGTCA
CCGTGTCCTCTGCTTCTACAAAGGGCCCCTCTGTGTTCCCACTGGC
TCCTAGCTCTAAGTCCACCTCTGGTGGAACCGCTGCTCTGGGCTGT
CTGGTCAAGGATTACTTCCCTGAGCCTGTGACCGTGTCTTGGAAC
TCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGC
AGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTC
TAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCTTCCAATACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCT
GCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTG
CTGGCGGACCCTCCGTTTTCCTGTTTCCACCTAAGCCTAAGGACAC
CCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGA
TGTGTCTCACGAGGACCCAGAAGTGAAGTTTAATTGGTACGTGGA
CGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAAC
AGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTACC
ACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC
AACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCTAAGGCT
AAGGGCCAGCCTCGCGAGCCTCAGGTTTACACACTGCCTCCATCT
CGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTT
AAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAAT
GGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGAC
19
TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAG
TCTCGGTGGCAGCAGGGCAACGTGTTCTCTTGTAGTGTGATGCAC
GAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGAGC
CCCGGCAAGTAATGA
20
5. Heavy Chain Amino Acid Sequence of A3 H11A4.1(mAb):
MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFT
FSNYAMYWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRD
NSKNTLYLQMNSLRTEDTAVYYCASGSDYGDYLLVYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLS
LSPGK
6. Light Chain Nucleotide Sequence of B14 H11A4.1(mAb):
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTGATATCCAGATGACCCAGTCTCCTTCCAGCCTGTC
TGCCTCTGTGGGCGACAGAGTGACAATCACCTGTCAGGCCAGCCA
GGACATCACCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCA
AGGCCCCTAAGCTGCTGATCTACGCCGCCTCTAATCTGGAAACAG
GCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCAC
CTTTACCATCTCTGGCCTGCAGCCTGAGGATATCGCCACCTACTAC
TGCCAGCAGTACGACAACCTGCCTCTGACCTTTGGCGGAGGCACC
AAGGTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATC
TTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTC
GTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAG
TGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCT
GTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCC
ACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTA
CGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAA
GTCTTTCAACAGAGGCGAGTGCTAATGA
7. Light Chain Amino Acid Sequence of B14 H11A4.1(mAb)
MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCQASQDI
TNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISG
LQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
21
8. Heavy Chain Nucleotide Sequence of B14 H11A4.1(mAb):
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAGTCTGGCGGCGGACTTG
TGAAGCCTGGTGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTT
CACCTTCTCCGACTACTACATGTCCTGGATCAGACAGGCCCCTGG
CAAAGGACTGGAATGGGTGTCCTACATCACCTACTCCGGCTCCAC
CATCTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCCAG
AGACAACGCCAAGTCCAGCCTGTACCTGCAGATGAACTCTCTGAG
AGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGACAGAGGCA
CCACAATGGTGCCCTTCGATTATTGGGGCCAGGGCACACTGGTCA
CCGTGTCCTCTGCTTCTACAAAGGGCCCCTCTGTGTTCCCACTGGC
TCCTAGCTCTAAGTCCACCTCTGGTGGAACCGCTGCTCTGGGCTGT
CTGGTCAAGGATTACTTCCCTGAGCCTGTGACCGTGTCTTGGAAC
TCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGC
AGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTC
TAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCTTCCAATACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCT
GCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTG
CTGGCGGACCCTCCGTTTTCCTGTTTCCACCTAAGCCTAAGGACAC
CCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGA
TGTGTCTCACGAGGACCCAGAAGTGAAGTTTAATTGGTACGTGGA
CGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAAC
AGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTACC
ACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC
AACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCTAAGGCT
AAGGGCCAGCCTCGCGAGCCTCAGGTTTACACACTGCCTCCATCT
CGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTT
AAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAAT
GGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGAC
TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAG
TCTCGGTGGCAGCAGGGCAACGTGTTCTCTTGTAGTGTGATGCAC
GAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGAGC
CCCGGCAAGTAATGA
22
9. Heavy Chain Amino Acid Sequence of B14 H11A4.1(mAb):
MGWSCIILFLVATATGVHSQVQLVESGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNA
KSSLYLQMNSLRAEDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLSLS
PGK
10. Nucleotide Sequence of H11A4.1 (Monomer)
ATGGGCTGGTCTTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCCTCTGACAAGACCCACACCTGTCCTCCATGTCCTG
CTCCAGAAGCTGCTGGCGGACCCTCCGTTTTCCTGTTTCCACCTAA
GCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTG
CGTGGTGGTGGATGTGTCTCACGAGGACCCAGAAGTGAAGTTTAA
TTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC
CTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGC
TGAACCCTTACCACCAGGATTGGCTGAACGGCAAAGAGTACAAG
TGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGAC
CATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAGGTTTACAC
ACTGCCTCCATCTCGGGAAGAGATGACCAAGAACCAGGTGTCCCT
GACCTGCCTGGTTAAGGGCTTCTACCCTTCCGATATCGCCGTGGA
ATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACAACCC
CTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCT
GACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCTTG
TAGTGTGATGCACGAGGCCCTGAGATGGCACTACACACAGAAGT
CCCTGTCTCTGAGCCCCGGCAAGTAATGA
23
11. Amino Acid Sequence of H11A4.1 (Monomer)
MGWSCIILFLVATATGVHSSDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALR
WHYTQKSLSLSPGK
12. Nucleotide Sequence of W11A4.1 (Dimer)
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCCTCTGATAAGACACACACCTGTCCTCCATGTCCTG
CTCCAGAAGCTGCTGGCGGACCCTCTGTGTTCCTGTTTCCTCCAAA
GCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTG
CGTGGTGGTGGATGTGTCTCACGAGGACCCAGAAGTGAAGTTCAA
TTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC
CTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGC
TGAACCCTTACCACCAGGACTGGCTGAACGGCAAAGAGTACAAG
TGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGAC
CATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAGGTTTACAC
ACTGCCTCCATCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCT
GACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGA
ATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACAC
CTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCT
GACCGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTG
CTCTGTGATGCACGAGGCCCTGAGATGGCACTACACACAGAAGTC
CCTGTCTCTGTCCCCTGGCAAAGGCGGAGGTTCTGGCGGTGGCTC
TGGTGGCGGATCTGGCGGAGGATCTTCAGGCGGTGGTTCTAGCTC
CGACAAGACCCACACAAGCCCACCTTCTCCAGCACCTGAAGCAGC
AGGCGGCCCATCAGTCTTTCTGTTCCCACCTAAGCCAAAGGATAC
ACTCATGATCAGCAGAACACCCGAAGTGACATGTGTCGTCGTGGA
CGTGTCCCATGAAGATCCCGAAGTCAAGTTTAATTGGTATGTCGA
TGGCGTCGAGGTCCACAATGCTAAGACAAAGCCACGGGAAGAAC
AGTATAACAGCACCTACCGGGTCGTGTCTGTCCTGAATCCATATC
ATCAGGATTGGCTCAATGGGAAAGAATACAAATGTAAAGTCTCTA
ACAAGGCTCTCGGAGCCCCAATCGAGAAAACCATCAGCAAGGCC
AAGGGACAGCCCAGAGAACCCCAGGTGTACACTCTGCCACCTAG
24
CAGGGACGAACTCACCAAGAATCAAGTGTCTCTCACATGCCTCGT
GAAGGGGTTTTACCCCAGCGACATTGCCGTCGAGTGGGAGTCTAA
CGGACAACCCGAAAACAATTATAAGACAACCCCACCAGTGCTGG
ATAGCGACGGCTCATTTTTCTTGTATAGCAAGCTCACCGTCGATA
AGAGCCGGTGGCAACAGGGAAATGTGTTCAGCTGCAGCGTGATG
CATGAAGCTCTGCGGTGGCATTATACCCAGAAAAGCCTGAGCCTG
TCTCCAGGCAAGTAATGA
13. Amino Acid Sequence of W11A4.1 (Dimer)
MGWSCIILFLVATATGVHSSDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALR
WHYTQKSLSLSPGKGGGSGGGSGGGSGGGSSGGGSSSDKTHTSPPSP
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLNPYHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALRWHYTQKSLSLSPGK
14. Glycine-Serine linker
GGGSGGGSGGGSGGGSSGGGSS
15. Heavy Chain Amino Acid Sequence of A3H11A4.0 (mAb)
MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFT
FSNYAMYWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRD
NSKNTLYLQMNSLRTEDTAVYYCASGSDYGDYLLVYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLNPYHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
25
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLS
LSPGK
16. Light Chain Amino Acid Sequence of A3H11A4.0 (mAb)
MGWSCIILFLVATATGVHSQSALTQPASVSGSPGQSITISCTGTSSDVG
GYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLT
ISGLQSEDEADYYCNSLTSISTWVFGGGTKLTVLGQPKANPTVTLFPP
SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK
QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
17. Heavy Chain Amino Acid Sequence of B14H11A4.0 (mAb)
MGWSCIILFLVATATGVHSQVQLVESGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNA
KSSLYLQMNSLRAEDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LNPYHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLSLSP
GK
18. Light Chain Amino Acid Sequence of B14H11A4.0 (mAb)
MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCQASQDI
TNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISG
LQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
19. Nucleotide Sequence of H11A4.2:
ATGGGCTGGTCTTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCCTCTGATAAGACACACACCTGTCCTCCATGTCCTG
CTCCAGAACTGCTCGGCGGACCCTCTGTGTTCCTGTTTCCTCCAAA
GCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACCTG
TGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAA
TTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC
CTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGC
TGAACCCTTACCACCAGGATTGGCTGAACGGCAAAGAGTACAAG
26
TGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACC
ATCTCCAAGGCTAAGGGCCAGCCTCGGGAACCTCAGGTTTACACA
CTGCCTCCATCTCGGGACGAGCTGACCAAGAATCAGGTGTCCCTG
ACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAA
TGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACACC
TCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTG
ACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGT
TCTGTGATGCACGAGGCCCTGAGATGGCACTACACACAGAAGTCC
CTGTCTCTGAGCCCCGGCAAGTAATGA
20 Amino Acid Sequence of H11A4.2:
MGWSCIILFLVATATGVHSSDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLNPYHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALR
WHYTQKSLSLSPGK
21 Light Chain Nucleotide Sequence of A3H11A4.2:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAATCTGCTTTGACTCAGCCTGCCTCCGTGTCTGG
CTCTCCTGGCCAGTCTATCACCATCTCTTGTACCGGCACCTCTAGC
GACGTCGGCGGCTACAATTACGTGTCCTGGTATCAGCAGCACCCC
GGCAAGGCTCCCAAGCTGATGATCTACGATGTGTCCAAGCGGCCC
TCCGGCGTGTCCAACAGATTCTCTGGCTCTAAGTCTGGCAACACC
GCCAGCCTGACAATCTCTGGCCTGCAGTCTGAGGACGAGGCCGAC
TACTACTGCAACTCCCTGACCTCCATCTCCACCTGGGTTTTCGGCG
GAGGCACAAAGCTGACAGTGCTGGGCCAGCCTAAGGCCAATCCT
ACCGTGACACTGTTCCCTCCATCCTCCGAGGAACTGCAGGCTAAC
AAGGCTACCCTCGTGTGCCTGATCTCCGATTTTTACCCTGGCGCTG
TGACCGTGGCTTGGAAGGCTGATGGATCTCCTGTGAAGGCCGGCG
TGGAAACCACCAAGCCTAGCAAGCAGTCCAACAACAAATACGCC
GCCTCCTCCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCAC
CGGTCCTACTCTTGCCAAGTGACCCATGAGGGCTCCACCGTGGAA
AAGACAGTGGCCCCTACCGAGTGCTCTTAATGA
22 Light Chain Amino Acid Sequence of A3H11A4.2:
27
MGWSCIILFLVATATGVHSQSALTQPASVSGSPGQSITISCTGTSSDVG
GYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLT
ISGLQSEDEADYYCNSLTSISTWVFGGGTKLTVLGQPKANPTVTLFPP
SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK
QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
23 Heavy Chain Nucleotide Sequence of A3H11A4.2:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGG
TGCAGCCTGGAAGAAGTCTGAGACTGTCTTGTGCCGCCTCCGGCT
TCACCTTCTCCAACTACGCTATGTACTGGGTCCGACAGGCCCCTG
GCAAAGGACTGGAATGGGTCGCCGTGATCTCCTACGACGGCTCCA
ACAAGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTC
GGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGA
GAACCGAGGACACCGCCGTGTACTACTGTGCCTCTGGCTCTGACT
ACGGCGACTACCTGCTGGTGTATTGGGGACAGGGAACCCTGGTCA
CCGTGTCCTCTGCTTCTACAAAGGGCCCCTCTGTGTTCCCACTGGC
TCCTAGCTCTAAGTCCACCTCTGGTGGAACCGCTGCTCTGGGCTGT
CTGGTCAAGGATTACTTCCCTGAGCCTGTGACCGTGTCTTGGAAC
TCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGC
AGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTC
TAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCTTCCAATACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCT
GCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGC
TCGGCGGACCCTCTGTGTTCCTGTTTCCTCCAAAGCCTAAGGACA
CCCTGTACATCACCCGCGAGCCTGAAGTGACCTGTGTGGTGGTGG
ATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGG
ACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAA
CAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTAC
CACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTC
CAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGC
TAAGGGCCAGCCTCGGGAACCTCAGGTTTACACACTGCCTCCATC
TCGGGAAGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTGGT
CAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAA
TGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGA
CTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAA
28
GTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCA
CGAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGAG
CCCCGGCAAGTAATGA
24 Heavy Chain Amino Acid Sequence of A3H11A4.2:
MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFT
FSNYAMYWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRD
NSKNTLYLQMNSLRTEDTAVYYCASGSDYGDYLLVYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLNPYHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLS
LSPGK
25 Light Chain Nucleotide Sequence of B14H11A4.2:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTGATATCCAGATGACCCAGTCTCCTTCCAGCCTGTC
TGCCTCTGTGGGCGACAGAGTGACAATCACCTGTCAGGCCAGCCA
GGACATCACCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCA
AGGCCCCTAAGCTGCTGATCTACGCCGCCTCTAATCTGGAAACAG
GCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCAC
CTTTACCATCTCTGGCCTGCAGCCTGAGGATATCGCCACCTACTAC
TGCCAGCAGTACGACAACCTGCCTCTGACCTTTGGCGGAGGCACC
AAGGTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATC
TTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTC
GTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAG
TGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCT
GTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCC
ACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTA
CGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAA
GTCTTTCAACAGAGGCGAGTGCTAATGA
26 Light Chain Amino acid Sequence of B14H11A4.2:
MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCQASQDI
TNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISG
29
LQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
27 Heavy Chain Nucleotide Sequence of B14H11A4.2:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAGTCTGGCGGCGGACTTG
TGAAGCCTGGTGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTT
CACCTTCTCCGACTACTACATGTCCTGGATCAGACAGGCCCCTGG
CAAAGGACTGGAATGGGTGTCCTACATCACCTACTCCGGCTCCAC
CATCTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCCAG
AGACAACGCCAAGTCCAGCCTGTACCTGCAGATGAACTCTCTGAG
AGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGACAGAGGCA
CCACAATGGTGCCCTTCGATTATTGGGGCCAGGGCACACTGGTCA
CCGTGTCCTCTGCTTCTACAAAGGGCCCCTCTGTGTTCCCACTGGC
TCCTAGCTCTAAGTCCACCTCTGGTGGAACCGCTGCTCTGGGCTGT
CTGGTCAAGGATTACTTCCCTGAGCCTGTGACCGTGTCTTGGAAC
TCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGC
AGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTC
TAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCTTCCAATACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCT
GCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGC
TCGGCGGACCCTCTGTGTTCCTGTTTCCTCCAAAGCCTAAGGACA
CCCTGTACATCACCCGCGAGCCTGAAGTGACCTGTGTGGTGGTGG
ATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGG
ACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAA
CAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTAC
CACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTC
CAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGC
TAAGGGCCAGCCTCGGGAACCTCAGGTTTACACACTGCCTCCATC
TCGGGAAGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTGGT
CAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAA
TGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGA
CTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAA
GTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCA
30
CGAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGAG
CCCCGGCAAGTAATGA
28 Heavy Chain Amino Acid Sequence of B14H11A4.2:
MGWSCIILFLVATATGVHSQVQLVESGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNA
KSSLYLQMNSLRAEDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
NPYHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLSLSPG
K
29 Light Chain Nucleotide Sequence of A3 H11A4.3:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAATCTGCTTTGACTCAGCCTGCCTCCGTGTCTGG
CTCTCCTGGCCAGTCTATCACCATCTCTTGTACCGGCACCTCTAGC
GACGTCGGCGGCTACAATTACGTGTCCTGGTATCAGCAGCACCCC
GGCAAGGCTCCCAAGCTGATGATCTACGATGTGTCCAAGCGGCCC
TCCGGCGTGTCCAACAGATTCTCTGGCTCTAAGTCTGGCAACACC
GCCAGCCTGACAATCTCTGGCCTGCAGTCTGAGGACGAGGCCGAC
TACTACTGCAACTCCCTGACCTCCATCTCCACCTGGGTTTTCGGCG
GAGGCACAAAGCTGACAGTGCTGGGCCAGCCTAAGGCCAATCCT
ACCGTGACACTGTTCCCTCCATCCTCCGAGGAACTGCAGGCTAAC
AAGGCTACCCTCGTGTGCCTGATCTCCGATTTTTACCCTGGCGCTG
TGACCGTGGCTTGGAAGGCTGATGGATCTCCTGTGAAGGCCGGCG
TGGAAACCACCAAGCCTAGCAAGCAGTCCAACAACAAATACGCC
GCCTCCTCCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCAC
CGGTCCTACTCTTGCCAAGTGACCCATGAGGGCTCCACCGTGGAA
AAGACAGTGGCCCCTACCGAGTGCTCTTAATGA
30 Light Chain Amino Acid Sequence of A3 H11A4.3:
MGWSCIILFLVATATGVHSQSALTQPASVSGSPGQSITISCTGTSSDVG
GYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLT
ISGLQSEDEADYYCNSLTSISTWVFGGGTKLTVLGQPKANPTVTLFPP
31
SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK
QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
31 Heavy Chain Nucleotide Sequence of A3H11A4.3:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGG
TGCAGCCTGGAAGAAGTCTGAGACTGTCTTGTGCCGCCTCCGGCT
TCACCTTCTCCAACTACGCTATGTACTGGGTCCGACAGGCCCCTG
GCAAAGGACTGGAATGGGTCGCCGTGATCTCCTACGACGGCTCCA
ACAAGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTC
GGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGA
GAACCGAGGACACCGCCGTGTACTACTGTGCCTCTGGCTCTGACT
ACGGCGACTACCTGCTGGTGTATTGGGGACAGGGAACCCTGGTCA
CCGTGTCCTCTGCTTCTACCAAGGGACCCTCTGTGTTCCCACTGGC
TCCTAGCTCTAAGTCCACCTCTGGTGGAACCGCTGCTCTGGGCTGT
CTGGTCAAGGATTACTTCCCTGAGCCTGTGACCGTGTCTTGGAAC
TCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGC
AGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTC
TAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCTTCCAATACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCT
GCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTG
CTGGCGGTCCCTCCGTTTTCCTGTTTCCACCTAAGCCTAAGGACAC
CCTGTACATCACTCGGGAGCCTGAAGTGACCTGCGTGGTGGTGGA
TGTGTCTCACGAGGACCCAGAAGTGAAGTTTAATTGGTACGTGGA
CGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAAC
AGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTACC
ACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC
AACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCTAAGGCT
AAGGGCCAGCCTCGCGAGCCTCAGGTTTACACACTGCCTCCATCT
CGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTT
AAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAAT
GGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGAC
TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAG
TCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCAC
GAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGAGC
CCCGGCAAGTGATGA
32
32 Heavy Chain Amino Acid Sequence of A3H11A4.3:
MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFT
FSNYAMYWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRD
NSKNTLYLQMNSLRTEDTAVYYCASGSDYGDYLLVYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLS
LSPGK
33 Light Chain Nucleotide Sequence of B14 H11A4.3:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTGATATCCAGATGACCCAGTCTCCTTCCAGCCTGTC
TGCCTCTGTGGGCGACAGAGTGACAATCACCTGTCAGGCCAGCCA
GGACATCACCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCA
AGGCCCCTAAGCTGCTGATCTACGCCGCCTCTAATCTGGAAACAG
GCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCAC
CTTTACCATCTCTGGCCTGCAGCCTGAGGATATCGCCACCTACTAC
TGCCAGCAGTACGACAACCTGCCTCTGACCTTTGGCGGAGGCACC
AAGGTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATC
TTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTC
GTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAG
TGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCT
GTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCC
ACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTA
CGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAA
GTCTTTCAACAGAGGCGAGTGCTAATGA
34 Light Chain Amino Acid Sequence of B14 H11A4.3:
MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCQASQDI
TNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISG
LQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
33
35 Heavy Chain Nucleotide Sequence of B14 H11A4.3:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCTCAGGTTCAATTGGTTGAGTCTGGCGGCGGACTTG
TGAAGCCTGGTGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTT
CACCTTCTCCGACTACTACATGTCCTGGATCAGACAGGCCCCTGG
CAAAGGACTGGAATGGGTGTCCTACATCACCTACTCCGGCTCCAC
CATCTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCCAG
AGACAACGCCAAGTCCAGCCTGTACCTGCAGATGAACTCTCTGAG
AGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGACAGAGGCA
CCACAATGGTGCCCTTCGATTATTGGGGCCAGGGCACACTGGTCA
CCGTGTCCTCTGCTTCTACCAAGGGACCCTCTGTGTTCCCTCTGGC
TCCTTCCAGCAAGTCTACCTCTGGTGGAACCGCTGCTCTGGGCTG
CCTGGTCAAGGATTACTTTCCTGAGCCTGTGACCGTGTCTTGGAA
CTCTGGTGCTCTGACCTCTGGCGTGCACACCTTTCCAGCTGTGCTG
CAGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTT
CTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACA
AGCCTTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCC
TGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCT
GCTGGCGGTCCCTCCGTTTTCCTGTTTCCACCTAAGCCTAAGGACA
CCCTGTACATCACTCGGGAGCCTGAAGTGACCTGCGTGGTGGTGG
ATGTGTCTCACGAGGACCCAGAAGTGAAGTTTAATTGGTACGTGG
ACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAA
CAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGAACCCTTAC
CACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTC
CAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCTAAGGC
TAAGGGCCAGCCTCGCGAGCCTCAGGTTTACACACTGCCTCCATC
TCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGT
GAAGGGATTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCTAA
CGGCCAGCCAGAGAACAACTACAAGACAACCCCTCCTGTGCTGG
ACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACA
AGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGC
ACGAGGCCCTGAGATGGCACTACACACAGAAGTCCCTGTCTCTGA
GCCCCGGCAAGTGATGA
36 Heavy Chain Amino Acid Sequence of B14 H11A4.3:
MGWSCIILFLVATATGVHSQVQLVESGGGLVKPGGSLRLSCAASGFT
34
FSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNA
KSSLYLQMNSLRAEDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRWHYTQKSLSLS
PGK
37 Nucleotide Sequence of H11A4.3:
ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCG
GCGTGCACTCCTCTGATAAGACACACACCTGTCCTCCATGTCCTG
CTCCAGAAGCTGCTGGCGGACCCTCTGTGTTCCTGTTTCCTCCAAA
GCCTAAGGACACCCTGTACATCACTCGGGAGCCTGAAGTGACCTG
CGTGGTGGTGGATGTGTCTCACGAGGACCCAGAAGTGAAGTTCAA
TTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC
CTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGC
TGAACCCTTACCACCAGGACTGGCTGAACGGCAAAGAGTACAAG
TGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGAC
CATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAGGTTTACAC
ACTGCCTCCATCTCGGGAAGAGATGACCAAGAACCAGGTGTCCCT
GACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGA
ATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACAC
CTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCT
GACCGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTG
CTCTGTGATGCACGAGGCCCTGAGATGGCACTACACACAGAAGTC
CCTGTCTCTGTCCCCTGGCAAGTGATGA
38 Amino Acid Sequence of H11A4.3:
MGWSCIILFLVATATGVHSSDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLNPYHQDWLNGKEYKCKVSNKALGAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALR
WHYTQKSLSLSPGK
35
The Fc variant(s) of the present invention can be present in IgG1, IgG2, IgG3, IgG4 or
IgG2/G4 isotypes, preferably the IgGl isotype. Fc variant(s) constructs of the IgG4 isotype
may further contain single amino acid substitution (i.e., S228P) in the hinge region of Fc
variant(s) to reduce the disruption of disulphide bond between two Fc chains 6.
In one of the aspects, the Fc variant(s) of the present invention may include one or more
non-natural amino acid(s) at one or more position. Introduction of such non-natural amino
acids in peptides are well known to the skilled person. Methods of making and introducing
a non-naturally-occurring amino acid into a protein are known from for example, US Patent
Nos. 7,083,970 and 7,524,647. In one of the aspects, the Fc variant(s) according to the
present invention has higher FcRn binding and higher half-life relative one or more protein
variant(s) known in the art.
Nucleic acid molecules encoding Fc variant(s), vectors and host cells
In one embodiment, the present invention provides nucleic acid molecules encoding the Fc
variant(s) or Fc variant(s) containing protein containing suitable expression vector
comprising such nucleic acids and suitable host cell comprising such nucleic acid molecules
encoding Fc variant(s) from the expression vectors. Suitable vectors to produce Fc variant(s)
or Fc variant(s) containing protein of the present invention by recombinant method are
known in the art for a person skilled in the art. Examples of such known vectors are
described in patent documents WO 2007 / 017903, WO 2012 / 046255, which are
incorporated herein. The host cell according to the present invention may be a prokaryotic
cell such as E-coli or a eukaryotic cell such as CHO cell.
Pharmaceutical compositions
A pharmaceutical composition, containing Fc variant(s) or Fc variant containing protein(s)
or Fc variant containing molecule(s) of the present invention, formulated together with
suitable pharmaceutically acceptable carrier(s) can be developed following suitable
methods well known in the art. Such compositions may also include one or a combination
of (e.g., two or more different) Fc variant(s) or Fc variant(s) containing protein, or
immunoconjugate or bispecific molecule(s) of the invention. For example, a pharmaceutical
composition of the invention can comprise a combination of Fc variant(s) or Fc variant(s)
containing protein(s) and antibody (or immunoconjugate or bispecific) that binds to
different epitopes on one or more of the target antigen.
Therapeutic Uses
36
The Fc variant(s) or Fc variant(s) containing protein or Fc variant containing molecule(s)
can be used for the treatment of diseases involving therapeutic methods that require binding
of the drug to the FcRn.
In one embodiment of the present invention, the Fc variant(s) or Fc variant(s) containing
protein of the present invention can be used to inhibit FcRn in vivo in subjects, including
humans, suffering from disease such as, but not limited to, autoimmune disease and
inflammations where the binding leads to a reduction in endogenous IgG levels.
In one embodiment of the present invention, the Fc variant(s) or Fc variant(s) containing
protein(s) of the present invention can be used to increase the circulating half of the
therapeutic drug that it is a part of making the drug available in the body for a long period
of time leading to reduced dosing frequency, increased compliance and better therapeutic
effect such as in cancer, infectious disease, autoimmune disease etc.
In one of the embodiment of the present invention, the Fc variant(s) or Fc variant(s)
containing protein(s) of the present invention can be used to target an antigen to dendritic
cells or other APCs in order to make a better prophylactic or therapeutic vaccine such as in
infectious disease or cancer and such.
In certain embodiment, the Fc variant(s) or Fc variant(s) containing protein(s) or Fc
variant(s) containing molecule(s) used to treat disease selected from Autoimmune hemolytic
anemia, Pernicious anemia, Idiopathic thrombocytopenic purpura, Goodpasture's syndrome,
Bullous pemphigoid, Pemphigous vulgaris, Hashimoto's thyroiditis, ANCA vasculitis,
Insulin-dependent diabetes mellitus (IDDM), Graves disease, Myasthenia gravis, CIDP
Diabetes, Antianemics (Beta-Thalassemia), Hematopoietic Agents, Ophthalmic Drugs,
Cold agglutinin disease, Antiphospholipid syndrome (APS), Lupus nephritis, Bone
Diseases, Neurological Genetic Disorders, Complement 3 glomerulopathy, Age-related
macular degeneration, Immunoglobulin A Nephropathy (IgAN) disease Diabetic
Retinopathy, Macular diseases, Non-Small Cell Lung Cancer Therapy, Ocular Genetic
Disorders, Hemophilia B, Agents for (Coagulation factor IX deficiency) Cardiovascular
Diseases, Type 2 Diabetes, Immunosuppressants, Rheumatoid Arthritis, Treatment of
Transplant Rejection, Brain Cancer, Breast Cancer , Colorectal Cancer , Diabetic
Retinopathy, Digestive / Gastrointestinal Cancer, Endocrine Cancer , Female Reproductive
System Cancer , Gastric Cancer, Genitourinary Cancer, Macular diseases, Melanoma ,
Multiple Myeloma , Myelodysplastic Syndrome Therapy, Myeloid Leukemia Therapy,
Non-Hodgkin's Lymphoma Therapy, Non-Small Cell Lung Cancer , Ovarian Cancer ,
Pancreatic Cancer , Prostate Cancer Therapy, Renal Cancer Therapy, Retinopathy, Aplastic
anemia, Analgesic Drugs, atypical hemolytic uremic syndrome Hematologic Genetic
Disorders, Multisystem Genetic Disorders, Osteoarthritis, Scleroderma, Treatment of
37
Autoimmune Diseases, Treatment of Gout, Urticaria, Ankylosing Spondylitis, Asthma
Therapy, Dermatologic Drugs, Idiopathic Inflammatory Myopathies, Immunosuppressants,
Inflammatory Bowel Disease, Interstitial Lung Diseases, Multiple Myeloma Therapy,
Multiple Sclerosis, Nephritis, Psoriatic Arthritis, Systemic Lupus Erythematosus, Agents
for Acute Alcoholic Hepatitis, Alzheimer's Dementia, Antiallergy / Antiasthmatic Drugs,
Antiarthritic Drugs, Antipsoriatics, Breast Cancer Therapy, Cancer Associated Disorders,
Dermatologic Drugs, Heart Failure Therapy, Lymphoma Therapy, Nephritis, Neurologic
Drugs (Miscellaneous), Respiratory Disorders, Therapy of Inborn Errors of Metabolism.
Preferred embodiments of the present invention comprises
1. An FcRn-antagonists that comprises of a Fc variant, wherein the Fc variant comprises the
L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino acid substitutions
as per EU numbering.
2. The FcRn-antagonists as claimed in claim 1 are monoclonal antibodies, monomers, dimers
or multimers.
3. The FcRn-antagonist as claimed in claim 2 is monomer having amino acid sequence as set
forth in SEQ ID NO. 1 or SEQ ID NO. 11.
4. The FcRn-antagonist as claimed in 2 is monoclonal antibody comprising amino acid
sequences as set forth in SEQ ID No. 3 and SEQ ID No. 5.
5. The FcRn-antagonist as claimed in 2 is monoclonal antibody comprising amino acid
sequences as set forth in SEQ ID No. 7 and SEQ ID No. 9.
6. The FcRn-antagonist as claimed in claim 2 is dimer having an amino acid sequence as set
forth in SEQ ID NO. 13.
7. The FcRn-antagonists as claimed in claim 1 further comprising M252Y, S254T, T256E
amino acid substitutions as per EU numbering.
8. The FcRn-antagonists as claimed in claim 7 are monoclonal antibodies, monomers, dimers
or multimers.
9. The FcRn-antagonist as claimed in claim 8 is monomer having an amino acid sequence as
set forth in SEQ ID NO. 38.
10. The FcRn-antagonist as claimed in 8 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 30 and SEQ ID No. 32.
11. The FcRn-antagonist as claimed in 8 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 34 and SEQ ID No. 36.
12. An FcRn-antagonists that comprise of an Fc variant, wherein the Fc variant comprises the
T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions
as per EU numbering.
38
13. The FcRn-antagonists as claimed in claim 12 are monoclonal antibodies, monomers, dimers
or multimers.
14. The FcRn-antagonist as claimed in claim 13 is monomer having amino acid sequence as set
forth in SEQ ID NO. 20.
15. The FcRn-antagonist as claimed in claim 13 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 22 and SEQ ID No. 24.
16. The FcRn-antagonist as claimed in 13 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 26 and SEQ ID No. 28.
17. The FcRn-antagonists as claimed in claims 1-16, having a KD in the range between 10-11 M
to 10-8 M for FcRn.
18. The FcRn-antagonists as claimed in claim 1-17, those cross-react with FcRn of mouse,
monkey and human.
19. A composition comprising FcRn-antagonist as claimed in any of claims 1-18 and an
acceptable carrier.
Examples
The following examples are put forth so as to provide those of ordinary skills in the art with
a disclosure and description of how the methods and Fc variant(s)s claimed herein are
performed. They are intended to purely exemplify only and are not intended to limit the
scope of the disclosure. Other Fc variant(s)s of the present invention can be developed using
methods as described in provided examples with suitable modifications as are well known
in the art and well within the capabilities of a skilled person. Such modifications are known
to the persons skilled in the art.
Example 1: Construction of full-length antibody with Fc variant(s)s,
pDGV/A3H11A4.1 (mAb) and pDGV/B14H11A4.1 (mAb).
An Fc variant(s) of the present invention, H11A4.1, was prepared as full antibody molecule
by fusing H11A4.1 fragment with heavy chain variable region (not cross reactive to human
/ mice / NHP proteins or antigens) of anti-Cov2 Spike monoclonal antibodies in
combination with light chain (not cross reactive to human / mice / NHP proteins or antigens)
of anti-Cov2 Spike monoclonal antibodies. (Patent publication no.WO2022101839;
PCT/IB2021/060495).
Briefly, Anti -CoV2 Spike monoclonal antibodies A3 (Sequence Id. Nos. 15 and 16) and
B14 (Sequence Id. Nos. 17 and 18) genes were chemically synthesized with L234A, L235A
and P329G mutations. Further, the H11A4 mutations, viz., T307N, V308P, L309Y, H433R
and N434W were incorporated into the A3 and B14 Fc regions using mutation inducing
39
oligonucleotides in sequential overlapping polymerase chain reactions. ApaI restriction site
at 5’ end and EcoR1 restriction site at 3’end were also incorporated using PCR to facilitate
cloning in frame with heavy chain variable region of pDGV/A3 (pDual cov2 A3) and
pDGV/ B14 (pDual cov2 B14) vectors.
The amplified ~ 1.0 kb heavy chain fragment (Constant region) was cloned in pDGV/A3
and pDGV/B14 plasmids digested with ApaI and EcoRI to replace the Fc region with the
H11A4.1 mutant Fc. After ligation, the reaction mixtures were transformed into E. coli
Top10F' cells and transformants were scored based on the carbenicillin resistance. The
sequences of pDGV/A3 H11A4.1 (mAb) (Sequence Id nos 2 and 3 for light chain; Sequence
Id nos. 4 and 5 for heavy chain) and pDGV/B14 H11A4.1 (mAb) (Sequence Id nos. 6 and
7 for light chain; Sequence Id. nos. 8 and 9 for heavy chain) plasmid vectors were confirmed
by restriction digestion and Sanger sequencing. The vectors were linearized with PvuI for
transfection in CHO-GS cells (Lonza).
Example 2: Construction of monomer pXC 17.4/H11A4.1 (Monomer) Fc variant(s).
The H11A4.1 (Monomer) variant(s) (Sequence Id. 10 and 11) comprises of the CH2-CH3
domains of IgG1 Fc with the following mutations - L234A, L235A T307N, V308P, L309Y,
P329G, H433R and N434W. A PCR was performed to incorporate a signal peptide upstream
to CH2 domain of pDGV/A3 H11A4.1 heavy chain constant region (Example 1). The PCR
also added a HindIII restriction site at 5’ end and an EcoRI restriction site at 3’ end to
facilitate cloning in pXC17.4 vector (Lonza).
The HindIII and EcoRI digested PCR amplicon of ~ 0.75 kb was cloned in HindIII and
EcoRI digested pXC-17.4 vector (Lonza). After ligation, the reaction mixture was
transformed in E. coli Top10F’ and transformants were scored on the basis of carbenicillin
resistance. The clones were analysed by restriction digestion with HindIII and EcoRI. The
sequence of pXC17.4/H11A4.1 (Monomer) clone was confirmed by restriction digestion
and Sanger sequencing. The vector was linearized with PvuI for transfection into CHO-GS
cells (Lonza).
Example 3: Construction of multimer (dimer) pXC 17.4/W11A4.1 (dimer) Fc
variant(s).
In W11A4.1 variant(s) (dimer; SEQ ID NO. 13) an Fc variant(s) dimer was formed by
linking the two chains of Fc variant(s) monomer (H11A4.1) via Glycine-Serine linker (SEQ
ID NO. 14: GGGSGGGSGGGSGGGSSGGGSS) and further, to prevent self-dimerization
C226S and C229S mutations were incorporated in one of the two chains of the Fc variant(s).
A codon optimized gene for the Fc variant(s) dimer with L234A, L235A and P329G
40
mutations was chemically synthesized at GeneArt, Germany. W11A4 (dimer) gene was
isolated from pMA Geneart construct by restriction digestion with HindIII and EcoRI. The
HindIII and EcoRI digested Fc variant(s) W11A4.1 (dimer) of ~ 1.5 kb was ligated to
HindIII and EcoRI digested pXC-17.4 vector (Lonza). The ligation mixture was transformed
in E. coli Top10F’ and transformants were scored on the basis of carbenicillin resistance.
The clones were analysed by restriction digestion with HindIII and EcoRI. The sequence of
pXC17.4/W11A4.1 (dimer) clone (SEQ ID 12 and 13) was confirmed by restriction
digestion and Sanger sequencing. The vector was linearized with PvuI for transfection in
CHO-GS cells (Lonza).
Example 4: Construction of monomer pXC 17.4/H11A4.2 Fc variant(s).
Amino acid substitutions were carried out at position M252Y, S254T and T256E residues
of H11A4.0 to generate H11A4.2 variant(s) (SEQ ID. No. 19 and 20). Briefly sequential
overlapping PCR was carried out with pXC17.4/H11A4.0 as template to incorporate desired
substitutions at amino acid position 252, 254 and 256 (EU numbering). The ~ 0.75 kb PCR
amplicon was digested with HindIII and EcoRI and cloned in pXC-17.4 plasmid (Lonza).
After ligation, the reaction mixture was transformed into E. coli Top10F’ and transformants
were scored on the basis of carbenicillin resistance. The clones were analysed by restriction
digestion with HindIII and EcoRI. The sequence of pXC17.4/H11A4.2 plasmid was
confirmed by restriction digestion and Sanger sequencing. The vector was linearized with
PvuI for transfection into CHO-GS cells (Lonza).
Example 5: Construction of full-length antibodies with Fc variant(s)s,
pDGV/A3H11A4 .2(mAb) and pDGV/B14 H11A4.2 (mAb).
An Fc variant(s) of the present invention, H11A4.2 was incorporated into a full antibody
format replacing constant regions of A3H11A4.0 and B14H11A4.0 (Patent publication no.
WO2022101839; PCT/IB2021/060495) with H11A4.2 fragment.
Briefly, for construct preparation, an ApaI restriction site at 5’ end and an EcoR1 restriction
site at 3’end were incorporated into the H11A4.2 nucleotide sequence flanking the coding
regions for amino acids 20 -247 (SEQ ID No : 20) by PCR to facilitate cloning in frame
with heavy chain variable regions of pDGV/A3 and pDGV/ B14 vectors. The amplified ~
1.0 kb H11A4.2 fragment was digested with ApaI and EcoRI and cloned in pDGV/A3 and
pDGV/B14 plasmids. The transformants in E. coli Top10F’ were scored on the basis of
carbenicillin resistance. The sequences of pDGV/A3 H11A4.2 (SEQ ID nos. 21 and 22 for
light chain; SEQ ID. Nos. 23 and 24 for heavy chain) and pDGV/B14 H11A4.2 (SEQ ID
nos. 25 and 26 for light chain; SEQ ID. nos. 27 and 28 for heavy chain) plasmid vectors
41
were confirmed by restriction digestion and Sanger sequencing. The vectors were linearized
with PvuI for transfection in CHO-GS cells (Lonza).
Example 6: Construction of full-length antibodies with Fc variant(s)s,
pDGV/A3H11A4.3 and pDGV/B14H11A4.3
Amino acid substitutions were carried out at positions M252Y, S254T and T256E residues
ofH11A4.1 to generate the H11A4.3 variant(s). The pDGV/A3 and pDGV/B14 constructs
encoding H11A4.3 in mAb format were generated by sequential overlapping polymerase
chain reactions to incorporate desired substitutions at amino acid positions 252, 254 and
256 (EU numbering) . Subsequently, MluI and EcoR1 restriction sites were also added at 5’
end and at 3’end to facilitate the insertion of the PCR amplified fragment in to pDGV/A3and
pDGV/B14 vectors.
The PCR amplicon containing the desired substitutions was digested with MluI and EcoR1
restriction enzymes and subsequently cloned in pDGV/A3 and pDGV/B14 plasmids
digested with MluI and EcoRI respectively. After ligation, the reaction mixtures were
transformed into E. coli Top10F' cells and transformants were scored based on the
carbenicillin resistance. The sequences of pDGV/A3 H11A4.3 (SEQ ID nos. 29 and 30 for
light chain; SEQ ID nos. 31 and 32 for heavy chain) and pDGV/B14 H11A4.3 (SEQ ID
nos. 33 and 34 for light chain; SEQ ID. Nos. 35 and 36 for heavy chain) plasmid vectors
were confirmed by restriction digestion and Sanger sequencing. The vectors were linearized
with PvuI for transfection in CHO-GS cells (Lonza).
Example 7: Construction of monomer pXC 17.4/H11A4.3 variant(s).
A nucleotide sequence encoding the secretory signal peptide was incorporated upstream to
the nucleotide sequence encoding for H11A4.3 by PCR. This was followed by another PCR
to add a HindIII restriction site at 5’ end and a EcoRI restriction site at 3’ end to facilitate
cloning in pXC17.4 vector (Lonza). The resulting amplicon of ~ 0.75 kb (SEQ ID. 37)
encoding H11A4.3 (SEQ ID 38) was digested with HindIII and EcoRI and ligated with
HindIII and EcoRI digested pXC-17.4 vector (Lonza). After ligation, the reaction mixture
was transformed in E. coli Top10F’ and transformants were scored on the basis of
carbenicillin resistance. The clones were confirmed by restriction digestion with HindIII
and EcoRI. The sequence of pXC17.4/H11A4.3 construct was confirmed by restriction
digestion and Sanger sequencing. The vectors were linearized with PvuI for transfection in
CHO-GS cells (Lonza).
Example 8 Generation of Fc variant(s) proteins
42
All vector constructs as described in examples 1 to 7 were used for transfections. All the
plasmids generated in above examples 1 to 7 were linearized with PvuI restriction enzyme
and used for transfection. Chinese Hamster ovary (CHO) cells were used as host for
expression of recombinant proteins. CHO cells were seeded ~24 hours prior to transfection
at a density of 0.5 million / mL to have cells in the exponential phase. Transfections were
performed using Neon Transfection system (Invitrogen) by electroporation technique
following manufacturer’s instructions. Post-transfection, cells were plated in 24 well cell
culture plates containing 1 mL of pre-warmed ProCHO5 Serum Free media (Lonza,
Switzerland) containing 25μM MSX and incubated in a humidified incubator at 37 °C in
presence of 5 % CO2. The cell numbers of all the transfected pool(s) (i.e., a heterogeneous
mixture of differently expressing cells) were regularly monitored and regular media
exchanges were given. Once the cells recovered from transfection, cells were further
expanded sequentially to 6 well culture plates, T-flasks and culti-tubes (TPP).
Fed-batch cultures were performed for transfected pools of all Fc variant(s) candidates in
culti-tubes (TPP) for recombinant protein production. Cells were seeded at a density of
0.3×106 cells / mL in ActiPro production medium from HycloneTm, Cytiva. Culti tubes were
incubated in a humidifed Kuhner shaker at 37 °C temperature, 5 % CO2 level with shaking
speed of 230 RPM. A fixed daily feeding regimen was followed during the culture for all
the pools using chemically defined feeds from Hyclone, Cytiva. After 72 hours of culture,
feeding was initiated and continued till the batch was harvested.
Upon harvest, the candidate proteins were isolated from the culture supernatants by Protein
A affinity chromatography. These proteins were further tested in various in-vitro assays to
analyse their various properties.
Example 9: Determination of kinetic rate constants of binding of Fc variant(s) to
recombinant human neonatal Fc receptor (rhFcRn) at pH 6.0
The kinetic constants for the binding of Fc variant(s) candidates (expressed in example 8)
to recombinant human neonatal Fc receptor (rhFcRn) were determined by Surface Plasmon
Resonance-based measurement using the Biacore 8K+ instrument (Cytiva). The CM5
sensor chip was activated by immobilizing anti-Histidine antibody on its surface using the
standard amine coupling chemistry followed by blocking with 1 M ethanolamine solution,
pH 8.0 for 7 min. Recombinant human FcRn receptor (rhFcRn) at a concentration of 0.1
μg/mL was captured onto the anti-Histidine antibody immobilized chip surface by injecting
for 60 s at a flow rate of 10 μL/min. Five dilutions (in the range of 55 nM – 0.68 nM) of Fc
variant(s)s samples in dPBS, pH 6.0 containing 0.05 % (v/v) polysorbate 20, were injected
over the captured FcRn protein on chip at flow rate of 30 μL / min with an association time
43
of 60 s and dissociation time of 60 s at 25 °C. Following each sample run, the chip surface
was regenerated with 10 mM glycine solution, pH 1.5 for 30 s. The rate constant values, kd
(dissociation constant) and ka (association constant) were calculated using 1:1 binding
model in Biacore Insight Evaluation software (v 3.0.12.15655). The KD values (affinity
constant) were calculated as the ratio of kd to ka, i.e., KD = kd / ka. Affinity constants for
binding of Fc variant(s) candidates to rhFcRn were measured at pH 6.0 and are shown in
table 3.
Table 3: Kinetic rate constant FcRn binders at pH 6.0
Sample details ka (1/Ms) kd (1/s) KD (M)
H11A4.1 (Monomer) 4.89E+06 2.73E-03 5.59E-10
A3 H11A4.1 (mAb) 1.79E+06 3.44E-03 1.93E-09
B14 H11A4.1 (mAb) 1.37E+06 3.92E-03 2.85E-09
W11A4.1 (Dimer) 2.27E+06 3.81E-03 1.68E-09
H11A4.2 (monomer) 3.53E+06 1.03E-03 2.92E-10
A3 H11A4.2 (mAb) 1.43E+06 1.49E-03 1.04E-09
B14 H11A4.2 (mAb) 1.01E+06 1.75E-03 1.74E-09
H11A4.3 (monomer) 1.78E+06 5.13E-04 2.88E-10
A3 H11A4.3 (mAb) 1.25E+06 1.01E-03 8.03E-10
B14 H11A4.3 (mAb) 1.00E+06 1.23E-03 1.23E-09
Example 10: Determination of kinetic rate constants of binding of Fc variant(s) to
recombinant human neonatal Fc receptor (rhFcRn) at pH 7.4
Samples were analyzed for FcRn binding at pH 7.4 to check the effect of pH on their binding
to and dissociation from, FcRn. In this experiment, affinity constants for the binding of Fc
variant(s) candidates to recombinant human neonatal Fc receptor (rhFcRn) were determined
by Surface Plasmon Resonance-based measurement using the Biacore 8K+ instrument
(Cytiva). The rhFcRn was captured on anti-histidine antibody immobilized on CM5 sensor
chip as mentioned in Example 9. The running buffer having pH 7.4 was used to carry out
the kinetic measurements. To measure the affinity constant, five dilutions (in the range of
550 nM – 6.79 nM) of Fc variant(s)s were prepared and injected at a flow rate of 30 μL/min
with an association time of 60 s and dissociation time of 60 s. All the reactions were carried
at 25°C. Affinity constants for the binding of Fc variant(s) candidates to rhFcRn were
measured at pH 7.4 and are shown in table 4.
44
Table 4: Affinity constant of FcRn binders at pH 7.4
Sample details ka (1/Ms) kd (1/s) KD (M)
H11A4.1 (Monomer) 4.31E+06 6.54E-02 1.52E-08
A3 H11A4.1 (mAb) 1.61E+06 4.77E-02 2.96E-08
B14 H11A4.1 (mAb) 1.44E+06 4.91E-02 3.41E-08
H11A4.2 (monomer) 3.00E+06 1.05E-02 3.51E-09
A3 H11A4.2 (mAb) 8.79E+05 9.83E-03 1.12E-08
H11A4.3 (monomer) 1.25E+06 9.52E-03 7.62E-09
A3 H11A4.3 (mAb) 4.23E+05 7.97E-03 1.88E-08
B14 H11A4.3 (mAb) 6.44E+05 9.74E-03 1.51E-08
As it can be seen from tables 3 and 4, the Fc variant(s) of the present invention can bind to
FcRn with higher affinity at pH 6.0 as compared to pH 7.4. Thus, the Fc variant(s) of the
present invention have retention of pH dependence (higher affinity at pH 6.0 than at near-
neutral pH) characteristic of FcRn interactions like wild type Fc.
Example 11: Determination of kinetic rate constants of binding of Fc variant(s) to
recombinant neonatal Fc receptor (rFcRn) of different species
In this experiment, binding of the Fc variant(s) candidates of the present invention to FcRn
of different species was determined by Surface Plasmon Resonance-based measurement
using the Biacore 8K+ instrument (Cytiva). To check kinetic rate constants, the experiment
was conducted using recombinant FcRn of mouse, monkey and human for binding with Fc
variant(s) candidates. FcRn (of Monkey and Human species) was captured on CM5 chip as
mentioned in Example 9. Mouse FcRn was immobilized on a CM5 sensor chip surface using
a standard amine coupling chemistry, as per the manufacturer’s protocol. 10 mM phosphate
buffered saline (PBS) (10 mM phosphate buffer, pH 6.0, 150 mM NaCl, 0.005 % Tween
20) was used as the running buffer to carry out the kinetic measurements. To measure the
association rate constant (ka) and dissociation rate constant (kd), five dilutions (in the range
of 55nM – 0.027nM) of Fc muteins were prepared in above mentioned running buffer and
injected at a flow rate of 30 μL/min with an association time of 60 s. Dissociation time was
kept up to 1800s. All the reactions were carried at 25 °C. After each sample run, the chip
surface was regenerated using Glycine pH 1.5 /3M MgCl2. The data, in the form of
sensorgrams, were analyzed using the Biacore Insight Evaluation software (v 3.0.12.15655).
45
The kinetic constants of binding of Fc variant(s) to the FcRn receptors of different species
are shown in table 5.
Table 5: Kinetic constant of Fc variants binding to FcRn of different species
Sample details Type of FcRn ka kd KD
H11A4.1
(Monomer)
Human FcRn 3.00E+06 2.10E-03 7.00E-10
Mouse FcRn 1.21E+06 1.09E-04 9.18E-11
Monkey FcRn 3.19E+06 2.97E-03 9.28E-10
A3 H11A4.1
(mAb)
Human FcRn 1.40E+06 3.50E-03 2.60E-09
Mouse FcRn 1.02E+06 1.69E-04 1.65E-10
Monkey FcRn 1.31E+06 4.08E-03 3.13E-09
B14 H11A4.1
(mAb)
Human FcRn 1.20E+06 3.90E-03 3.30E-09
Mouse FcRn 8.39E+05 1.58E-04 1.89E-10
Monkey FcRn 1.17E+06 5.00E-03 4.26E-09
H11A4.2
(Monomer)
Human FcRn 3.53E+06 1.03E-03 2.92E-10
Mouse FcRn 7.19e+05 3.99e-05 5.56e-11
Monkey FcRn 2.95E+06 1.75E-03 5.92E-10
A3 H11A4.2
(mAb)
Human FcRn 1.43E+06 1.49E-03 1.04E-09
Mouse FcRn 2.94e+05 3.31e-05 1.13e-10
Monkey FcRn 2.96E+06 2.70E-03 9.14E-10
H11A4.3
(Monomer)
Human FcRn 1.78E+06 5.13E-04 2.88E-10
Mouse FcRn 4.71E+04 3.95E-05 8.39E-10
Monkey FcRn 1.60E+06 2.21E-03 1.38E-09
A3 H11A4.3
(mAb)
Human FcRn 1.25E+06 1.01E-03 8.03E-10
Mouse FcRn 6.01E+04 1.55E-04 2.57E-09
Monkey FcRn 8.10E+05 3.05E-03 3.76E-09
B14 H11A4.3
(mAb)
Human FcRn 1.00E+06 1.23E-03 1.23E-09
Mouse FcRn 9.46E+04 7.40E-05 7.82E-10
Monkey FcRn 1.33E+06 3.26E-03 2.45E-09
As it can be seen from table 5, Fc variants of the present invention can binds to not only
human FcRn, but also mouse FcRn as well as monkey FcRn in-vitro. The advantageous
characteristics of cross-reactivity with monkey and mouse FcRn infer that the Fc variant(s)
of the present invention can be tested in non-human primates and mice and the resulting
46
data can be very useful in predicting their pharmacokinetics, pharmacodynamics and
toxicity profiles in humans.
Example 12: Effect of Fc variant(s)s on total serum IgG levels in wild-type C57BL/6
mice
In the part 1 of present study, two Fc variant(s)s of the present invention in full length
antibody format namely, A3H11A4.1 (Sequence ID nos. 3 and 5 i.e. light chain amino acid
sequence and heavy chain amino acid sequence respectively) and B14H11A4.1 (Sequence
ID nos. 7 and 9 i.e. light chain amino acid sequence and heavy chain amino acid sequence
respectively) were compared with two full length antibodies namely A3H11A4.0 (Sequence
ID nos. 15 and 16 i.e. heavy chain amino acid sequence and light chain amino acid sequence
respectively) and B14H11A4.0 (Sequence ID nos. 17 and 18 i.e. heavy chain amino acid
sequence and light chain amino acid sequence respectively).
The antibodies of the present invention A3H11A4.1 and B14H11A4.1 have been compared
to A3H11A4.0 and B14H11A4.0 respectively to determine the effect of Fc variant(s) of the
present invention on total serum IgG levels in circulation. Wild-type C57BL/6 mice were
randomized based on their body weight. At Day -2 (i.e., 48 hours pre-dose), blood collection
was performed. On Day 0 (0 hour), dose of antibodies as mentioned in Table 6 were
administered via intra-peritoneal route to respective groups of study animals (6 mice per
group).
Table 6: Administration of antibodies via intra-peritoneal route
Sr. No. Treatment Group Dose
1 Normal Control (Placebo) NA
2 A3H11A4.0 3 mg / 20 gm of mice
3 A3H11A4.1 3 mg / 20 gm of mice
4 B14H11A4.0 3 mg / 20 gm of mice
5 B14H11A4.1 3 mg / 20 gm of mice
Blood samples were collected at time points: Day 1, Day 2, Day 5 and Day 8. Endogenous
serum IgG levels were determined using ELISA from the collected samples at mentioned
time points. All the four treatment groups [A3H11A4.1, B14H11A4.1, A3H11A4.0 and
B14H11A4.0] showed reduction in mouse IgG concentration as compared to the normal
control (Placebo group). Upon comparison between the treatment groups, A3H11A4.1 and
B14H11A4.1 showed higher IgG reduction than A3H11A4.0 and B14H11A4.0 across the
47
study duration. The graphical presentation of the results with relative IgG concentration (%)
with respect to Pre-dose levels across all treatment groups are shown in Figure-3.
In part 2 of the present study, two Fc variants of the present invention in full-length antibody
format namely, A3H11A4.2 (SEQ IDs 22 and 24 i.e. light chain amino acid sequence and
heavy chain amino acid sequence respectively) and A3H11A4.3 (SEQ IDs 30 and 32 i.e.
light chain amino acid sequence and heavy chain amino acid sequence respectively) were
compared with the full-length antibody namely A3H11A4.0 (SEQ IDs 15 and 16 i.e. heavy
chain amino acid sequence and light chain amino acid sequence respectively). The full-
length amino acid and nucleotide sequences of said antibodies and Fc format candidates are
described in table 2.
The antibodies of the present invention A3H11A4.2 and A3H11A4.3 have been compared
to A3H11A4.0 to determine the effect of Fc variants of the present invention on total serum
IgG level in circulation. Wild-type C57BL/6 mice were randomized based on their body
weight. At Day -2 (i.e., 48 hours pre-dose), blood collection was performed. On Day 0 (0
hour), dose of candidates as mentioned in Table 7 were administered via intra-peritoneal
route to respective group of study animals (6 mice per group).
Table 7: Administration of antibodies via intra-peritoneal route
Sr. No. Treatment Group Dose
1 Normal control (Placebo) NA
2 A3H11A4.0 3 mg / 20 gm of mice
3 A3H11A4.2 3 mg / 20 gm of mice
4 A3H11A4.3 3 mg / 20 gm of mice
Blood samples were collected at time points: Day 1, Day 2, Day 5 and Day 8. Endogenous
serum IgG levels were determined using ELISA from the collected samples at mentioned
time points. All the three treatment groups [A3H11A4.0, A3H11A4.2 and A3H11A4.3]
showed reduction in mouse IgG concentration as compared to the normal control (Placebo
group). Upon comparison between the antibodies tested in the study, the treatment groups,
A3H11A4.2 and A3H11A4.3, showed higher IgG reduction than A3H11A4.0. The
graphical presentation of the results with relative IgG concentration (%) with respect to pre-
dose levels across different antibody candidates are shown in Figure-4.
In part 3 of the present study, three Fc variants of the present invention in Fc format namely,
H11A4.1, H11A4.2 and H11A4.3 were evaluated to check their effect on total serum IgG
48
levels in circulation. The full-length amino acid and nucleotide sequences of said Fc format
candidates are described in table 2.
Wild-type C57BL/6 mice were randomized based on their body weight. At Day -2 (i.e., 48
hours pre-dose), blood collection was performed. On Day 0 (0 hour), dose of candidates as
mentioned in Table 8 were administered via intra-peritoneal route to respective group of
study animals (6 mice per group).
Table 8: Administration of antibodies via intra-peritoneal route
Sr. No. Treatment Group Dose
1 Normal control (Placebo) NA
2 H11A4.1 1 mg / 20 gm of mice
3 H11A4.2 1 mg / 20 gm of mice
4 H11A4.3 1 mg / 20 gm of mice
Blood samples were collected at time points: Day 1, Day 2, Day 5 and Day 8. Endogenous
serum IgG levels were determined using ELISA from the collected samples at mentioned
time points. All the three treatment groups [H11A4.1, H11A4.2 and H11A4.3] showed
reduction in mouse IgG concentration as compared to the normal control (Placebo group),
with the highest IgG depletion observed on day 2. The graphical presentation of the results
with relative IgG concentration (%) with respect to pre-dose levels across different Fc
candidates are shown in Figure-5.
Example 13: Effect of Fc variants on total serum IgG levels in rhesus monkeys
The effect of Fc variant(s) of the present invention in the Fc format namely, H11A4.1
(Sequence ID nos. 1 and Sequence ID nos. 11) on total serum IgG level in circulation was
determined by a single dose study in rhesus monkeys. H11A4.1 was administered at 10
mg/kg dose via intravenous infusion route to the animals (N=2). Blood samples were
collected at time points: pre-dose, 6 hr, 24 hr (Day 1), Day 2, 3, 4, 5, 7, 9, 11, 14, 17, 21,
28, 35, 42, 49 and 56. Endogenous serum IgG levels were determined using ELISA based
method from the collected samples at mentioned time points. Relative endogenous IgG
reduction as compared to pre-dose values was observed in both animals with highest IgG
depletion observed on day 5.
The graphical presentation of the results with relative IgG concentration (%) with respect
to pre-dose levels are shown in Figure-6.
49
Example 14: Effect of Fc variants on total serum IgG levels in rhesus monkeys
The effect of an Fc variant(s) of the present invention in full-length antibody format namely,
A3H11A4.1 (Sequence ID nos. 3 and 5 i.e. light chain amino acid sequence and heavy chain
amino acid sequence respectively) on total serum IgG level in circulation was determined
by a single dose study in rhesus monkeys. A3H11A4.1 was administered at 30 mg/kg dose
via intravenous infusion route to the animals (N=2). Blood samples were collected at time
points: pre-dose, 6 hr, 24 hr (Day 1), Day 2, 3, 4, 5, 7, 9, 11, 14, 17, 21, 28, 35, 42, 49 and
56. Endogenous serum IgG levels were determined using ELISA based method from the
collected samples at mentioned time points. Relative endogenous IgG reduction as
compared to pre-dose values was observed in both animals with highest IgG depletion
observed on day 9.
The graphical presentation of the results with relative IgG concentration (%) with respect
to pre-dose levels are shown in Figure-7.
Example 15: Effect of Fc variants on total serum IgG levels in rhesus monkeys
The effect of an Fc variants of the present invention in full-length antibody format namely,
B14H11A4.1 (Sequence ID nos. 7 and 9 i.e. light chain amino acid sequence and heavy
chain amino acid sequence respectively) on total serum IgG level in circulation was
determined by a single dose study in rhesus monkeys. B14H11A4.1 was administered at 30
mg/kg dose via intravenous infusion route to the animals (N=2). Blood samples were
collected at time points: pre-dose, 6 hr, 24 hr (Day 1), Day 2, 3, 4, 5, 7, 9, 11, 14, 17, 21,
28, 35, 42, 49 and 56. Endogenous serum IgG levels were determined using ELISA based
method from the collected samples at mentioned time points. Relative endogenous IgG
reduction as compared to pre-dose values was observed in both animals with highest IgG
depletion observed on day 4. The graphical presentation of the results with relative IgG
concentration (%) with respect to pre-dose levels are shown in Figure-8.
Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to
herein is incorporated by reference for all purposes.
50
Equivalents
The invention may be embodied in other specific forms without departing from the spirit or
essential characteristics thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting the invention described herein. Scope of the
invention is thus indicated by the appended claims rather than by the foregoing description,
and all changes that come within the meaning and range of equivalency of the claims are
intended to be embraced therein.
References incorporated in present patent application:
1. Leona E. Ling1, Jan L. Hillson, M281, an Anti-FcRn Antibody: Pharmacodynamics,
Pharmacokinetics, and Safety Across the Full Range of IgG Reduction in a First-in-Human
Study, Clinical Pharmacology & Therapeutics, Volume 105 Number 4, April 2019
2. Peter Kiessling et al., The FcRn inhibitor rozanolixizumab reduces human serum IgG
concentration: A randomized phase 1 study, Sci. Transl. Med. 9, eaan1208, November 2017
3. Deisenhofer, Crystallographic refinement and atomic models of a human Fc fragment and
its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-.ANG.
resolution, Biochemistry 20:2361-2370, 1981
4. Edelman, G.M. et al., The covalent structure of an entire gamma G immunoglobulin
5 molecule.Proc. Natl. Acad. USA, 63, 78-85, 1969
5. Roux et al., Comparisons of the Ability of Human IgG3 Hinge Mutants, IgM, IgE, and IgA2,
to Form Small Immune Complexes: A Role for Flexibility and Geometry, J. Immunol. 161:
4083, 1998
6. Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. A
single amino acid substitution abolishes the heterogeneity of chimeric mouse / human
(IgG4) antibody. Mol Immunol. 30(1): 105-8, 1993.
51
We claim:
1. An FcRn-antagonists that comprises of a Fc variant, wherein the Fc variant comprises the
L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino acid substitutions
as per EU numbering.
2. The FcRn-antagonists as claimed in claim 1 are monoclonal antibodies, monomers, dimers
or multimers.
3. The FcRn-antagonist as claimed in claim 2 is monomer having amino acid sequence as set
forth in SEQ ID NO. 1 or SEQ ID NO. 11.
4. The FcRn-antagonist as claimed in 2 is monoclonal antibody comprising amino acid
sequences as set forth in SEQ ID No. 3 and SEQ ID No. 5.
5. The FcRn-antagonist as claimed in 2 is monoclonal antibody comprising amino acid
sequences as set forth in SEQ ID No. 7 and SEQ ID No. 9.
6. The FcRn-antagonist as claimed in claim 2 is dimer having an amino acid sequence as set
forth in SEQ ID NO. 13.
7. The FcRn-antagonists as claimed in claim 1 further comprising M252Y, S254T, T256E
amino acid substitutions as per EU numbering.
8. The FcRn-antagonists as claimed in claim 7 are monoclonal antibodies, monomers, dimers
or multimers.
9. The FcRn-antagonist as claimed in claim 8 is monomer having an amino acid sequence as
set forth in SEQ ID NO. 38.
10. The FcRn-antagonist as claimed in 8 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 30 and SEQ ID No. 32.
11. The FcRn-antagonist as claimed in 8 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 34 and SEQ ID No. 36.
12. An FcRn-antagonists that comprise of an Fc variant, wherein the Fc variant comprises the
T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions
as per EU numbering.
13. The FcRn-antagonists as claimed in claim 12 are monoclonal antibodies, monomers, dimers
or multimers.
14. The FcRn-antagonist as claimed in claim 13 is monomer having amino acid sequence as set
forth in SEQ ID NO. 20.
15. The FcRn-antagonist as claimed in claim 13 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 22 and SEQ ID No. 24.
16. The FcRn-antagonist as claimed in 13 is monoclonal antibody comprising amino acid
sequences set forth in SEQ ID No. 26 and SEQ ID No. 28.
52
17. The FcRn-antagonists as claimed in claims 1-16, having a KD in the range between 10-11 M
to 10-8 M for FcRn.
18. The FcRn-antagonists as claimed in claim 1-17, those cross-react with FcRn of mouse,
monkey and human.
19. A composition comprising FcRn-antagonist as claimed in any of claims 1-18 and an
acceptable carrier.