FIELD OF THE INVENTION
The present disclosure relates to antibodies that bind human CD19 ("anti-human CD19
antibodies" or "anti-CD19 antibodies"), compositions comprising such anti-human CD19
antibodies, and methods of using such anti-human CD19 antibodies.
BACKGROUND
B cells can produce antibodies against various antigens and thus play an important role in
the humoral immune response. Additionally, B cells also function as antigen presenting cells
(APC) and secrete cytokines. B cells express B cell receptor (BCR) on their cell membranes; and
BCR allows the B cell to bind a specific antigen, against which it will initiate an antibody
response. Dysregulation of B cells is associated with a variety of disorders.
Human B-lymphocyte antigen CD19 (also known as Cluster of Differentiation 19, Blymphocyte
surface antigen B4, T -Cell Surface Antigen Leu-12, or CVID3) is a transmembrane
protein widely expressed during all phases of B cell development until its terminal differentiation
into plasma cells. Thus, human CD19 is a pan-B cell marker. CD19 is found in association with
CD21, a complement receptor, and with CD81, a member of the tetraspan family (Carter, et al.,
Immunol. Res. 26: 45-54, 2002). CD19 is a co-receptor for the BCR and acts as an adaptor
protein to recruit cytoplasmic signaling proteins to the BCR complex. CD 19 is required for
normal B cell function and is involved in diverse B cell responses, including B cell survival,
proliferation, activation, and differentiation.
Anti-human CD19 antibodies have been described previously and are being tested in
clinical trials. Many known anti-human CD19 antibodies are B cell-depleting antibodies; for
example, inebilizumab (also known as MEDI-551) and tafasitamab (also known as MOR208 or
XmAb5574) in Phase III clinical trials, loncastuximab in Phase II clinical trial, 4G7SDIE and
DI-B4 in Phase I clinical trial, were all reported to be B cell-depleting antibodies (Cree, et al.,
Lancet. 394(10206):1352-1363, 2019; Kellner, et al., Leukemia. 27(7):1595-1598, 2013;
Kaplon, et al., MAbs. 12(1): 1703531, 2020; Seidel, et al., Mol. Ther. 24(9): 1634-43, 2016;
W02007076950).
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Obexelimab (also known as XmAb5871) is an Fc-engineered antibody that binds both
CD19 and FcyRIIb, and inhibits B cell function by engaging the inhibitory FcyRIIb receptor
signaling (Szili, et al., MAbs. 6(4): 991-999, 2014). However, high affinity binding of
obexelimab to FcyRIIb could lead to non-specific binding to other cell types. In human patients,
obexelimab was reported to have a short half-life, with T 112 of 3. 5 ± 1. 0 days; and reduce
peripheral human B cell counts, with a mean reduction of about 30-40% of the baseline level
(Jaraczewska-Baumann, et al., European League Against Rheumatism (EULAR) 2015 Annual
Meeting Poster: A Phase Jb/2a Study of the Safety, Tolerability, Pharmacokinetics and
Pharmacodynamics of XmAb®5871 in Patients with Rheumatoid Arthritis, June 12, 2015,
available at https://investors.xencor.com/static-files/00 17 dcfl-deb2-46eb-93ff-90ba164ec50c ).
Therefore, there remains a need for alternative anti-human CD19 antibodies that
specifically bind and inhibit human B cells, without depleting them, for treating B cell associated
disorders.
DETAILED DESCRIPTION
Provided herein are antibodies that bind human CD19 and inhibit B cell responses (e.g.,
proliferation, activation, and differentiation) without depleting the B cells (i.e., "non-depleting
anti-human CD19 antibodies"). Such non-depleting anti-human CD19 antibodies can be used to
treat B cell associated disorders, e.g., autoimmune diseases, without destroying those important
immune cells, and thus avoid problematic concurrent immunocompromise, long-term immune
suppression, and other complications resulting from B cell depletion. The anti-human CD19
antibodies provided herein have one or more of the following properties: 1) bind human CD19
(and sometimes cynomolgus monkey CD19) with desirable binding affinities and/or association
and dissociation rates, 2) bind human B cells specifically and inhibit primary human B cell
proliferation, activation and/or differentiation, 3) do not deplete human B cells, 4) being
internalized into human B cells, 5) low immunogenicity risk, 6) low hydrophobicity, and/or 7)
good stability, solubility, viscosity, and pharmacokinetic characteristics for development and use
in the treatment of autoimmune disorders. As described below, in a side-by-side comparison,
one such anti-human CD19 antibody CB3fis shown to bind human B cells with high specificity
in a whole blood assay, whereas obexelimab shows non-specific binding to human neutrophils in
addition to B cell binding. Since neutrophil is the most abundant type of white blood cells in
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human, the nonspecific binding to neutrophils might explain the short half-life of obexelimab
observed in human patients. Additionally, in an in vitro B cell apoptosis assay, obexilimab is
shown to induce primary human B cell apoptosis in a dose-dependent manner, whereas the antihuman
CD 19 antibody CB3f does not induce apoptosis of primary human B cells in the same
assay.
In one aspect, provided herein are novel non-depleting anti-human CD19 antibodies. In
some embodiments, the anti -human CD 19 antibodies are fully human antibodies.
In some embodiments, provided herein are antibodies that bind human CD19, wherein
the antibodies comprise a heavy chain variable region (VH) and a light chain variable region
(VL), wherein the VH comprises heavy chain complementarity determining regions (HCDR)
HCDRI, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining
regions (LCDR) LCDRI, LCDR2, and LCDR3, wherein the HCDRI comprises SEQ ID NO: 29,
the HCDR2 comprises SEQ ID NO: 30, the HCDR3 comprises SEQ ID NO: 31, the LCDRI
comprises SEQ ID NO: 32, the LCDR2 comprises SEQ ID NO: 33, and the LCDR3 comprises
SEQ ID NO: 34. In some embodiments, the anti-human CD19 antibodies comprise a VH
comprising SEQ ID NO: 37 and a VL comprising SEQ ID NO: 41.
In some embodiments, the anti-human CD19 antibody has a human IgG1 or IgG4
isotype. In some embodiments, the anti-human CD19 antibody has a human IgG4 isotype. In
some embodiments, the anti-human CD19 antibody has a modified human IgG4 hinge region
comprising a S228P mutation (according to the EU Index Numbering), which reduces the IgG4
Fab-arm exchange in vivo (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767). In some
embodiments, the anti-human CD19 antibody has a human IgG1 isotype. In some embodiments,
the anti-human CD19 antibody has a modified human IgG 1 Fe region that has reduced or
eliminated Fe effector functions such as antibody-dependent cell cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC). Such an antibody is termed an "IgG !-effector null"
antibody.
In some embodiments, the anti-human CD19 antibody has a human IgG4 isotype. For
example, in some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID
NO: 35 and a light chain (LC) comprising SEQ ID NO: 39. In some embodiments, the antibody
has a human IgG 1 isotype. For example, in some embodiments, the antibody comprises a HC
comprising SEQ ID NO: 50 and a LC comprising SEQ ID NO: 39. In some embodiments, the
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antibody is an IgG !-effector null antibody. For example, in some embodiments, the antibody
comprises a HC comprising SEQ ID NO: 52 and a LC comprising SEQ ID NO: 39.
In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that
bind human CD19, wherein the antibody fragments comprise a VH and a VL, wherein the VH
comprises HCDRI, HCDR2, and HCDR3, and the VL comprises LCDRI, LCDR2, and LCDR3,
wherein the HCDRI comprises SEQ ID NO: 29, the HCDR2 comprises SEQ ID NO: 30, the
HCDR3 comprises SEQ ID NO: 31, the LCDRI comprises SEQ ID NO: 32, the LCDR2
comprises SEQ ID NO: 33, and the LCDR3 comprises SEQ ID NO: 34. In some embodiments,
the antibody fragments comprise a VH comprising SEQ ID NO: 37 and a VL comprising SEQ
IDNO: 41.
Provided herein are also antibodies that bind human CD19, wherein the antibodies
comprise a VH and a VL, wherein the VH comprises HCDRI, HCDR2, and HCDR3, and the VL
comprises LCDRI, LCDR2, and LCDR3, wherein the HCDRI comprises SEQ ID NO: 29, the
HCDR2 comprises SEQ ID NO: 30, the HCDR3 comprises SEQ ID NO: 31, the LCDRI
comprises SEQ ID NO: 43, the LCDR2 comprises SEQ ID NO: 44, and the LCDR3 comprises
SEQ ID NO: 45. In some embodiments, the anti-human CD19 antibodies comprise a VH
comprising SEQ ID NO: 37 and a VL comprising SEQ ID NO: 48. In some embodiments, the
antibody has a human IgG4 isotype. For example, in some embodiments, the antibody
comprises a HC comprising SEQ ID NO: 35 and a LC comprising SEQ ID NO: 46. In some
embodiments, the antibody has a human IgGl isotype. For example, in some embodiments, the
antibody comprises a HC comprising SEQ ID NO: 50 and a LC comprising SEQ ID NO: 46. In
some embodiments, the antibody is an IgGl-effector null antibody. For example, in some
embodiments, the antibody comprises a HC comprising SEQ ID NO: 52 and a LC comprising
SEQ ID NO: 46.
In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that
bind human CD19, wherein the antibody fragments comprise a VH and a VL, wherein the VH
comprises HCDRI, HCDR2, and HCDR3, and the VL comprises LCDRI, LCDR2, and LCDR3,
wherein the HCDRI comprises SEQ ID NO: 29, the HCDR2 comprises SEQ ID NO: 30, the
HCDR3 comprises SEQ ID NO: 31, the LCDRI comprises SEQ ID NO: 43, the LCDR2
comprises SEQ ID NO: 44, and the LCDR3 comprises SEQ ID NO: 45. In some embodiments,
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the antibody fragments comprise a VH comprising SEQ ID NO: 37 and a VL comprising SEQ
IDNO: 48.
Also provided herein are antibodies that bind human CD19, wherein the antibodies
comprise a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL
comprises LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 15, the
HCDR2 comprises SEQ ID NO: 16, the HCDR3 comprises SEQ ID NO: 17, the LCDR1
comprises SEQ ID NO: 18, the LCDR2 comprises SEQ ID NO: 19, and the LCDR3 comprises
SEQ ID NO: 20. In some embodiments, the anti-human CD19 antibodies comprise a VH
comprising SEQ ID NO: 23 and a VL comprising SEQ ID NO: 27. In some embodiments, the
antibody has a human IgG4 isotype. For example, in some embodiments, the antibody
comprises a HC comprising SEQ ID NO: 21 and a LC comprising SEQ ID NO: 25. In some
embodiments, the antibody has a human IgG 1 isotype. In some embodiments, the antibody is an
IgG 1-effector null antibody. For example, in some embodiments, the antibody comprises a HC
comprising SEQ ID NO: 54 and a LC comprising SEQ ID NO: 25.
In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that
bind human CD19, wherein the antibody fragments comprise a VH and a VL, wherein the VH
comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3,
wherein the HCDR1 comprises SEQ ID NO: 15, the HCDR2 comprises SEQ ID NO: 16, the
HCDR3 comprises SEQ ID NO: 17, the LCDR1 comprises SEQ ID NO: 18, the LCDR2
comprises SEQ ID NO: 19, and the LCDR3 comprises SEQ ID NO: 20. In some embodiments,
the antibody fragments comprise a VH comprising SEQ ID NO: 23 and a VL comprising SEQ
IDNO: 27.
Also provided herein are antibodies that bind human CD19, wherein the antibodies
comprise a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL
comprises LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the
HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDRI
comprises SEQ ID NO: 4, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises
SEQ ID NO: 6. In some embodiments, the anti-human CD19 antibodies comprise a VH
comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 13. In some embodiments, the
antibody comprises a HC comprising SEQ ID NO: 7 and a LC comprising SEQ ID NO: 11.
.,., .
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In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that
bind human CD19, wherein the antibody fragments comprise a VH and a VL, wherein the VH
comprises HCDRI, HCDR2, and HCDR3, and the VL comprises LCDRI, LCDR2, and LCDR3,
wherein the HCDRl comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the
HCDR3 comprises SEQ ID NO: 3, the LCDRl comprises SEQ ID NO: 4, the LCDR2 comprises
SEQ ID NO: 5, and the LCDR3 comprises SEQ ID NO: 6. In some embodiments, the antibody
fragments comprise a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 13.
In another aspect, provided herein are nucleic acids encoding a heavy chain or light
chain, or a VH or VL, of the novel anti-human CD19 antibodies described herein, and vectors
comprising such nucleic acids.
In some embodiments, provided herein are nucleic acids encoding a heavy chain or light
chain of the anti-human CD19 antibodies described herein. In some embodiments, provided
herein are nucleic acids comprising a sequence encoding SEQ ID NO: 35, 52, 50, 39, 46, 21, 54,
25, 7 or 11. In some embodiments, provided herein are nucleic acids comprising a sequence
encoding an antibody heavy chain that comprises SEQ ID NO: 35, 52, 50, 21, 54, or 7. For
example, the nucleic acid can comprise a sequence selected from SEQ ID NO: 36, 53, 51, 22, 55,
or 8. In some embodiments, provided herein are nucleic acids comprising a sequence encoding
an antibody light chain that comprises SEQ ID NO: 39, 46, 25, or 11. For example, the nucleic
acid can comprise a sequence selected from SEQ ID NO: 40, 47, 26, or 12.
Also provided herein are nucleic acids encoding a VH or VL of the anti-human CD19
antibodies described herein. In some embodiments, provided herein are nucleic acids comprising
a sequence encoding SEQ ID NO: 37, 23, 27, 9, 41, 48, 13. In some embodiments, provided
herein are nucleic acids comprising a sequence encoding an antibody VH that comprises SEQ ID
NO: 37, 23, 27, 9. For example, the nucleic acid can comprise a sequence selected from SEQ ID
NO: 38, 24, 28, 10. In some embodiments, provided herein are nucleic acids comprising a
sequence encoding an antibody VL that comprises SEQ ID NO: 41, 48, 13. For example, the
nucleic acid can comprise a sequence selected from SEQ ID NO: 42, 49, 14.
Provided herein are also vectors comprising a nucleic acid sequence encoding an
antibody heavy chain or light chain. For example, such vectors can comprise a nucleic acid
sequence encoding SEQ ID NO: 35, 52, 50, 39, 46, 21, 54, 25, 7, or 11. In some embodiments,
the vector comprises SEQ ID NO: 36, 53, 51, 40, 47, 22, 55, 26, 8, or 12 .
•... t:
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Provided herein are also vectors comprising a nucleic acid sequence encoding an
antibody VH or VL. For example, such vectors can comprise a nucleic acid sequence encoding
SEQ ID NO: 37, 23, 27, 9, 41, 48, or 13. In some embodiments, the vector comprises SEQ ID
NO: 38, 24, 28, 10, 42, 49, or 14.
Provided herein are also vectors comprising a first nucleic acid sequence encoding an
antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In
some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 35,
50, or 52, and a second nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some
embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 21 or 54,
and a second nucleic acid sequence encoding SEQ ID NO: 25.
In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ
ID NO: 35 and a second nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments,
the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 52 and a second nucleic
acid sequence encoding SEQ ID NO: 39. In some embodiments, the vector comprises a first
nucleic acid sequence encoding SEQ ID NO: 50 and a second nucleic acid sequence encoding
SEQ ID NO: 39. In some embodiments, the vector comprises a first nucleic acid sequence
encoding SEQ ID NO: 35 and a second nucleic acid sequence encoding SEQ ID NO: 46. In some
embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 52 and a
second nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the vector
comprises a first nucleic acid sequence encoding SEQ ID NO: 50 and a second nucleic acid
sequence encoding SEQ ID NO: 46. In some embodiments, the vector comprises a first nucleic
acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID
NO: 25. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ
ID NO: 54 and a second nucleic acid sequence encoding SEQ ID NO: 25. In some
embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 7 and a
second nucleic acid sequence encoding SEQ ID NO: 11.
Also provided are compositions comprising a first vector comprising a nucleic acid
sequence encoding an antibody heavy chain, and a second vector comprising a nucleic acid
sequence encoding an antibody light chain. In some embodiments, the composition comprises a
first vector comprising a nucleic acid sequence encoding SEQ ID NO: 35, 52, or 50, and a
second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some
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embodiments, the composition comprises a first vector comprising a nucleic acid sequence
encoding SEQ ID NO: 21 or 54, and a second vector comprising a nucleic acid sequence
encoding SEQ ID NO: 25.
In some embodiments, the composition comprises a first vector comprising a nucleic acid
sequence encoding SEQ ID NO: 35 and a second vector comprising a nucleic acid sequence
encoding SEQ ID NO: 39. In some embodiments, the composition comprises a first vector
comprising a nucleic acid sequence encoding SEQ ID NO: 52 and a second vector comprising a
nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the composition
comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 50 and a
second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39. In some
embodiments, the composition comprises a first vector comprising a nucleic acid sequence
encoding SEQ ID NO: 35 and a second vector comprising a nucleic acid sequence encoding SEQ
ID NO: 46. In some embodiments, the composition comprises a first vector comprising a nucleic
acid sequence encoding SEQ ID NO: 52 and a second vector comprising a nucleic acid sequence
encoding SEQ ID NO: 46. In some embodiments, the composition comprises a first vector
comprising a nucleic acid sequence encoding SEQ ID NO: 50 and a second vector comprising a
nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the composition
comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 and a
second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25. In some
embodiments, the composition comprises a first vector comprising a nucleic acid sequence
encoding SEQ ID NO: 54 and a second vector comprising a nucleic acid sequence encoding SEQ
ID NO: 25. In some embodiments, the composition comprises a first vector comprising a nucleic
acid sequence encoding SEQ ID NO: 7 and a second vector comprising a nucleic acid sequence
encoding SEQ ID NO: 11.
Nucleic acids of the present disclosure may be expressed in a host cell, for example, after
the nucleic acids have been operably linked to an expression control sequence. Expression
control sequences capable of expression of nucleic acids to which they are operably linked are
well known in the art. An expression vector may include a sequence that encodes one or more
signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectors
containing a nucleic acid of interest (e.g., a nucleic acid encoding a heavy chain or light chain of
an antibody) may be transferred into a host cell by well-known methods, e.g., stable or transient
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transfection, transformation, transduction or infection. Additionally, expression vectors may
contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase,
to aide in detection of host cells transformed with the desired nucleic acid sequences.
In another aspect, provided herein are cells, e.g., host cells, comprising the nucleic acids,
vectors, or nucleic acid compositions described herein. A host cell may be a cell stably or
transiently transfected, transformed, transduced or infected with one or more expression vectors
expressing all or a portion of an antibody described herein. In some embodiments, a host cell
may be stably or transiently transfected, transformed, transduced or infected with an expression
vector expressing HC and LC polypeptides of an antibody of the present disclosure. In some
embodiments, a host cell may be stably or transiently transfected, transformed, transduced or
infected with a first vector expressing HC polypeptides and a second vector expressing LC
polypeptides of an antibody described herein. Such host cells, e.g., mammalian host cells, can
express the anti -human CD 19 antibodies described herein. Mammalian host cells known to be
capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NSO cells.
In some embodiments, the cell, e.g., host cell, comprises a vector comprising a first
nucleic acid sequence encoding SEQ ID NO: 35, 50, or 52, and a second nucleic acid sequence
encoding SEQ ID NO: 39 or 46. In some embodiments, the cell, e.g., host cell, comprises a
vector comprising a first nucleic acid sequence encoding SEQ ID NO: 21 or 54, and a second
nucleic acid sequence encoding SEQ ID NO: 25.
In some embodiments, the cell, e.g., host cell, comprises a first vector comprising a
nucleic acid sequence encoding SEQ ID NO: 35, 52, or 50, and a second vector comprising a
nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some embodiments, the cell, e.g., host
cell, comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 or 54,
and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25.
The present disclosure further provides a process for producing an anti-human CD19
antibody described herein by culturing the host cell described above, e.g., a mammalian host cell,
under conditions such that the antibody is expressed and recovering the expressed antibody from
the culture medium. The culture medium, into which an antibody has been secreted, may be
purified by conventional techniques. Various methods of protein purification may be employed,
and such methods are known in the art and described, for example, in Deutscher, Methods in
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Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd
Edition, Springer, NY (1994).
Also provided are antibodies produced by any of the processes described herein.
In another aspect, provided herein are pharmaceutical compositions comprising an
antibody, nucleic acid, or vector described herein. Such pharmaceutical compositions can also
comprise one or more pharmaceutically acceptable excipient, diluent or carrier. Pharmaceutical
compositions can be prepared by methods well known in the art (e.g., Remington: The Science
and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).
The anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical compositions
described herein can be used for treating B cell associated disorders. Because of their critical role
in regulating the immune system, dysregulation ofB cells is associated with a variety of
disorders. B cell associated disorders include autoimmune diseases, which are caused by
activation of self-reactive B cells and T cells, and lymphomas and leukemias, which are caused
by excessive and/or uncontrolled B cell proliferation. Examples ofB cell associated autoimmune
diseases include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Sjogren's
syndrome, idiopathic thrombocytopenia purpura, Type 1 diabetes, Pemphigus vulgaris,
Neuromyelitis optica, ANCA vasculitis (anti-neutrophil cytoplasmic antibodyassociated
vasculitis), Myasthenia gravis.
Given the anti-human CD19 antibodies described herein do not deplete B cells, they offer
advantages over the B cell depleting antibodies for treating autoimmune diseases and can avoid
problematic concurrent immunocompromise, long-term immune suppression, and other
complications resulting from B cell depletion. As shown below, the anti-human CD19
antibodies described herein are internalized in primary human B cells. Thus, the anti-human
CD 19 antibodies described herein can also be used to deliver other therapeutic agents into human
B cells.
In some embodiments, provided herein are methods of treating a B cell associated
disorder, e.g., an autoimmune disease, in a subject (e.g., a human patient) in need thereof, by
administering to the subject a therapeutically effective amount of an anti-human CD19 antibody,
a nucleic acid encoding such an anti-human CD19 antibody, a vector comprising such a nucleic
acid, or a pharmaceutical composition comprising such an anti-human CD19 antibody, nucleic
acid or vector, as described herein. The antibodies, nucleic acids, vectors, or pharmaceutical
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compositions described herein may be administered by parenteral routes (e.g., subcutaneous and
intravenous). In some embodiments, the B cell associated disorder is selected from systemic
lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, idiopathic
thrombocytopenia purpura, Type 1 diabetes, Pemphigus vulgaris, Neuromyelitis optica, ANCA
vasculitis, Myasthenia gravis.
Also provided are anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical
compositions described herein for use in therapy. Furthermore, the present disclosure also
provides anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical compositions
described herein for use in the treatment of a B cell associated disorder, e.g., e.g., an autoimmune
disease, e.g., systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjogren's
syndrome, idiopathic thrombocytopenia purpura, Type 1 diabetes, Pemphigus vulgaris,
Neuromyelitis optica, ANCA vasculitis, Myasthenia gravis.
Provided herein are also use of the anti-human CD19 antibodies, nucleic acids, vectors,
or pharmaceutical compositions described herein in the manufacture of a medicament for the
treatment of a B cell associated disorder, e.g., an autoimmune disease, e.g., systemic lupus
erythematosus, multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, idiopathic
thrombocytopenia purpura, or Type 1 diabetes, Pemphigus vulgaris, Neuromyelitis optica,
ANCA vasculitis, Myasthenia gravis.
As used herein, the term "a," "an," "the" and similar terms used in the context of the
present disclosure (especially in the context of the claims) are to be construed to cover both the
singular and plural unless otherwise indicated herein or clearly contradicted by the context.
The term "antibody," as used herein, refers to an immunoglobulin molecule that binds an
antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody,
human antibody, humanized antibody, chimeric antibody, or conjugated antibody. The antibodies
can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3,
IgG4).
An exemplary antibody is an immunoglobulin G (IgG) type antibody comprised of four
polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via
inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains
includes a variable region of about 100-125 or more amino acids primarily responsible for
antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains
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contains a constant region primarily responsible for effector function. Each heavy chain is
comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light
chain is comprised of a light chain variable region (VL) and a light chain constant region. The
IgG isotype may be further divided into subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).
The VH and VL regions can be further subdivided into regions of hyper-variability,
termed complementarity determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein
and are important regions of the antibody for antigen binding specificity. Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the
heavy chain are referred to as "HCDRl, HCDR2, and HCDR3" and the three CDRs of the light
chain are referred to as "LCDR1, LCDR2 and LCDR3". The CDRs contain most of the residues
that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs
may be done according to the well-known schemes, including those described in Kabat (Kabat et
al., "Sequences ofProteins oflmmunological Interest," National Institutes of Health, Bethesda,
Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of
immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al.,
"Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular
Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop
Conformations", Journal of Molecular Biology, 406, 228-256 (2011 )), or IMGT (the
international ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al.,
Nucleic Acids Res. 1999; 27:209-212). The North CDR definitions are used for the anti-human
CD 19 antibodies described herein.
Exemplary embodiments of antibodies of the present disclosure also include antibody
fragments or antigen-binding fragments, which comprise at least a portion of an antibody
retaining the ability to specifically interact with an antigen such as Fab, Fab', F(ab')2, Fv
fragments, scFv, scFab, disulfide-linked Fvs (sdFv), a Fd fragment and linear antibodies.
The terms "bind" and "binds" as used herein are intended to mean, unless indicated
otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction
with another protein or molecule, which results in proximity of the two proteins or molecules as
determined by common methods known in the art.
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The term "CD 19" as used herein, unless stated otherwise, refers to human B-lymphocyte
antigen CD19 (also known as Cluster of Differentiation 19, B-lymphocyte surface antigen B4, TCell
Surface Antigen Leu-12, or CVID3). The amino acid sequence of human CD19 is known in
the art, e.g., NCBI Reference Sequence NP _ 001171569.1 (isoform 1, SEQ ID NO: 56) or
NP _001761.3 (isoform 2, SEQ ID NO: 57). Isoform 2 is a splice variant ofisoform 1 and is one
amino acid shorter than isoform 1 in the intracellular domain. The term "CD19" is used herein to
refer collectively to all known human CD 19 isoforms and polymorphic forms.
The term "Fe region" as used herein refers to a region of an antibody, which comprises
the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fe region may include a
portion of the hinge region or the entire hinge region of the antibody heavy chain.
The term "non-depleting antibody" as used herein refers to an antibody that does not
significantly reduce B cell numbers in a subject after treatment, as compared to the B cell
numbers before the treatment. B cell number can be measured using well-known assays such as
those described in the Examples. A non-depleting antibody typically does not induce antibody
dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP),
complement dependent cellular cytotoxicity (CDC), or apoptosis of the B cells.
The terms "nucleic acid" or "polynucleotide", as used interchangeably herein, refer to
polymers of nucleotides, including single-stranded and I or double-stranded nucleotidecontaining
molecules, such as DNA, eDNA and RNA molecules, incorporating native, modified,
and I or analogs of, nucleotides. Polynucleotides of the present disclosure may also include
substrates incorporated therein, for example, by DNA or RNA polymerase or a synthetic
reaction.
The term "subject", as used herein, refers to a mammal, including, but are not limited to,
a human, chimpanzee, ape, monkey, cattle, horse, sheep, goat, swine, rabbit, dog, cat, rat, mouse,
guinea pig, and the like. Preferably the subject is a human.
The term "therapeutically effective amount," as used herein, refers to an amount of a
protein or nucleic acid or vector or composition that will elicit the biological or medical response
of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate
symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In a
non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of a
protein or nucleic acid or vector or composition that, when administered to a subject, is effective
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to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a
disease.
As used herein, "treatment" or "treating" refers to all processes wherein there may be a
slowing, controlling, delaying or stopping of the progression of the disorders or disease disclosed
herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total
elimination of all disorder or disease symptoms. Treatment includes administration of a protein
or nucleic acid or vector or composition for treatment of a disease or condition in a patient,
particularly in a human.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGs. lA-lB show the surface hydrophobicity profiles of the anti-human CD19 parental
antibody C323 (FIG. IA) and the optimized anti-human CD19 antibody CB3f(FIG. lB).
FIG. 2 shows the binding of anti-human CD19 antibodies to human CD19 in an ELISA
assay.
FIG. 3 shows the inhibition of primary B cell proliferation by the anti-human CD19 mAb
CB3f.
FIG. 4 shows the inhibition ofB cell activation in whole blood by the anti-human CD19
mAb CB3f.
FIG. 5 shows the inhibition ofB cell differentiation into plasmablasts by the anti-human
CD19 mAb CB3f.
FIGs. 6A-6B show the anti-human CD19 mAb CB3flacks CDC (FIG. 6A) and ADCC
(FIG. 6B) activities.
FIG. 7 shows binding specificity of anti-human CD19 mAb CB3f and obexelimab to
cells in human whole blood.
FIG. 8 shows difference in the induction ofB cell apoptosis by obexilimab and antihuman
CD19 mAb CB3f.
FIGs. 9A-9B show the reduction of human IgM in NSG mice treated with anti-human
CD19 mAb CB3f on Day 6 (FIG. 9A) and Day 10 (FIG. 9B) after treatment.
FIG. 10 shows the reduction of CD86 expression on human B cells in NSG mice treated
with anti-human CD19 mAb CB3f.
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FIG. 11 shows the frequency ofB cells in NSG mice treated with anti-human CD19 mAb
CB3f.
FIG. 12 shows the internalization of anti-human CD19 mAb1 (CB3f) and anti-human
CD19 mAb2 (C323.C1) in primary human B cells.
FIGs. 13A-13B show comparison of the efficacy of a non-depleting CD19 surrogate Ab
and a depleting CD20 surrogate Ab in the mouse collagen-induced arthritis (CIA) model.
FIG.13A shows treatment with the non-depleting CD19 surrogate Ab in semi-established mode
reduced clinical score greater than the depleting CD20 surrogate Ab in the mouse CIA model.
Clinical scores of mice between Day 21 and Day 42 of the study (n = 12/group except n = 5 for
Isotype control treated no disease control group). Symbols represent mean of group and error
bars represent standard error ofthe mean (SEM). Animals were dosed starting Day 19.
FIG.13B shows treatment with the non-depleting CD 19 surrogate Ab reduced clinical score
AUC (Days 24 to 42) greater than the depleting CD20 surrogate Ab in the mouse CIA model.
Clinical score AUCs of mice between Day 24 and Day 42 of the study (n = 12/group except n =
5 for isotype control treated no disease control group). Bars represent mean of group and error
bars represent standard error of the mean (SEM). Mice were dosed starting Day 19. Bars that do
not share a common letter are significantly different from each other (p<.05 one-way analysis of
variance (ANOVA) Tukey's post-hoc).
FIG. 14 shows treatment with the non-depleting CD19 surrogate Ab in semi-therapeutic
mode delayed and reduced incidence of diabetes greater than the depleting CD20 surrogate Ab in
the NOD model of Type 1 diabetes. Incidence of diabetes (mice with blood glucose levels above
240 mg/dl were considered as diabetic>. n = 1 0/group except n = 9 for untreated group).
Animals were dosed starting at 12 weeks of age.
FIG. 15 shows treatment with the non-depleting CD19 surrogate Ab in semi-therapeutic
mode reduced clinical score greater than the depleting CD20 surrogate Ab in the mouse EAE
model. Clinical scores of mice between Day 6 and Day 42 of the study (n = 12/group). Symbols
represent mean of group and error bars represent standard error of the mean (SEM). Animals
were dosed starting Day 6. * p<0.05 vs same day isotype control.
EXAMPLES
The following examples are offered to illustrate, but not to limit, the claimed invention.
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Example 1. Generation of antibodies that bind human CD19 (anti-human CD19 antibodies).
Anti-human CD19 antibody C323 is discovered from a phage display library using cellbased
panning against human embryonic kidney (HEK) cells co-transfected with human CD19
(SEQ ID NO: 56) and its co-receptor CD21. A negative panning against parental HEK-293 cells
is used to remove non-specific cell binders. CD 19-specific binding is confirmed using CD 19
extracellular domain (ECD) protein by ELISA. Following conversion to IgG format and
purification, cell binding is confirmed using Daudi human Burkitt's lymphoma cell line and
isolated primary human B cells by F ACS.
Hydrophobic interaction chromatography (HIC) is a technique for separation of proteins
commonly used for characterization of antibody variants; and the retention time to the HIC
column of a protein of interest reflects its overall hydrophobicity (J Pharm Biomed Anal. 2016
130:3-18). C323 is deemed to be hydrophobic due to prolonged HIC column retention time.
Antibody hydrophobicity can cause manufacture problems such as poor expression and protein
aggregation. Targeted and random mutagenesis is used to enhance the biophysical properties of
C323 and increase its affinity and potency. A homology model of the variable region of the C323
anti-human CD19 parental mAb is created. A spatial aggregation propensity algorithm is then
applied to the model to identify surface exposed hydrophobic patches. This process identifies
eight surface-exposed hydrophobic residues within LCDR1, HCDR2 and HCDR3: LCDR1:
Y31, Y32; HCDR2: I52, I53 and F54; HCDR3: F97, Y99 and YlOOa (Kabat numbering) (FIG.
1A). These residues are targeted for mutagenesis. Libraries are created to include more
hydrophilic (polar or charged) amino acids using VVK codon-based mutagenic oligonucleotides
and incorporated using Kunkel mutagenesis into an uracil-containing single-stranded DNA
template encoding the original C323 parental antibody with the selected CDR sequences deleted.
Phage-expressed Fabs are screened using biotinylated human CD19 ECD antigen. Affinity
neutral mutations identified in this manner are DNA sequenced and unique clones expressed as
peri plasmic Fab in E. coli and analyzed by titration ELISA. This process identified three affinity
neutral but more hydrophilic amino acid substitutions: LCDR1: Y31H, HCDR2: F54Y and
HCDR3: Y99K (Kabat numbering).
In parallel, optimization to increase affinity is done using NNK codon-based mutagenic
oligonucleotides targeting all six CDRs and incorporated using Kunkel mutagenesis into an
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uracil-containing single-stranded DNA template with the selected CDRs deleted. Screening of
phage-expressed Fab variants is done by capture-lift (Anal Biochem. 1998 256(2):169-77) and
ELISA using a biotinylated human CD19 ECD antigen. In this manner, CDR substitutions
leading to increased affinity are identified: HCDRl: F29I, 134Y; HCDR2: G55D, HCDR3:
G100bA; LCDR1: G27aK, A34H, LCDR2: S52R, A55P, LCDR3: N93Q (Kabat numbering) and
combined, leading to the generation of C323 .C 1.
The three hydrophilic substitutions described above (Y31H, F54Y and Y99K) are added
to C323.C1, generating a Fab template that is used for a final round of CDR randomization using
NNK codon-based mutagenesis. Beneficial CDR mutations are combined in a library allowing
all the beneficial mutations to be randomly combined or back-mutated to wild type sequence.
This process identifies a high-affinity variant termed CB3. CB3 contains additional CDR residue
substitutions: HCDR1: G27H, HCDR2: G50D, I53A, D55G, T56S, A57P; LCDR1: K27aH,
H34A, LCDR3: L95Q (Kabat numbering).
Using CB3 as a template, additional CDR changes are made to revert specific residues to
human IGKV3-20 germline identity. One residue in LCDR1: N29S and five residues in LCDR2:
A50G, T51A, R52S, T53S, P55A are simultaneously reverted with minimal impact on the
antibody's functions. This resulted in a final molecule called CB3f.
Several versions of the CB3f are generated, including (1) an IgG4 isotype comprising a
HC of SEQ ID NO: 35 and a LC of SEQ ID NO: 39; (2) an IgG 1 isotype comprising a HC of
SEQ ID NO: 50 and a LC of SEQ ID NO: 39, and (3) an IgG1 effector null antibody comprising
a HC of SEQ ID NO: 52 and a LC of SEQ ID NO: 39. Unless otherwise specified, "CB3f'
refers to the IgG4 isotype comprising a HC of SEQ ID NO: 35 and a LC of SEQ ID NO: 39.
FIGs.1A-1B show the improved surface hydrophobicity profile of CB3f compared to the
parental antibody C323. The retention time of CB3f on a HIC column was reduced more than
two folds when compared to the parental antibody C323. At the same time, the CB3f antibody
titer from a transient CHO expression has increased more than two fold when compared to the
parental C323 antibody titer.
FIG. 2 shows the improved binding to human CD 19 by the affinity engineered antibodies
CB3 and CB3f, when compared to C323 and C323.C1, in an ELISA assay. The ELISA assay is
performed as follows. A 96 well microtiter plate is coated overnight at 4°C using 50 !J.Liwell of
goat anti-human kappa polyclonal antibody diluted to 5 11g/mL in phosphate buffered saline
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(PBS). Following overnight incubation, the plate is aspirated and blocked using 200 !J.L of casein
buffer for 1 hour at room temperature. The plate is washed three times using PBST (PBS with
0.1% Tween). The anti-human CD19 antibodies are diluted to 5 11g/mL in PBS casein and 50 !J.L
added to each column of the anti-human kappa coated casein blocked plate for 1 hour at 37°C.
The plate is washed three times using PBST. Biotinylated human CD19 extracellular domain
protein is serially diluted from 20 11g/mL to 9 ng/mL and 50 !J.L added to the plates and
incubated for 1 hour at 37°C. The plate is washed three times using PBST and then transferred to
a beaker containing 1liter ofPBST and incubated with stirring overnight (approx. 16 hr) at
37°C. The wash buffer is aspirated and 50 !J.L ofneutravidin alkaline phosphatase-conjugate
diluted 1:1000 in casein buffer is added and incubated for 1 hour at 37°C. The plate is washed
three times using PBS 0.1% Tween. Fifty !J.L of AMP-PMP substrate diluted 1:35 in deionized
water is added and the absorbance at 560 nm read on a Spectramax plate reader.
As shown below, CB3f shows high affinity binding to human CD19 expressing CHO
cells and has unexpectedly acquired binding to cynomolgus monkey CD19 expressing CHO
cells. CB3fhas 92% and 96% identity to human IGHV1-69 and IGKV3-20 germlines,
respectively. Hence the anti-human CD19 antibody CB3fhas high affinity, lcnv hydrophobiciry,
and high percentage of human germline identity, i.e., low immunogenicity risk.
The anti-human CD 19 antibodies described herein, including but not limited to, CB3f,
can be expressed in a mammalian cell line such as HEK293 or CHO, either transiently or stably
transfected with an expression system for secreting the antibody using an optimal predetermined
HC:LC vector ratio or a single vector system encoding both HCs and LC. Clarified media, into
which the antibody has been secreted, can be purified using the commonly used techniques. The
purity of the antibody, after these chromatography steps, may achieve a value greater than 99.0%
(monomer).
Example 2. Characterization of the anti-human CD19 antibodies.
Binding Affinity to CD 19
The solution-phase equilibrium binding affinities ofthe anti-human CD19 mAb CB3fto
membrane-bound human CD19 and cynomolgus monkey CD19 stably expressed on CHO cells
are measured by an MSD solution equilibrium titration (MSD-SET) assay at 37°C. Additionally,
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the monovalent affinity and kinetics ofCB3fFab fragment binding to CD19 are measured by
surface plasmon resonance (SPR) at 37°C.
An MSD SI6000 instrument (Meso Scale Discovery, Rockville, MD) is used for reading
MSD plates. MSD assay plates are prepared as follows. A multi-array 96-well plate (Meso
Scale Discovery, PIN L15XA-3) is coated overnight at 4°C with a 1 ~g/mL solution of a goat
anti-human Fe capture antibody (Jackson ImmunoResearch, PIN 109-005-098) in PBS. Plates
are washed 3X in TBS + 0.1% Tween-20 (TBST) following coating.
CHO cells stably expressing human and cynomolgus monkey CD 19 cells are fixed in 1%
paraformaldehyde in PBS for 5 minutes at room temperature. Paraformaldehyde is removed by
washing with PBS, and the cells are stored at 4°C in 1% Blocker A (diluted from 3% Blocker A,
Meso Scale Discovery, PIN R93AA-1) with 0.05% (w/v) sodium azide.
For the human CD19 affinity measurement, samples are prepared in duplicates with a
fixed antibody concentration of 400 pM, 80 pM, 16 pM and 3.2 pM. For the cynomolgus
monkey CD19 affinity measurement, samples are prepared in duplicates with a fixed antibody
concentration of 10 nM, 2 nM, 400 pM, and 80 pM. Fixed human CD19 CHO cells are pelleted
by centrifugation and resuspended in 3% Blocker A solution at 22 x 106 cells/mL. Fixed
cynomolgus monkey CD19 CHO cells are pelleted by centrifugation and resuspended in 3%
Blocker A solution at 200 x 106 cells/mL. The cells are serially diluted 2. 5-fold in a conicalbottom
96-well plate in 3% Blocker A down to approximately 900 cells/mL for human CD 19
and 8,400 cell/mL for cynomolgus monkey CD19 for a total of 12 cell dilutions each. These are
mixed 1:1 with previously prepared antibody dilutions. For human CD19, the final antibody
concentrations are 200 pM, 40 pM, 8 pM and 1.6 pM, and the final cell dilutions of 11 x 106
cells/mL down to approximately 450 cells/mL. For cynomolgus monkey CD19, the final
antibody concentrations are 5 nM, 1 nM, 200 pM and 40 pM, and the final cell dilutions of 100 x
106 cells/mL down to approximately 4,200 cells/mL. The plate is incubated on a plate shaker at
37°C for 3-4 days for human CD19 and 2 days for cynomolgus monkey CD19 to allow binding
to reach equilibrium.
Following incubation, cells are pelleted by centrifugation. One hundred microliters of the
clarified supernatant from the plate is transferred to the prepared MSD plate and incubated on a
plate shaker at room temperature for 60 minutes. Following incubation, the plate is washed 3X
with TBST, then 100 ~L of 1 ~g/mL biotinylated goat anti-human IgG primary antibody
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(Southern Biotech, Catalog 20 I 0-08) in I% Blocker A is added to all wells. This is incubated on
a plate shaker at room temperature for 60 minutes. The plate was washed 3X with TEST, then
100 !JL of I!Jg/mL SULFO-TAG streptavidin (Meso Scale Discovery, PIN R32AD-I) in I%
Blocker A was added to all wells. This was incubated on a plate shaker at room temperature for
60 minutes. The plate is washed 3x with TEST, then IX Read Buffer T (Meso Scale Discovery,
P/NR92TC-I) is added immediately before reading the plate. Dissociation constant (Ko) and a
least common multiplier (LCM) to account for unknown antigen concentrations on cells are
globally fit from the MSD-SET data to an equilibrium binding equation (see Darling and Brault,
2005, Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions. Assay
and Drug Development Technologies 2: 647-657) using non-linear regression in GraphPad
Prism.
Biacore T200 instrument (GE Healthcare Life Sciences), reagents, and Biacore T200
Evaluation Software Ver 3 .I are used for the surface plasmon resonance analysis of human,
mouse and cynomolgus monkey CDI9 binding to the Fab fragment ofCB3f. Recombinant
CDI9-Fc proteins are purchased from R&D Systems. A protein A sensor chip (GE protein A
chip PIN 29I2755) is used. Running buffer is IX HBS-EP+ (Teknova PIN H8022), and running
temperature is 37°C.
CD I9 F c fusion proteins are diluted to 3 !JglmL in running buffer, and approximately 60
RU each of human, cynomolgus monkey, and mouse proteins are captured in flow cells (Fe) 2, 3
and 4, respectively. CB3fFab fragment is diluted to 1000 nM in running buffer and then 5-fold
serially diluted in running buffer to I.6 nM for a total of 5 dilutions. Fab or buffer blank is
injected at 50 !JLimin for 300 seconds followed by a 900 second dissociation
phase. Regeneration is performed by injecting 10 mM glycine pH 1.5 for 30 seconds at 50
!JLimin over all Fe. Reference-subtracted data is collected as Fc2-Fci, Fc3-Fci and Fc4-Fci, and
then reference-subtracted data is blank subtracted. The on-rate (kon) and off-rate (korr) are fit
using the "1: I Binding" model. The affinity (Ko) is calculated from the binding kinetics
according to the relationship: Ko = korrlkon.
CB3fbinds to human and cynomolgus monkey CDI9 expressed on CHO cells with an
affinity (Ko) of3.35 pM and 45.2 pM, respectively (Table I).
The Fab fragment ofCB3fbinds to human CDI9 with an on-rate (kon) of3.20 x 106 M"1s·
\an off-rate (korr) of2.35 x 10"4 s·\ and an affinity (Ko) of76.8 pM (Table 2). The Fab
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fragment ofCB3fbinds to cynomolgus monkey CD19 with an on-rate (kon) of 1.04 x 106 M"1s"an off-rate (korr) of9.78 x 10"2 s·1
, and an affinity (KD) of94.5 nM (Table 2). No binding to
mouse CD19 is observed at I 11M Fab concentration.
Table 1: In vitro binding of CB3fto fixed CHO cells expressing either human or
cynomolgus monkey CD19.
[Measured by MSD-SET at 37°C. Results are reported as the geometric mean of the KD from 3
independent replicates. Error estimate is calculated as geometric mean x standard deviation logw
KD x ln 10]
Species mAb Affinity (pM)
HumanCD19 3.35 ± 0.56
Cynomolgus monkey CD19 45.2 ± 2.8
Table 2: In vitro binding parameters of the Fab fragment of CB3f to human, cynomolgus
monkey, and mouse CD19 Fe fusion proteins.
[Measured by Surface Plasmon Resonance (SPR) at 37°C. Results are reported as the mean±
standard deviation of 3 independent replicates.]
Species Fab On-Rate (kon) Fab Off-Rate (korr) Fab Affinity (K»)
(M-1s·1 x 106) (s·1 x 10"4) (pM)
HumanCD19 3.20±1.15 2.35 ± 0.37 76.8 ± 16.0
Cynomolgus monkey CD19 1.04 ± 0.03 978 ± 79 94,500 ± 7,900
Mouse CD19 No binding observed at 1 11M Fab concentration
Stability
Stability ofCB3fis assessed at a high concentration (approximately 100 mg/mL) in 5
mM histidine buffer (pH 6.0) with excipients. Concentrated samples are incubated for a period
of 4 weeks at 5°C and 35°C. Following incubation, samples are analyzed for the percentage of
high molecular weight (%HMW) with size exclusion chromatography (SEC), for fragmentation
by capillary electrophoresis (CE-SDS), and for chemical modification (for example deamidation,
isomerization, or oxidation) by LC-MS peptide mapping. After 4 weeks at 35°C, CB3f exhibits
Ll%HMW of 0.5%, L1%fragments of 0.6%, and no CDR chemical modifications greater than
0.2%.
Freeze/thaw stability under the same conditions is evaluated using a 3 repeated slow,
controlled temperature cycle which mimics the freeze/thaw conditions of large volumes of bulk
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drug substance placed at -70°C. CB3f exhibits L1%HMW of 1.9% as measured by SEC after the
3 freeze-thaw cycles. Other excipients can further reduce %HMW growth (data unshown).
These results indicate CB3f possesses good physical and chemical stabilities.
Solubility
Solubility is assessed by concentrating 1 OOmg of CB3f with a 30 kDa molecular weight
cut-off centrifugal filter (for example, Amicon U.C. filters, Millipore, catalog# UFC903024) to a
volume of approximately 0.5 mL. The final concentration of the sample is measured by UV
absorbance at 280 nm using a Solo VPE spectrophotometer (C Technologies, Inc).
CB3f displays a solubility of greater than or equal to 183 mg/mL in 5 mM histidine pH 6
buffer and greater than or equal to 170 mg/mL in PBS (phosphate-buffered saline) pH
7.4. These results indicate that CB3f exhibits high solubility.
Viscosity
Viscosity of CB3f is analyzed at 15°C at an approximate concentration of 125 mg/mL in
5 mM histidine at pH 6.0 with different excipients. Viscosity measurements are made with a
VROC Initium (RheoSense). CB3f exhibited viscosities of 14.2 cP at 131 mg/mL in 5mM
histidine at pH 6 + 280mM mannitol, 9.5 cP in 5mM histidine at pH 6.0 + 150 mM sodium
chloride, and 6.1 cP viscosity in 5mM histidine pH 6.0 + 280 mM arginine. These results
indicate that CB3f exhibits low viscosity, which could enable high concentration dosing.
Pharmacokinetics (PK)
PK properties of CB3f is studied in cynomolgus monkeys following a single
subcutaneous administration of0.03, 0.3, 1 and 10 mg/kg ofCB3f, or a single intravenous bolus
administration of 1 mg/kg of CB3f. CB3f shows linear PK over the subcutaneous dose range
examined, with the terminal half-life (T 112) ranging from 182 to 301 hours. The T 112 after a single
intravenous bolus administration of 1 mg/kg of CB3f is 324 hours.
It has been reported that obexelimab has an average T112 of3.5 ± 1.0 days, which equal to
60-108 hours (Jaraczewska-Baumann, et al., European League Against Rheumatism (EULAR)
2015 Annual Meeting Poster: A Phase 1 b/2a Study of the Safety, Tolerability, Pharmacokinetics
and Pharmacodynamics of XmAb®5871 in Patients with Rheumatoid Arthritis, June 12, 2015,
availab 1 e at https ://investors .xencor. com/static-fil es/00 17 dcfl-deb 2-46eb-93 ff-90ba 164ec5 Oc ).
Therefore, CB3 f has a better T 112 than obexelimab.
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Example 3. In vitro functional characterization of the anti-human CD19 antibodies.
In vitro inhibition of B cell proliferation by the anti-human CD19 mAb CB3f.
The ability of the anti-human CD19 mAb CB3fto inhibit proliferation of primary human
B cells is tested in an in vitro B cell proliferation assay.
Primary human B cells are isolated from healthy donor PBMCs by negative selection
using a B cell isolation kit (Stemcell Technologies). Human primary B cells are re-suspended at
1 x 106 cells/mL and cultured at 37°C in polystyrene 96-well, u-bottom plates in complete
medium (RPMI-1640 containing 10% Fetal bovine serum, 1x MEM- nonessential amino acids,
1 mM Sodium Pyruvate, lx Penicillin-Streptomycin Solution (all from Coming) and lx
Glutamax (Gibco), 0.1% B-mercaptoethanol (Life Technologies). Cells are pre-treated with antihuman
CD19 antibody for 1 hour and stimulated with mouse anti-human IgM (21-lg/mL-Southem
Biotech) plus rabbit anti -mouse IgG (121-lg/mL-Thermo Fisher) for 2 days at 37° and 5% COz.
Cells are then pulsed with [3H]-thymidine (11-lCi thymidine/well, PerkinElmer, Boston, MA) for
18 hours of cell culture. The level of incorporation of [3H]-thymidine is measured by 2450
Microplate Counter (MicroBeta2 serial number: 5129186, PerkinElmer, Boston, MA) and
expressed as a cell count per minute ( c.c.p.m).
The anti-human CD19 mAb CB3finhibits the proliferation of primary human B cells in a
dose dependent manner, while the isotype control does not demonstrate inhibition at any tested
concentration (FIG. 3). The inhibitory function ofCB3fis tested using B cells obtained from 12
different donors, and the average ICso for CB3f is 0.007 nM (Table 3). The data show that the
anti-human CD19 mAb CB3f can inhibit proliferation of primary human B cells in a dose
dependent manner, while B cell proliferation is not affected by the isotype control antibody.
Table 3. Inhibition of primary B cell proliferation by the anti-human CD19 mAb CB3f
ICso Values (nM)
Donor 1 0.006
Donor 2 0.009
Donor 3 0.003
Donor 4 0.006
Donor 5 0.004
Donor 6 0.003
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Donor 7 0.003
Donor 8 0.005
Donor 9 0.005
Donor 10 0.028
Donor 11 0.004
Donor 12 0.005
Average 0.007
In vitro inhibition of B cell activation in human whole blood
The ability of the anti -human CD 19 mAb CB3f to inhibit activation of primary human B
cells in whole blood is tested in an in vitro whole blood activation assay.
EDTA treated human whole blood (Healthy donors, TSRI Normal Blood Donor Services,
San Diego, CA) is cultured in polystyrene 96-well, u-bottom plates and pre-incubated with CB3f
or the isotype control antibody for 30 minutes to one hour at 37 oc and 5% C02. 7 nM of each
antibody is used as the highest concentration with 4 fold dilution and 12 points titration in
complete medium (RPMI-1640 containing 10% fetal bovine serum, 1x MEM- nonessential
amino acids, 1mM sodium pyruvate, 1x penicillin-streptomycin Solution (all from Coming) and
1x Glutamax (Gibco), 0.1% ~-mercaptoethanol (Life Technologies). Whole blood is stimulated
with 2.5 11g/mL of TLR9ligand for 24 hours at 37 oc and 5% C02 (CpG ODN 7909 (InvivoGen,
San Diego, CA)). B cell activation profile is measured by flow cytometry and data is analyzed
using FlowJo software.
Flow cytometry is performed as follows. Treated and activated human whole blood is
lysed with RBC lysis buffer (Fisherscientific, USA). Cells are stained with the appropriate
combination of fluorochrome-conjugated antibodies for 30 min at 4°C to identify B cell
activation markers: CD69 BV605 (cat #310938) from BioLegend. Cells are also stained with
CD3 FITC (cat# 300306), CD19 APC (cat# 363006) all from BioLegend, CD20 PerCP-Cy5.5
(cat# 560736) from BD Pharminogen and fixable viability dye eFluor™ 780 ( eBioscience ). At
least 25,000-50,000 events gated on living cells are analyzed for each sample. Samples are
acquired on a BD Fortessa X-20 and results are analyzed using FlowJo Software.
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The anti-human CD19 mAb CB3finhibits activation of primary human B cells in a dose
dependent manner, which is measured by the reduction of CD69+ B cells. The assay is
performed using blood obtained from three different donors and representative results are shown
in FIG. 4. The average ICso for CB3f in this assay is 0.008 nM (Table 4). The isotype control
antibody did not demonstrate inhibition of CD69 expression at any tested concentration. The data
show that anti-human CD19 mAb CB3f can inhibit activation of primary human B cells in whole
blood in a dose dependent manner, while B cell activation is not affected by the isotype control
antibody.
Table 4. Inhibition ofB cell activation in whole blood by anti-human CD19 mAb CB3f
ICso Values (nM)
Donor 1 0.015
Donor 2 0.0006
Donor 3 0.008
Average 0.008
In vitro inhibition of B cell differentiation into plasmablasts
Memory human B cells are isolated from healthy donor PBMCs using a Memory B cell
Isolation Kit (Miltenyi Biotec). Human primary memory B cells are re-suspended at 1 x 106
cells/mL and cultured at 37 oc in polystyrene 96-well, u-bottom plates in complete medium
(RPMI-1640 containing 10% fetal bovine serum, 1x MEM- nonessential amino acids, 1mM
sodium pyruvate, 1x penicillin-streptomycin solution (all from Corning) and 1x Glutamax
(Gibco), 0.1% ~-mercaptoethanol (Life Technologies). Cells are pre-treated with anti-human
CD19 mAb CB3ffor 1 hour and stimulated with 50 ng/mL anti-CD40, 200 ng/mL BAFF, 1
ng/mL IL-2, 100 ng/mL IL-21 (all from R&D) for 5 days. Cells are washed and stained with the
appropriate combination of fluorochrome-conjugated antibodies for 30 min at 4 °C to identify
differentiation of memory B cells into plasmablasts: CD38 PE, CD3 FITC (cat# 300306), CD19
APC (cat# 363006) all from BioLegend, CD20 PerCP-Cy5.5 (cat.# 560736) from BD
Pharminogen and fixable viability dye eFluor™ 780, eBioscience. At least 25,000-50,000 events
gated on living cells are analyzed for each sample. Samples are acquired on a BD Fortessa X-20
and results are analyzed using FLowJo Software. Percent of plasmablasts is defined as% of
CD38bright/CD2010w B cells.
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The anti-human CD19 mAb CB3finhibits differentiation of primary human memory B
cells into plasmablasts in a dose dependent manner (FIG. 5). The experiment is repeated three
times and the representative data is shown. The isotype control antibody does not demonstrate
inhibition of plasmablast differentiation at any tested concentration. The data show that the CB3f
can inhibit differentiation of primary human memory B cells into plasmablasts in a dose
dependent manner, while the differentiation is not affected by the isotype control antibody.
CD19 mAb CB3fis a non-depleting mAb and lacks activity in in vitro CDC andADCC assays.
The anti-human CD19 mAb CB3fis a B cell inhibitory antibody, which is designed to
inhibit B cell function without causing B cell depletion. In vitro assays are performed to confirm
CB3flacks Complement-Dependent Cytotoxicity (CDC) and Antibody-Dependent Cellular
Cytotoxicity (ADCC) activities, which imply that it does not have depleting function.
Wil2-s cell line expressing CD19 and CD20 are used as target cells, and Jurkat cell lines
expressing functional FcyRIIIa (V158)-NFAT-Luc (Eli Lilly and Company) are used as the
effector cell line. CB3f is tested and an IgG 1 antibody that is a known potent inducer of ADCC
and CDC is used as a positive control.
CB3f is serially-diluted in triplicates starting at 10 11g/mL and 1 11g/mL test
concentrations for the CDC and ADCC assay, respectively. 50 11Liwell test compound or assay
buffer are added to 96-well plate (Costar 3916). Wil2-s cells are diluted to a concentration of
1x106 cells/mL and added 50 11Liwell to plate. CDC and ADCC plates are incubated for 1 hour
at 37°C. Next, Jurkat V158 cells are diluted to a concentration of3x106 cells/mL and added 50
11Liwell for ADCC plate, or 50 11Liwell of pre-diluted complement from human serum (Qui del
A113) for CDC plate. CDC plates are incubated at 37°C for 2 hours followed by addition of 100
11Liwell Cell-Titre Glo (Prom ega G7571). The ADCC plates are incubated at 37°C for 4 hours
followed by addition of 100 11Liwell ONE-Glo (Prom ega E8130). The contents of the plates are
mixed using a plate shaker at low speed, and luminescence signal is read on an Envision 11
multi-mode plate reader using 0.2 cps integration. Data is analyzed using GraphPad Prism v8.2.
The results of the CDC and ADCC assays are shown in FIGs. 6A-6B (representative
results from three independent plate runs). All response levels are classified relative to the
positive control IgG1 antibody. The anti-human CD19 mAb CB3fhas no CDC activity (FIG.
6A) nor ADCC activity (FIG. 6B) at the indicated concentrations.
Specificity of anti-human CD19 mAb binding to B cells in human whole blood.
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The anti-human CD19 mAb CB3fis a B cell inhibitory antibody, which acts by
suppressing B cell function. Obexelimab is an antibody that binds both CD19 and FcyRIIb.
However, high affinity binding of obexelimab to FcyRIIb could lead to binding to other cell
types in a CD19-independent manner. Therefore, the binding specificity of obexelimab and antihuman
CD19 mAb CB3fis compared using a human whole blood binding assay.
EDTA treated human whole blood (Healthy donors, TSRI Normal Blood Donor Services,
San Diego, CA) is plated in polystyrene 96-well and stained with different concentration of
Alexa Flour® 647 conjugated CB3f, Alexa Flour® 647 conjugated and Alexa Flour® 647
conjugated isotype control plus appropriate combination of fluorochrome-conjugated
extracellular antibodies to detect lymphocyte/granulocyte population. 7 nM of each antibody is
used as the highest concentration with 3-fold dilution and 8 points titration in DPBS lx no Ca2
+,
no Mg2+ (Dulbecco's Phosphate-Buffered Saline) supplemented with 2% ofFetal Bovine Serum
(FBS-heat inactivated) both from Coming®. The cocktail includes CD20 PerCP-Cy5.5 (cat#
560736), CD45 BV421 (cat# 563879), CD66 FITC (cat# 555724) all from BD Biosciences.
CD3 BV605 (cat# 317322), CD11b PE-Cy7 (cat# 101216) all from BioLegend and fixable
viability dye eFluor™ 780, (cat# 65-0865-14) from eBioscience. Whole blood plus antibodies
are stained for 30 min at 4°C in the dark. Dead cells are excluded by viability dye and at least
25,000-50,000 events gated on living cells are analyzed. Samples are acquired on BD Fortessa
X-20 and results are analyzed using FlowJo Software.
Flow cytometry analysis demonstrates that CB3fbinds exclusively to B cells in human
whole blood at all tested concentrations (FIG. 7). Obexelimab shows binding to human B cells as
well as a population ofCD20-negative cells (i.e., non-B cells), which express CD66 and CD11b
and thus are identified as neutrophils (FIG. 7). Therefore, the data indicate the anti-human CD19
mAb CB3fhas highly specific binding to human B cells in human whole blood, whereas
obexelimab shows non-specific binding to human neutrophils in addition to B cell binding. Since
neutrophil is the most abundant type of white blood cells in human, the nonspecific binding to
neutrophils might explain the short half-life of obexelimab observed in human patients. The
experiment is repeated using blood from four different donors, representative results are shown
in FIG. 7.
In vitro apoptosis assay.
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As described above, obexilimab was shown to reduce B cell counts in human patients
during clinical studies (Jaraczewska-Baumann, et al., EULAR 2015 Annual Meeting Poster: A
Phase 1 b/2a Study of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of
XmAb®5871 in Patients with Rheumatoid Arthritis, June 12, 2015).
The ability of the anti-human CD19 mAb CB3f and obexilimab to induce apoptosis of
primary human B cells is tested using an in vitro B cell apoptosis assay. Human primary B cells
are isolated from healthy donor PBMCs by negative selection using B cell isolation kit (Stem cell
Technologies). Human primary B cells are re-suspended at 1 x 106 cells/mL and cultured at 37
oc in polystyrene 96-well, u-bottom plates in complete medium (RPMI-1640 containing 10%
Fetal bovine serum, 1x MEM- nonessential amino acids, lmM sodium pyruvate, 1x penicillinstreptomycin
solution (all from Corning) and lx Glutamax (Gibco), 0.1% ~-mercaptoethanol
(Life Technologies). Cell are treated with indicated concentrations ofCB3f, obexilimab, or an
isotype control antibody for 24 hours at 37 °C and 5% C02. B cell apoptosis is measured by
flow cytometry using Annexin V staining in combination with viability dye. Cells are stained
with the appropriate combination of fluorochrome-conjugated antibodies for 30 min at 4 °C to
identify B cell activation markers: CD19 APC (Biolegend), CD20 PerCP-Cy5.5 (BD
Pharminogen), Annexin V (Invitrogen) and fixable viability dye eFluor™ 780 (eBioscience).
Apoptotic cells are defined as live Annexin v+ cells. Samples are acquired on a BD Fortessa X-
20 and results are analyzed using FlowJo Software.
The experiment is repeated using B cells obtained from three different donors and
representative data is shown in FIG. 8. Obexilimab induces B cell apoptosis in a dose-dependent
manner (FIG. 8), which may explain the reduction ofB cell counts in human patients observed in
the clinical studies. In contrast, CB3f induces very little to no apoptosis of human B cells when
compared to the isotype control (FIG. 8). The data indicates that, in contrast to obexilimab, the
anti -human CD 19 mAb CB3f does not induce apoptosis of primary human B cells ex vivo,
suggesting a different mechanism of action for CB3f. This data further indicates that the antihuman
CD19 mAb CB3f acts by inhibiting B cell function without depleting B cells or
significantly reducing B cell numbers.
Example 4. In vivo functional characterization of the anti-human CD19 antibodies.
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Female NSG mice (NOD.Cg-Prkdcscid 112rgtm1Wjl/SzJ, JAX Labs, Stock# 05557) are
housed 3 per cage at 72° C under a 12 hour light: dark cycle and allowed food and water ad
libitum (n=33). Human peripheral blood mononuclear cells (PBMCs) are isolated from LRS
tubes obtained the San Diego Blood Bank (San Diego CA) using SepMate 50 Ficol preparation
tubes according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC).
Freshly isolated PBMCs are suspended in PBS at 1.2 e8 cells/mL and mice are en grafted with
100 11L PBMCs suspension intravenously on day 0 (1.2e7/mouse, n=29); 4 mice are not
administered PBMCs as non-engrafted controls. On Day 1, mice are divided into 3 weight
matched groups and dosed with human lgG4 isotype control or CB3f at 0.01 or 1.0 mg/kg
subcutaneously (200 11Limouse, n= 10, 10, and 9 respectively). Dosing continues once weekly
for the remainder of the experiment. Health checks and body weight measurements are
performed routinely. Blood is collected by tail snip into heparin coated capillary tubes on Days
6 and 10. On Day 15, blood is collected by cardiac puncture under isoflurane anesthesia into
EDTA tubes for F ACS analysis and clarified by centrifugation for plasma analyses. Spleens are
harvested and processed to single cell suspensions for F ACS analyses.
Mice are weighed in a BSL2 hood and assessed for clinical signs of distress 2-3
times/week. Clinical signs common to this model are scruffy hair, hunched body, wasting, and
labored breathing or movement. Body weight change is calculated as a percentage of their
baseline weight: (Day (x) weight/Day 0 weight)* 100.
Blood from the cardiac puncture is collected into EDTA coated tubes, clarified by
centrifugation, and the resultant plasma is stored at -80°C for future processing. Plasma IgM
levels are measured using the Mesoscale Discovery Human Isotyping panel (Rockville
Maryland) according to the manufacturer's instructions.
Single cell suspensions of mouse spleens are used for F ACS analysis. Cells are plated in
96-well plates and stained with the appropriate combination offluorochrome-conjugated
antibodies for 30 min at 4°C to identify B cell activation markers: hCD45-BV421, CD86
BV650, CD3 APC, CD19 FTIC all from BioLegend, CD20 PerCP-Cy5.5 and fixable viability
dye eFluor™ 780. At least 250,000 events gated on living cells are analyzed for each sample.
Samples are acquired on a BD Fortessa X-20 and results are analyzed using FlowJo Software.
Data is graphed and statistics are calculated using Prism Software (GraphPad, San Diego, CA).
Differences in weights between groups are determined by 2-way RM-ANOVA with Tukey's
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post hoc test. Differences in plasma IgM levels are determined by 1-way ANOVA with Tukey's
post hoc test and considered significant if p < 0. 05.
Injection of human PBMCs into NSG mice results in marked engraftment of functional B
cells as measured by secretion of human IgM into the periphery. Human IgM in the IgG4
isotype control treated animals increases rapidly from non-detectable to 1.3 ± 0.1 and 48.8 ± 6.2
11g/mL at Days 6 and 10 post engraftments, respectively. CB3f dosed at 0.01 and 1.0 mg/kg/wk
significantly attenuates the secretion oflgM by 60% and 78% on Day 6 as shown in FIGs. 9A-
9B (p< 0.01 for both doses). A 57% reduction in circulating IgM is also observed on Day 10
with the 1.0 mg/kg/wk dose of CB3f (p <0.05). Mouse weights are not different between groups,
nor does any mouse display signs of GvHD.
Activation of human B cells in NSG mice is measured by the expression of activation
marker CD86. As demonstrated in FIG. 10, treatment with anti-human CD19 mAb CB3freduces
the expression of CD86 on human B cells in a dose-dependent manner.
Treatment with CB3f causes mild reduction in the percentage of splenic B cells, which is
probably due to reduced B cell activation and proliferation. Despite this fact, B cells are present
in spleens in mostly unaltered numbers (>50% of splenocytes), indicating non-depleting nature
of the anti-human CD19 mAb CB3f (FIG. 11).
Example 5. Internalization of the anti-human CD19 antibodies in primary human B cells
Peripheral blood mononuclear cells (PBMCs) are collected from LRS-WBC of healthy
volunteers using standard density gradient centrifugation method. Human primary B cells are
isolated from healthy donor PBMCs by negative selection using B cell isolation kit. For the
internalization study, B cells are resuspended at 4x106cells/mL and 50 11L is added to each well
in a 96 well plate. A labeled F(ab')2 targeting human Ig Fey fragment (F(ab')2-TAMRA-QSY7)
is used as a probe to track internalization. The test antibody is incubated with probe at 4 oc for
30 minutes to form complex and 50 11L is added to the B cells in each well. The final
concentration of the test antibody is 2 11g/mL. Cells are incubated for 24 hat 37 oc in a C02
incubator. Cells are then washed twice with 2% FBS PBS and resuspeneded in 2% FBS PBS
with a viability dye (SYTOX Green, Invitrogen). Data is collected on a BD Fortessa X-20 and
analyzed in FlowJo.Both anti-human CD19 mAbl (CB3f) and anti-human CD19 mAb2 (C323.Cl)
demonstrate internalization in primary human B cells at 24 hours (FIG. 12). An IgG 1 effector
null isotype control antibody is used as a negative control and does not show any significant
internalization as demonstrated by low percentage of TAMRA+ cells (FIG. 12). A positive
control IgG4 mAb demonstrates internalization comparable to the tested anti-human CD19
mAbs (FIG. 12).

CLAIMS:
1. An antibody that binds human CD19, wherein the antibody comprises a heavy chain variable
region (VH) and a light chain variable region (VL ), wherein the VH comprises heavy chain
complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises
light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein
the HCDR1 comprises SEQ ID NO: 29,
the HCDR2 comprises SEQ ID NO: 30,
the HCDR3 comprises SEQ ID NO: 31,
the LCDR1 comprises SEQ ID NO: 32,
the LCDR2 comprises SEQ ID NO: 33, and
the LCDR3 comprises SEQ ID NO: 34.
2. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 37 and the VL comprises
SEQ ID NO: 41.
3. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising
SEQ ID NO: 35 and a light chain (LC) comprising SEQ ID NO: 39.
4. The antibody of claim 1 or 2, wherein the antibody comprises a HC comprising SEQ ID NO:
52 and a LC comprising SEQ ID NO: 39.
5. An antibody that binds human CD19, wherein the antibody comprises a VH and a VL,
wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1,
LCDR2, and LCDR3, wherein
the HCDR1 comprises SEQ ID NO: 29,
the HCDR2 comprises SEQ ID NO: 30,
the HCDR3 comprises SEQ ID NO: 31,
the LCDR1 comprises SEQ ID NO: 43,
the LCDR2 comprises SEQ ID NO: 44, and
the LCDR3 comprises SEQ ID NO: 45.
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6. The antibody of claim 5, wherein the VH comprises SEQ ID NO: 37 and the VL comprises
SEQ ID NO: 48.
7. The antibody of claim 5 or 6, wherein the antibody comprises a HC comprising SEQ ID NO:
35 and a LC comprising SEQ ID NO: 46.
8. The antibody of claim 5 or 6, wherein the antibody comprises a HC comprising SEQ ID NO:
52 and a LC comprising SEQ ID NO: 46.
9. An antibody that binds human CD19, wherein the antibody comprises a VH and a VL,
wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1,
LCDR2, and LCDR3, wherein
the HCDR1 comprises SEQ ID NO: 15,
the HCDR2 comprises SEQ ID NO: 16,
the HCDR3 comprises SEQ ID NO: 17,
the LCDR1 comprises SEQ ID NO: 18,
the LCDR2 comprises SEQ ID NO: 19, and
the LCDR3 comprises SEQ ID NO: 20.
10. The antibody of claim 9, wherein the VH comprises SEQ ID NO: 23 and the VL comprises
SEQ ID NO: 27.
11. The antibody of claim 9 or 10, wherein the antibody comprises a HC comprising SEQ ID
NO: 21 and a LC comprising SEQ ID NO: 25.
12. The antibody of claim 9 or 10, wherein the antibody comprises a HC comprising SEQ ID
NO: 54 and a LC comprising SEQ ID NO: 25.
13. The antibody of any one of claims 1-12, wherein the antibody is a non-depleting antibody.
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14. A nucleic acid comprising a sequence encoding SEQ ID NO: 35, 52, 39, 46, 21, 54, or 25.
15. A vector comprising the nucleic acid of claim 14.
16. The vector of claim 15, wherein the vector comprises a first nucleic acid sequence encoding
SEQ ID NO: 35 or 52, and a second nucleic acid sequence encoding SEQ ID NO: 39 or 46.
17. The vector of claim 15, wherein the vector comprises a first nucleic acid sequence encoding
SEQ ID NO: 21 or 54, and a second nucleic acid sequence encoding SEQ ID NO: 25.
18. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ
ID NO: 35 or 52, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:
39 or 46.
19. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ
ID NO: 21 or 54, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:
25.
20. A cell comprising the vector of claim 16 or 17.
21. A cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:
35 or 52, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39 or
46.
22. A cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:
21 or 54, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25.
23. The cell of any one of claims 20-22, wherein the cell is a mammalian cell.
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24. A process of producing an antibody comprising culturing the cell of any one of claims 20-23
under conditions such that the antibody is expressed and recovering the expressed antibody from
the culture medium.
25. An antibody produced by the process of claim 24.
26. A pharmaceutical composition comprising the antibody of any one of claims 1-13 or 25, and
a pharmaceutically acceptable excipient, diluent or carrier.
27. A method of treating a B cell associated disorder in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of the antibody of any one of
claims 1-13 or 25, or the pharmaceutical composition of claim 26.
28. The method of claim 27, wherein the B cell associated disorder is an autoimmune disease.
29. The method of claim 27, wherein the B cell associated disorder is selected from systemic
lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, idiopathic
thrombocytopenia purpura, Type 1 diabetes, pemphigus vulgaris, meuromyelitis optica, ANCA
vasculitis, or myasthenia gravis.
30. The antibody of any one of claims 1-13 or 25 for use in therapy.
31. The antibody of any one of claims 1-13 or 25, or the pharmaceutical composition of claim
26, for use in the treatment of a B cell associated disorder.
32. The antibody or pharmaceutical composition of claim 31, wherein the B cell associated
disorder is an autoimmune disease.
33. The antibody or pharmaceutical composition of claim 31, wherein the B cell associated
disorder is selected from systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis,
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Sjogren's syndrome, idiopathic thrombocytopenia purpura, Type 1 diabetes, pemphigus vulgaris,
neuromyelitis optica, ANCA vasculitis, or myasthenia gravis.
34. Use of the antibody of any one of claims 1-13 or 25, in the manufacture of a medicament for
the treatment of a B cell associated disorder.
35. The use of claim 34, wherein the B cell associated disorder is an autoimmune disease.
36. The use of claim 34, wherein the B cell associated disorder is selected from systemic lupus
erythematosus, multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, idiopathic
thrombocytopenia purpura, Type 1 diabetes, pemphigus vulgaris, neuromyelitis optica, ANCA
vasculitis, or myasthenia gravis.