(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property

Organization
International Bureau
(10) International Publication Number
(43) International Publication Date WO 2015/169811 A2
12 November 2015 (12.11.2015) P O PCT

(51) International Patent Classification: (74) Agent: TTOFI, Evangelia; Medlmmune Limited, Milstein
C07K 16/28 (2006.01) Building, Granta Park, Cambridge Cambridgeshire CB21
6GH (GB).
(21) International Application Number:
PCT/EP2015/059876 (81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
(22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
5 May 20 15 (05.05.2015)
BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
(25) Filing Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
(26) Publication Language: English KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
(30) Priority Data: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
61/989,218 6 May 2014 (06.05.2014) US PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
(71) Applicant: MEDIMMUNE LIMITED [GB/GB]; M il TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
stein Building, Granta Park, Cambridge Cambridgeshire
CB21 6GH (GB). (84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
(72) Inventors: CARROLL, Danielle; c/o Medlmmune Lim GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
ited, Milstein Building, Granta Park, Cambridge Cam TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
bridgeshire CB21 6GH (GB). ROSSANT, Chris; c/o TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
Medlmmune Limited, Milstein Building, Granta Park, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
Cambridge Cambridgeshire CB21 6GH (GB). BARRY, Si¬ LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
mon, Thomas; c/o AstraZeneca R&D Alderley, Alderley SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
Park, Macclesfield Cheshire SK10 4TG (GB). GW, KM, ML, MR, NE, SN, TD, TG).
[Continued on next page]

(54) Title: ANTI-CXC CHEMOKINE RECEPTOR-2 BINDING MOLECULES AND USES THEREOF
(57) Abstract: This disclosure relates to an anti-CXCR2
binding molecules and uses thereof, in particular in treatment
of cancers and inflammatory diseases. Furthermore, the dis
closure provides compositions and methods for treating can
cers and inflammatory diseases.
 WO 2015/169811 A2 III III II II II I IIII
Published:
— without international search report and to be republished
upon receipt of that report (Rule 48.2(g))
ANTI-CXC CHEMOKINE RECEPTOR-2 BINDING MOLECULES AND USES
THEREOF

BACKGROUND

Field of the Disclosure

[0001] This disclosure relates to novel isolated humanized antibodies, or the derived
functional fragments capable of binding to and modulating signaling from CXC
chemokine receptor-2 (CXCR2) for the treatment of cancers and inflammatory diseases.
Particularly, the present invention relates to antibodies as well as their use for the
treatment of cancer and inflammatory disease. Pharmaceutical compositions composed
of such antibodies and a process for the selection of such antibodies are also covered.

Background of the Disclosure

[0002] Chemokines are a family of low molecular weight secreted proteins (8-15 kDa)
that control cellular trafficking, particularly the migration of leucocytes, under both
physiological and pathological conditions (Luster, N Engl J Med. 338(7):436-45 (1998)).
Chemokines can be classified into four distinct groups depending on the positioning of
the cysteine residues in the amino terminus of the protein. The family members comprise
CXC, CC, XC, and CX3C chemokines (Moser and Willimann, Ann Rheum Dis. Nov;63
Suppl 2:ii84-ii89 (2004)) of which CXC and CC are the largest and most characterized.
CXC and CC chemokines are distinguished by the arrangement of these cysteine residues,
either separated by a single amino acid (CXC) or in adjacent positions (CC) and they bind
receptors, designated CXCR and CCR respectively.
[0003] Chemokines mediate their biological effects via binding to cell surface molecules,
which belong to the superfamily of seven-transmembrane spanning receptors that signal
through coupling to heterotrimeric G proteins. The majority of chemokine receptors
recognize more than one chemokine but are generally restricted to a single subgroup
(CXC, CC, XC, or CX3C). Chemokine binding to its G-protein coupled receptor (GPCR)
induces a conformational change in the receptor resulting in dissociation of the receptor-
associated heterotrimeric G proteins. This in turn initiates a cascade of intracellular
events including modulation of adenylyl cyclase activity, cytosolic calcium levels and
 protein kinase activity. These intracellular events mediate a wide range of functions in
diverse leukocyte populations driving chemotaxis, degranulation, oxidative burst,
phagocytosis, and lipid mediator synthesis.
[0004] The chemokine receptor CXCR2 binds to a number of CXC chemokines with the
conserved glutamic acid-leucine-arginine (ELR+) sequence motif including interleukin-8
(IL-8/CXCL8), growth-related oncogenes (Gro-oc/CXCLl, Gro-p/CXCL2 and Gro-
Y/CXCL3), epithelial cell-derived neutrophil activating factor-78 (ENA-7 8/CXCL5 ),
granulocyte chemoattractant protein-2 (GCP-2/CXCL6) and neutrophil- activating
protein-2 (NAP-2/CXCL7). While IL-8 and GCP-2 are potent agonists of both the
CXCR1 and CXCR2 receptors, which share approximately 77% amino acid identity, the
other ligands show selectivity for CXCR2 over CXCR1. (Rajagopalan, and
Rajarathnam.,7 Biol Chem. 16; 279 (29):30000-8 (2004)). Both CXCR1 and 2 are
expressed at the highest levels on human neutrophils, but lower expression is observed
elsewhere on many other cell types including but not limited to monocytes, fibroblasts,
endothelial and tumor cells. Ligand binding to CXCR2 stimulates coupling with Gi
family of Guanine nucleotide binding proteins which results in the inhibition of adenylyl
cyclase activity and release of intracellular inositol phosphates leading to increases in
intracellular Ca2+ levels (Hall et al, Br J Pharmacol. Feb;126(3):810-8 (1999)).
[0005] Following agonist stimulation CXCR2 is phosphorylated and internalized through
arrestin/dynamin dependent mechanisms leading to receptor ligand uncoupling, and
receptor degradation or recycling (Feniger-Barish et ah, Cytokine. 11(12):996-1009
(1999), (Yang et al, J Biol Chem. 16; 274 (16): 11328-33 (1999)).
[0006] Under normal conditions CXCR2 regulates neutrophil homeostasis as well as
recruitment and release from the bone marrow as part of a response to acute inflammation
and injury [Eash et a . J Clin Invest. 120:2423-31 (2010). The CXCR2 axis (receptors
and ligands) have been implicated in a range of inflammatory disorders and has been
considered as a therapeutic target for multiple pathologies. CXCR2 has an important role
in acute lung injury (ALI), asthma, chronic obstructive pulmonary disease (COPD) and
cystic fibrosis (CF). In many of these conditions, CXC chemokines and CXCR2
expression are increased and important roles have been identified. For example, in
animal models of ALI, exposure to hyperoxic gas injury leads to neutrophil recruitment,
increased airway microvascular leakage and lung edema which can be reversed or
 inhibited using a CXCR2 neutralizing polyclonal antibody (Sue et al..; J Immunol.
172(6): :3860-890 (2004), Strieter et al. Curr Drug Targets Inflamm Allergy. ;4(3): 299-
303 (2005)). CXCR2 has been shown to be important in the response to viral and
bacterial infections during asthma exacerbations and may play a role in the development
of bronchial hyperresponsiveness and increased vascularity. In CF, where decreased lung
clearance is a clear cause of pathology and recurrent lung infections with bacterial
pathogens are common, studies indicate that CXCR2 may be involved in mucous
hypersecretion as well as proliferation of mucous-secreting cells. Other inflammatory
diseases where the CXCR2 axis is thought to play a role include: gram negative sepsis
where decreased expression of CXCR2 is observed alongside reduced neutrophil
migration and activation (Cummings et al., J Immunol. 162(4):2341-6 (1999), (Ness et
al., J Immunol.; 171(7):3775-84 (2003) inflammatory bowel disease (IBD) and arthritis
(Boppana et al., Exp Biol Med (Maywood). Mar 13 (2014)), and in the response to viral
infections (Londhe et al., J Inflamm (Lond). 2(1):4.(2005))
[0007] CXCR2 and its chemokine ligands have been implicated in multiple cancer types
(D. Raman et al., Cancer Lett. 256(2): 137-65. (2007), (Hertzer et al, Expert Opin Ther
Targets. 17(6):667-80 (2013)). CXCR2 and its ligands are over-expressed in a large
number of cancers including but not limited to colon (Li A, et al., Clin Cancer Res.
7(10):3298-304 (2001); Erreni M, et al., Methods Enzymol. 460:105-21 (2009)), breast
(Nannuru KC, et al., J Carcinog. 10:40 (2011)), prostate (Murphy C, et al., Clin Cancer
Res. 11(1 1):41 17-27 (2005)), lung , both small-cell- and non-small-cell- carcinoma
(Wislez M, et al., Cancer Res. 66(8):4198-207) (2006 ) (Yanagawa J, et al., Clin Cancer
Res. 15(22):6820-9 (2009)), ovary (Yang G, et al., Clin Cancer Res. 16(15):3875-86
(2010)), pancreas (Wente MN, et al., Cancer Lett. 241:221-7 (2006)), kidney (Mestas J,
et al., J Immunol. 175(8):535 1-7 (2005)), and brain cancer (Robinson S, et al.,
Neurosurgery 48(4):864-73 (2001)).
[0008] There is clear evidence that CXCR2 and its associated ligands are important for
multiple biological processes important in cancer cell biology. CXC chemokines and
CXCR2 modulate tumor behavior by several important mechanisms: recruitment of pro-
tumorigenic immune infiltrate (neutrophils, MDSCs, MCSs, Macrophages and CAFs). In
a breast cancer tumor model infiltrating macrophages have increased expression of
inflammatory chemokines that bind CXCR2 and these macrophages are capable of
 promoting tumor cell migration and invasion through the activation of CXCR2 (Bohrer
et al, Mol Cancer Res. 10(10): 1294-305 (2012). CXCR2 ligands and CXCR2 have been

implicated in chemoresistance and cancer cell survival mechanisms. In a breast cancer

model cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21
amplification are primed for survival in metastatic sites. Knocking down the expression
of CXCL1 and CXCL2 or CXCR2 blockage reduced tumor volume and lung metastasis
in both models and enhanced the efficacy of chemotherapy particularly against metastatic
lesions (Acharyya et al, Cell 150(1): 165-78 (2012)). Studies in non-small-cell lung
cancer (NSCLC) support a role for CXCR2 in tumor growth and angiogenesis in mice
(Keane et al. J Immunol.; 172:2853-60 (2004) as well as in non-pathological
angiogenesis (Strieter et al, J Biol Chem. 270(45):27348-57 (1995)). CXCR2 has also
been implicated in the activation of a tumor- specific immune response (Peng et al., Clin
Cancer Res. 16(22):5458-68 (2010)) and stimulation of tumor cell proliferation in an
autocrine or paracrine fashion (Murphy et al., Clin Cancer Res ll(ll):4l 17-4127
(2005) ) .
[0009] Inhibition of CXCR2 or CXCR2 gene ablation has demonstrated the potential
impact that selective CXCR2 antagonists can have on inflammatory disorders including
cancer. CXCR2 deficiency suppressed inflammation-driven tumorigenesis in both skin
and intestinal models, as well as spontaneous genetic driven cancer models similar results
were also obtained with a peptide antagonist (a class of cell-penetrating lipopeptides
based on the third intracellular loop of GPCRs that target chemokine receptors and
selectively inhibits signaling) (Jamieson et al, J. Clin. Invest. 122(9):3 127-44 (2012)).
Furthermore blockade of ELR+ CXC chemokines and CXCR2 reduces
tumorigenesis/angiogenesis in lung, esophageal, renal cell, colon and pancreatic cancers
(Keane et al. J Immunol.; 172:2853-60 (2004), Wislez et al, Cancer Res. 66(8):4198-207
(2006) ), Miyazaki et al.,Cancer R es.;66(8):4279-84 (2006)).
[0010] Chemokine receptors in general have proved to be difficult targets to antagonize
and it has taken a significant effort to identify potent, selective CXCR2 antagonists. Since
1998 many small molecular weight CXCR2 antagonist have been described, (Dwyer and
Yu Expert Opin Ther Pat. Feb 20 (2014) several of which have now progressed into
clinical trials. Nevertheless there remains a clear a need for better and more potent
selective antagonists of CXCR2 function including antagonistic monoclonal antibodies.
 Thus, there remains a need in the art for the development of novel strategies for the
identification of new CXCR2- specific binding molecules and therapeutic agents.

BRIEF SUMMARY

[0011] Novel binding molecules and antibodies or antigen-binding fragments thereof

which specifically bind to CXC chemokine receptor-2 (CXCR2), and methods of making
and using the same are provided herein.
[0012] In certain aspects, an isolated binding molecule or antigen-binding fragment
thereof which specifically binds to CXC chemokine receptor-2 (CXCR2), wherein the
binding molecule (a) can inhibit binding to interleukin 8 (IL-8), (b) can inhibit binding to
growth-related protein alpha (Gro-alpha), (c) can inhibit the IL-8 or Gro-cc induced
calcium responses, (d) can inhibit IL-8, Gro- γ , Gro- β , Gro- γ , ENA-78, GCP-2 and NAP-2
induced beta-arrestin recruitment in a mammalian cell, or (e) any combination thereof are
provided herein. In one aspect, the binding molecule for fragment thereof binds to
CXCR2 as expressed on the surface of a mammalian cell. In certain aspects, the binding
molecule or fragment thereof of comprises an antibody or antigen-binding fragment
thereof. In certain aspects, the antibody or antigen binding fragment thereof specifically
binds an epitope comprising amino acids 30-39 and amino acids 279-289 of SEQ ID
NO:37. In certain aspects, the antibody or antigen binding fragment thereof specifically
binds an epitope comprising amino acids 30-39 of SEQ ID NO:37. In certain aspects, the
antibody or antigen binding fragment thereof specifically binds an epitope comprising
amino acids 31-35 of SEQ ID NO:37. In certain aspects, the antibody or antigen binding
fragment thereof specifically binds an epitope comprising amino acids 34 and 35 of SEQ
ID NO:37. In one aspect, the antibody or antigen binding fragment thereof is in contact
with residues L34 and D35 of SEQ ID NO:37. In another aspect, the antibody or antigen-
binding fragment thereof specifically binds to the same CXCR2 epitope as an antibody or
antigen-binding fragment thereof comprising the heavy chain variable region (VH) and
i ht chain variable region (VL) region of HY29 or HY29GL or any combination thereof.
In a further aspect, the antibody or antigen-binding fragment thereof specifically binds to
 CXCR2, and competitively inhibits CXCR2 binding by an antibody o antigen-binding
fragment thereof comprising the V and VL of HY29, HY29GLor any combination
thereof.
[0013] In certain aspects, the antibody or antigen-binding fragment thereof comprises a

V and a VL, wherein the V comprises a V complementarity determining region- 1

(VHCDR 1) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 3 for HY29 or SEQ ID NO: 2 for HY29GL. In certain
aspects, the antibody or antigen-binding fragment thereof comprises a V and a VL,
wherein the V comprises a VHCDR 1 amino acid sequence identical to SEQ ID NO: 3
for HY29 or SEQ ID NO: 2 1 for HY29GL. I certain aspects, the antibody or antigen-
binding fragment thereof comprises a VH and a VL, wherein the V comprises a V
complementarity determining region-2 (VHCDR2) amino acid sequence identical, except
for four, three, two, or one amino acid substitutions to: SEQ ID NO: 4 for HY29 or SEQ
I NO: 22 for HY29GL. In certain aspects, the antibody or antigen-binding fragment
thereof comprises a V and a VL, wherein the V comprises a VHCDR2 amino acid
sequence identical to SEQ ID NO: 4 for HY29 or SEQ ID NO: 2 1 for HY29GL. In
certain aspects, the antibody or antigen-binding fragment thereof comprises a V and a
VL, wherein the V comprises a V complementarity determining region-3 (VHCDR3)
amino acid sequence identical, except for four, three, two, or one amino acid substitutions
to: SEQ ID NO: 5 for HY29 or SEQ ID NO: 23 for HY29GL. In certain aspects, the
antibody or antigen-binding fragment thereof comprises a V and a VL, wherein the V
comprises a VHCDR3 amino acid sequence identical to SEQ ID NO: 5 for HY29 or SEQ
ID NO: 22 for HY29GL.
[0014] In certain aspects, the antibody or antigen-binding fragment thereof comprises a

VI and a VL, wherein the VL comprises a VL complementarity determining region - 1

(VLCDR ) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 12 for HY29 or SEQ ID NO: 30 for HY29GL. In certain
aspects, the antibody or antigen-binding fragment thereof comprises a V and a VL,
wherein the VL comprises a VLCDR I amino acid sequence identical to SEQ D NO: 12
for HY29 or SEQ ID NO: 30 for HY29GL. In certain aspects, the antibody or antigen-
binding fragment thereof comprises a V and a VL, wherein the VL comprises a VL
complementarity determining region-2 (VLCDR2) amino acid sequence identical, except
fo fou , three, two, or one amino acid substitutions to: SEQ ID NO: 13 for HY29 or SEQ
ID NO: 3 1 for HY29GL. In certain aspects, the antibody or antigen-binding fragment

thereof comprises a VH and a VL, wherein the VL comprises a VLCDR2 amino acid
secjuence identical to SEQ ID NO: 3 for HY29 or SEQ ID NO: 3 1 for HY29GL. In

certain aspects, the antibody or antigen-binding fragment thereof comprises a V and a

VL, wherein the VL comprises a VL complementarity determining region-3 (VLCDR3)
amino acid sequence identical, except for four, three, two, or one amino acid substitutions
to: SEQ ID NO: 4 for HY29 or SEQ ID NO: 32 for HY29GL. I certain aspects, the
antibody or antigen-binding fragment thereof comprises a V and a VL, wherein the VL
comprises a VLCDR3 amino acid sequence identical to SEQ ID NO: 4 for HY29 or

SEQ ID NO: 32 for HY29GL.

In certain aspects, the antibody or antigen-binding fragment thereof comprises a

V and a VL, wherein the V comprises a VHCDR I, a VHCDR2, and a VHCDR3

identical, except for four, three, two, or one amino acid substitutions in one or more
VHCDRs to: SEQ ID NOs: 3, 4, and 5 for HY29, respectively or SEQ D NOs: 2 1, 22,
and 23 for HY29GL, respectively. I certain aspects, the antibody or antigen-binding
fragment thereof comprises a V and a VL, wherein the V comprises VHCDR 1,
VHCDR2, and VHCDR3 amino acid sequences identical to: SEQ ID NOs: 3 4, and 5 for
HY29, respectively or SEQ ID NOs: 2 1, 22, and 23 for HY29GL, respectively. In

certain aspects, the antibody or antigen-binding fragment thereof comprises a V I and a

VL, wherein the VL comprises a VLCDR 1, a VLCDR2, and a VLCDR3 identical, except
for four, three, two, or one amino acid substitutions in one or more VL CDRs to: SEQ ID
NOs: 2, 3, and 4 for HY29, respectively or SEQ ID NOs: 30, 3 1, and 32 for HY29GL,
respectively. I certain aspects, the antibody or antigen-binding fragment thereof
comprises a V and a VL, wherein the VL comprises VLCDR I, VLCDR2, and VLCDR3
amino acid sequences identical to: SEQ ID NOs: 2, 3, and 4 for HY29, respectively or
SEQ ID NOs: 30, 3 , and 32 for HY29GL, respectively. In certain aspects, the antibody
or antigen-binding fragment thereof of comprises a V and a VL comprising a VHCDR I,
a VHCDR2, a VHCDR3, a VLCDR 1, a VLCDR2, and a VLCDR 3 identical, except for
four, three, two, or one amino acid substitutions i one or more CDRs to: SEQ D NOs: 3,
4, 5, 2, 3, and 4 for HY29, respectively or SEQ ID NOs: 20, 2 1, 22, 30, 3 , and 32 for
HY29GL, respectively. In certain aspects, the antibody or antigen-binding fragment
 thereof comprises a and a L comprising VHCDR 1, VHCDR2, VHCDR3,
VLCDR1, VLCDR2, and VLCDR3 amino acid sequences identical to: SEQ ID NOs: 3,
4, 5, 12, 13, and 14 for HY29, respectively or SEQ ID NOs: 20, 2 1, 22, 30, 3 1, and 32 for
HY29GL, respectively.
[0016] In certain aspects, the antibody or antigen-binding fragment thereof comprises a
V I and a VL, wherein the V comprises an amino acid sequence at least 75%, 80%,
85%, 90%, or 95% identical to SEQ ID NO: 2 for HY29 or SEQ ID NO: 20 for HY29GL.
In certain aspects, the antibody or antigen-binding fragment thereof comprises a V and a
VL, wherein the V comprises the amino acid sequence of SEQ D NO: 2 for HY29 or
SEQ D NO: 20 for HY29GL. In certain aspects, the antibody or antigen-binding
fragment thereof comprises a V and a VL, wherein the VL comprises an amino acid
sequence at least 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1 I for HY29 or
SEQ D NO: 29 for HY29GL. In certain aspects, the antibody or antigen-binding

fragment thereof comprises a V and a VL, wherein the VL comprises the amino acid
sequence of SEQ ID NO: 1 for HY29 or SEQ ID NO: 29 for HY29GL. I certain
aspects, the antibody or antigen-binding fragment thereof comprises a V and a VL,
wherein the V and VL comprises the amino acid sequences of SEQ D NO: 2 and SEQ
NO: I 1 for HY29, respectively or the amino acid sequences of SEQ NO: 20 and

SEQ ID NO: 29 for HY29. 1_GL, respectively. In certain aspects, the antibody or
fragment thereof comprises a VH with the amino acid sequence SEQ D NO: 20 and a VL
with the amino acid sequence of SEQ D N O :29.
[0017] In certain aspects, the antibody or fragment thereof specifically binds to CXCR2
with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10 -2 M,
10 2 M, 5 x 10 3 M, 10 3 M, 5 x 10 4 M, 10 4 M, 5 x 10 5 M, 10 5 M, 5 x 10 6 M, 10 6 M, 5 x
10 7
M, 10 7 M, 5 X 10 8 M, 10 8 M. 5 x 10 9 M, 10 9 M, 5 x 10 10 M, 10 10 M, 5 x 10 11 M,
10 11 M, 5 X 10 12 M, 10 12 M, 5 x 10 13 M, 10 13 M, 5 x 10 14 M, 10 14 M, 5 x 10 15 M, or
10 15 M . In certain aspects, the antibody or fragment thereof is humanized or chimeric.
n certain aspects, the antibody or fragment thereof has V and VL domains that are fully
human and the antibody constant regions are derived non-human mammal. In certain

aspects, the antibody or fragment thereof is fully human. In certain aspects, the antibody
fragment is an Fab fragment, an Fab' fragment, a F(ab)2 fragment, or a single chain Fv
(scFv) fragment.
[0018] In certain aspects, the antibody or fragment thereof comprises a light chain
constant regions selected from the group consisting of a human kappa constant region and
a human lambda constant region. In certain aspects, the antibody or fragment thereof
comprises at a heavy chain constant region or fragment thereof. In one aspect, the heavy
chain constant region or fragment thereof is human IgGl.
[0019] In certain aspects, the antibody or a fragment thereof is monoclonal. In certain
aspects, the antibody or fragment thereof comprises a VH with the amino acid sequence
SEQ D O: and a VL with the amino acid sequence of SEQ D NO: 1. I certain
aspects, the antibody or fragment thereof comprises a human kappa light chain constant
region or fragment thereof and a human IgGl heavy chain constant region or fragment
thereof. I certain aspects, the antibody or fragment thereof comprises a human lambda
light chain constant region or fragment thereof and a human IgGl heavy chain constant
region or fragment thereof.
[0020] In certain aspects, the antibody or fragment thereof is conjugated to an agent
selected from the group consisting of antimicrobial agent, a therapeutic agent, a prodrug,
a peptide, a protein, an enzyme, a lipid, a biological response modifier, pharmaceutical
agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label,
polyethylene glycol (PEG), and a combination of two or more of any said agents. In
certain aspects, the detectable label is selected from the group consisting of an enzyme, a
fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or
a combination of two or more of any said detectable labels.
[0021] In certain aspects, a composition comprises the antibody or a fragment thereof and
a carrier. In certain aspects, a isolated polynucleotide comprises a nucleic acid encoding
the VH. In certain aspects, the polynucleotide further comprises a nucleic acid encoding
the VL where the binding molecule or antigen-binding fragment thereof expressed by the
polynucleotide specifically binds CXCR2.
[0022] In certain aspects, an isolated polynucleotide comprises a nucleic acid encoding
the VL of any one of claims 1 to 59. In certain aspects, the polynucleotide further
comprises a nucleic acid encoding the V where the binding molecule or antigen-binding
fragment thereof expressed by the polynucleotide specifically binds CXCR2.
[0023] In certain aspects, a vector comprises the polynucleotide. In certain aspects, the
vector further comprises one or more promoters operably associated with one or more of
 the nucleic acids. In certain aspects, the vector comprises one or more promoters

operably associated with said polynucleotide, wherein the vector can express a binding
molecule that specifically binds CXCR2 in a suitable host cell. In certain aspects, a host
cell comprises the polynucleotide or the vector.
[0024] In certain aspects, a method of producing a binding molecule or antigen-binding

fragment thereof that specifically binds CXCR2, comprising culturing the host cell, and
recovering the binding molecule or fragment thereof are described herein. I certain
aspects, a binding molecule or antigen-binding fragment thereof which specifically binds
CXCR2 is produced by the method.
[0025] In certain aspects, a method of treating cancer in a subject in need thereof,

comprising administering to the subject an effective amount of the binding molecule or

fragment thereof, the antibody or fragment thereof, the composition, the polynucleotide,
the vector, or the host cell are described herein. In certain aspects, the cancer is

lymphoma, colon cancer, lung cancer melanoma, or pancreatic cancer. In certain aspects,
the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0026] FIG. 1. Inhibition of IL-8 (a), Gro - (b) and ENA-78 (c) induced calcium flux by
ZY05KA-G08 Antibody.
[0027] FIG. 2 . Representative data showing binding of ZY05KA-G08 antibody to HEK
cells over-expressing human CXCR2 (a) cynomolgus CXCR2 (b), mouse CXCR2 (c) or
human CXCR 1 (d).
[0028] FIG. 3 . Binding of IIY29 to human neutrophils and neutrophils isolated from
cynomolgus monkeys. Antibodies HY29, 6C6 (CD 182) and matched isotype control
antibodies NIP228 and mouse IgG I were assessed for binding to human neutrophils (a
and b) and neutrophils isolated from cynomolgus monkeys (c and d). Unstained control
cells and cells stained with the secondary detection fluorophore only are included for
reference.
[0029] FIG 4 . Example of IL-8 (a) and Gro-cx (b) dose response curves in the presence of
increasing concentrations of HY29 Antibody.
[0030] FIG. 5 . Example of competition of antibodies with DyLight-649 labelled HY29
(a) and DyLight-650 labelled 6C6 (b) fo binding to HEK human CXCR2 cells.
[0031] FIG. 6 . Binding of HY29 Antibody to Structured Peptides (a) Binding to linear
and CLIPS peptides derived from N terminus, ECL1, ECL2 and ECU (b) CLIPS
peptides with highest antibody binding levels by ELISA (c) Linear mapping of HY29
binding to the CXCR2 N-terminus (d) Alanine mutation analysis of HY29 binding to
CXCR2 N-terminus and ECL3. Values o left are signal obtained in the assay.
[0032] FIG 7 . HY29 Inhibition of Gro-cx and IL-8 driven human neutrophil chemotaxis
[0033] FIG. 8 . Effect of HY29 on LPS-induced changes in BALF total cell (a) and
neutrophil (b). Groups of male or female wild type (wt) or hCXCR2 transgenic (Tg) mice
were dosed i.p. with vehicle or HY29. 24h post dose mice were challenged by intranasal
administration with vehicle (PBS) or LPS. A BAL was performed 24h after LPS
challenge. Data are presented as individual points for each animal with mean values
(horizontal lined, fold increase (parentheses), percent inhibition and respective p- values
shown.
[0034] FIG. 9 . Effect of HY29 on pro-inflammatory cytokines in BAL fluid (a) and lung
homogenates (b)
[0035] FIG. lO.Tumor growth inhibition (i) and neutrophil pharmacodynamics (ii) in
syngeneic mouse tumor models in human CXCR2 transgenic mice
[0036] FIG. 11. Tumor growth inhibition in KRAS mutant lung cancer patient derived
xenograft tumor model.
[0037] FIG. 2 . HY29 inhibition of BxPC3 pancreatic cancer cell invasion. The figure
shows cell counts of BxPC-3 cell invasion in response to CXCL ligands for ligand treated
and untreated controls. Pre-treatment with 2.8ug/ml HY29mAb inhibits BxPC-3 cell
invasion.
[0038] FIG. 3 . HY29 inhibition of pancreatic cancer cell growth i 3D. The figure
shows 4 day viability of four pancreatic tumor cell lines in 3 matrigel overlay culture.
Images were captured under phase contrast microscopy. Upper panel shows phase
contrast micrograph, lower panel shows viability (left hand panel ) and 3D structure
number (right panel ). Significance was tested by one way ANOVA, error bars represent
standard deviation.
[0039] FIG. 14. IL8rb humanization. Targeting strategy allows generation of humanized
allele. Mouse 1 8 b ex on 2 contains the open reading frame (ORF); ex on I is annotated as
a 5' non-coding exon. Mouse 1 8 b open reading frame in exon 2 has been replaced with
its human counterpart from translation initiation codon to termination codon. Puromycin
resistance cassette has been flanked b y F3 recombination sites and inserted in intron 1.
Humanized allele after Flp-mediated removal of selection marker. Human I18rb ORF has
been PGR amplified using human genomic DNA as template.

DETAILED DESCRIPTION

I. DEFINITIONS

[0040] It is to be noted that the term "a" or "an" entity refers to one or more of that entity;

for example, "a binding molecule which specifically binds to CXCR2," is understood to
represent one or more binding molecules which specifically bind to CXCR2. As such, the
terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0041] As used herein, the term "polypeptide" is intended to encompass a singular
"polypeptide" as well as plural "polypeptides," and refers to a molecule composed of
monomers (amino acids) linearly linked b y amide bonds (also known as peptide bonds).
The term "polypeptide" refers to any chain or chains of two or more amino acids, and
does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or
chains of two or more amino acids, are included within the definition of "polypeptide,"
and the term "polypeptide" can be used instead of, or interchangeably with any of these
terms. The term "polypeptide" is also intended to refer to the products of post-expression
modifications of the polypeptide, including without limitation glycosylation, acetylation,
phosphorylation, amidation, derivatization b y known protecting/blocking groups,
proteolytic cleavage, or modification b y non-naturally occurring amino acids. A
polypeptide can be derived from a natural biological source or produced by recombinant
technology, but is not necessarily translated from a designated nucleic acid sequence. It
can be generated in any manner, including b y chemical synthesis.
[0042] A polypeptide as disclosed herein can he of a size of about 3 or more, 5 or more,
10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more,
500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a
defined three-dimensional structure, although they do not necessarily have such structure.
Polypeptides with a defined three-dimensional structure are referred to as folded, and
polypeptides that do not possess a defined three-dimensional structure, but rather can
adopt a large number of different conformations, and are referred to as unfolded. As used
herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate
moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing
side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
[0043] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is
intended a polypeptide that is not in its natural milieu. No particular level of purification
is required. For example, an isolated polypeptide can be removed from its native or
natural environment. Recombinantly produced polypeptides and proteins expressed, e.g.,
from cDNA clones in non-native host cells are considered isolated as disclosed herein, as
are native or recombinant polypeptides which have been separated, fractionated, or
partially or substantially purified by any suitable technique.
[0044] Other polypeptides disclosed herein are fragments, derivatives, analogs, or
variants of the foregoing polypeptides, and any combination thereof. The terms
"fragment," "variant," "derivative" and "analog" when referring to a binding molecule
such as an antibody which specifically binds to CXCR2 as disclosed herein include any
polypeptides which retain at least some of the antigen-binding properties of the
corresponding native antibody or polypeptide. Fragments of polypeptides include, for
example, proteolytic fragments, as well as deletion fragments, in addition to specific
antibody fragments discussed elsewhere herein. Variants of a binding molecule, e.g., a
antibody that specifically binds to CXCR2 as disclosed herein include fragments as
described above, and also polypeptides with altered amino acid sequences due to amino
acid substitutions, deletions, or insertions. Variants can occur naturally or be non-
naturally occurring. Non-naturally occurring variants can be produced using art-known
mutagenesis techniques. Variant polypeptides can comprise conservative or non-
conservative amino acid substitutions, deletions or additions. Derivatives of a binding
molecule, e.g., an antibody that specifically binds to CXCR2 as disclosed herein are
 polypeptides that have been altered so as to exhibit additional features not found on the
native polypeptide. Examples include fusion proteins. Variant polypeptides ca also be
referred to herein as "polypeptide analogs." As used herein a "derivative" of a binding
molecule, e.g., an antibody that specifically binds to CXCR2 refers to a subject
polypeptide having one or more residues chemically derivatized by reaction of a
functional side group. Also included as "derivatives" are those peptides which contain one
or more naturally occurring amino acid derivatives of the twenty standard amino acids.
For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be
substituted for lysine: 3-methylhistidine ca be substituted for histidine: homoserine can
be substituted for serine; and ornithine can be substituted for lysine.
[0045] The term "polynucleotide" is intended to encompass a singular nucleic acid as
well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct,
e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise
a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond,
such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one
or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or
RNA, which has been removed from its native environment. For example, a recombinant
polynucleotide encoding a binding molecule, e.g., an antibody that specifically binds to
CXCR2 contained in a vector is considered isolated as disclosed herein. Further
examples of an isolated polynucleotide include recombinant polynucleotides maintained
in heterologous host cells or purified (partially or substantially) polynucleotides in

solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of

polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules
produced synthetically. In addition, polynucleotide or a nucleic acid can be or can

include a regulatory element such as a promoter, ribosome binding site, or a transcription

terminator.
[0046] As used herein, a "coding region" is a portion of nucleic acid that consists of
codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not
translated into an amino acid, it can be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites, transcriptional
terminators, nitrons, and the like, are not part of a coding region. Two or more coding
 regions can he present in a single polynucleotide construct, e.g., on a single vector, or in
separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any
vector can contain a single coding region, or can comprise two or more coding regions,
e.g., a single vector can separately encode an immunoglobulin heavy chain variable
region and an immunoglobulin light chain variable region. In addition, a vector,

polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or
unfused to a nucleic acid encoding an a binding molecule which specifically binds to
CXCR2, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof.
Heterologous coding regions include without limitation specialized elements or motifs,
such as a secretory signal peptide or a heterologous functional domain.
[0047] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of

DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide normally

can include a promoter and/or other transcription or translation control elements operably
associated with one or more coding regions. An operable association is when a coding
region for a gene product, e.g., a polypeptide, is associated with one or more regulatory
sequences i such a way as to place expression of the gene product under the influence or
control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding
region and a promoter associated therewith) are "operably associated" if induction of
promoter function results in the transcription of mRNA encoding the desired gene product
and if the nature of the linkage between the two DNA fragments does not interfere with
the ability of the expression regulatory sequences to direct the expression of the gene
product or interfere with the ability of the DNA template to be transcribed. Thus, a
promoter region would be operably associated with a nucleic acid encoding a polypeptide
if the promoter was capable of effecting transcription of that nucleic acid. The promoter
can be a cell-specific promoter that directs substantial transcription of the DNA only in
predetermined cells. Other transcription control elements, besides a promoter, for
example enhancers, operators, repressors, and transcription termination signals, can be
operably associated with the polynucleotide to direct cell-specific transcription. .Suitable
promoters and other transcription control regions are disclosed herein.
[0048] A variety of transcription control regions are known to those skilled in the art.
These include, without limitation, transcription control regions that function in vertebrate
cells, such as, but not limited to, promoter and enhancer segments from
 cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian
virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other
transcription control regions include those derived from vertebrate genes such as act in,
heat shock protein, bovine growth hormone and rabbit β -globin, as well as other
sequences capable of controlling gene expression in eukaryotic cells. Additional suitable
transcription control regions include tissue-specific promoters and enhancers as well as
lymphokine-inducible promoters (e.g., promoters inducible by interferons or
interleukins).
[0049] Similarly, those of ordinary skill in the art know a variety of translation control
elements. These include, but are not limited to ribosome binding sites, translation
initiation and termination codons, and elements derived from picornaviruses (particularly
an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
[0050] In other embodiments, a polynucleotide can be RNA, for example, in the form of

messenger RNA (i RNA).

[0051] Polynucleotide and nucleic acid coding regions ca be associated with additional
coding regions which encode secretory or signal peptides, which direct the secretion of a
polypeptide encoded by a polynucleotide as disclosed herein, e.g., a polynucleotide
encoding a binding molecule which specifically binds to CXCR2, e.g., an antibody, or
antigen-binding fragment, variant, or derivative thereof. According to the signal
hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory
leader sequence which is cleaved from the mature protein once export of the growing
protein chain across the rough endoplasmic reticulum has been initiated. Those of
ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally
have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from
the complete or "full length" polypeptide to produce a secreted or "mature" form of the
polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin
heavy chain or light chain signal peptide is used, or a functional derivative of that
sequence that retains the ability to direct the secretion of the polypeptide that is operably
associated with it. Alternatively, a heterologous mammalian signal peptide, or a
functional derivative thereof, can be used. For example, the wild-type leader sequence
can be substituted with the leader sequence of human tissue plasminogen activator (TP A )
or mouse β -glucuronidase.
[0052] Disclosed herein are certain binding molecules, or antigen-binding fragments,
variants, or derivatives thereof. Unless specifically referring to full -si zed antibodies such
as natural 1y-occurri ng antibodies, the term "binding molecule" encompasses full- s ized
antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such
antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered
antibody molecules or fragments that bind antigen in a manner similar to antibody
molecules.
[0053] As used herein, the term "binding molecule" refers in its broadest sense to a
molecule that specifically binds an antigenic determinant. A non-limiting example of an
antigen-binding molecule is an antibody or fragment thereof that retains antigen-specific
binding.
[0054] The terms "antibody" and "immunoglobulin" can be used interchangeably herein.
An antibody or a fragment, variant, or derivative thereof as disclosed herein comprises at
least the variable domain of a heavy chain and a least the variable domains of a heavy
chain and a light chain. Basic immunoglobulin structures i vertebrate systems are
relatively well understood. See, e.g., Harlow et a , Antibodies: A Laboratory Manual,
(Cold .Spring Harbor Laboratory Press, 2nd ed. 1988).
[0055] As will be discussed in more detail below, the term "immunoglobulin" comprises
various broad classes of polypeptides that can be distinguished biochemically. Those
skilled i the art will appreciate that heavy chains are classified as gamma, mu, alpha,
delta, or epsilon, (γ , µ , , δ , ε ) with some subclasses among them (e.g., γ 1-γ 4). It is the
nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or
IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., IgGi, IgG 2, IgG ,
IgGi, IgA,, etc., are well characterized and are known to confer functional specialization.
Modified versions of each of these classes and isotypes are readily discernible to the
skilled artisan i view of the instant disclosure and, accordingly, are within the scope of
this disclosure.
[0056] Light chains are classified as either kappa or lambda (κ , λ ) . Each heavy chain
class can be bound with either a kappa or lambda light chain. In general, the light and
heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy
chains are bonded to each other by covalent disulfide linkages or non-covalent linkages
when the immunoglobulins are generated either by hybridomas, B cells or genetically
 engineered host cells. In the heavy chain, the amino acid sequences run from an N-

terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each
chain.
[0057] Both the light and heavy chains are divided into regions of structural and
functional homology. The terms "constant" and "variable" are used functionally. In this
regard, it will be appreciated that the variable domains of both the light (VL) and heavy
(VH) chain portions determine antigen recognition and specificity. Conversely, the
constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer
important biological properties such as secretion, transplacental mobility, Fc receptor
binding, complement binding, and the like. By convention the numbering of the constant
region domains increases as they become more distal from the antigen binding site or
amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-
terminal portion is a constant region; the CH3 and CL domains actually comprise the
carboxy-terminus of the heavy and light chain, respectively.
[0058] As indicated above, the variable region allows the binding molecule to selectively
recognize and specifically bi d epitopes o antigens. That is, the L domain and V
domain, or subset of the complementarity determining regions (CDRs), of a binding
molecule, e.g., an antibody combine to form the variable region that defines a three
dimensional antigen binding site. This quaternary binding molecule structure forms the
antigen-binding site present a the end of each arm of the Y. More specifically, the
antigen-binding site is defined by three CDRs on each of the V and VL chains.
[0059] In naturally occurring antibodies, the si "complementarity determining regions"
or "CDRs" present in each antigen binding domain are short, non-contiguous sequences
of amino acids that are specifically positioned to form the antigen binding domain as the
antibody assumes its three dimensional configuration in an aqueous environment. The
remainder of the amino acids i the antigen binding domains, referred to as "framework"
regions, show less inter- molecular variability. The framework regions largely adopt a β -
sheet conformation and the CDRs form loops which connect, and in some cases form part
of, the β -sheet structure. Thus, framework regions act to form a scaffold that provides for
positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The
antigen-binding domain formed by the positioned CDRs defines a surface complementary
to the epitope on the immunoreactive antigen. This complementary surface promotes the
non-covalent binding of the antibody to its cognate epitope. The amino acids comprising
the CDRs and the framework regions, respectively, can be readily identified for any given
heavy or light chain variable region by one of ordinary skill in the art, since they have
been precisely defined (see, "Sequences of Proteins of Immunological Interest," Kabat,
E., et al., U.S. Department of Health and Human Services, ( 1983); and Chothia and Lesk,
./. Mol. Biol., 6:9 1-9 7 ( 1987), which are incorporated herein by reference in their
entireties).
In the case where there are two or more definitions of a term that is used and/or

accepted within the art, the definition of the term as used herein is intended to include all
such meanings unless explicitly stated to the contrary. A specific example is the use of the
term "complementarity determining region" ("CDR") to describe the non-contiguous
antigen combining sites found within the variable region of both heavy and light chain
polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)
and by Chothia et al., ./. Mol. Biol. 196:90 1-9 7 ( 1987), which are incorporated herein by
reference, where the definitions include overlapping or subsets of amino acid residues
when compared against each other. Nevertheless, application of either definition to refer
to a CDR of an antibody or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues that encompass the
CDRs as defined by each of the above cited references are set forth below in Table 1 as a
comparison. The exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the art can routinely
determine which residues comprise a particular CDR given the variable region amino acid
sequence of the antibody.

TABLE 1: CDR Definitions 1

'Numbering of all CDR definitions i Table 1 is according to the

 numbering conventions set forth by Kabat et al. (see below).

[0061] Kabat et al. also defined a numbering system for variable domain sequences that
is applicable to any antibody. One of ordinary skill in the art can unambiguously assign

this system of "Kabat numbering" to any variable domain sequence, without reliance o
any experimental data beyond the sequence itself. As used herein, "Kabat numbering"
refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human
Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid residue positions in a
binding molecule which specifically binds to CXC 2, e.g., an antibody, or antigen-
binding fragment, variant, or derivative thereof as disclosed herein are according to the
Kabat numbering system.
[0062] Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or
derivatives thereof include, but are not limited to, polyclonal, monoclonal, human,
humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments,
e.g.. Fab, Fab' and F(ab') 2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,
disulfide-1 inked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments
produced by a Fab expression library. ScFv molecules are known in the art and are
described, e.g., in US patent 5,892,019. Immunoglobulin or antibody molecules
encompassed by t is disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and
IgY), class (e.g., IgG I, IgG2, IgG3, IgG4, IgA 1 and IgA2) or subclass of
immunoglobulin molecule.
[0063] By "specifically binds," it is generally meant that a binding molecule, e.g., an
antibody or fragment, variant, or derivative thereof binds to a epitope via its antigen
binding domain, and that the binding entails some complementarity between the antigen
binding domain and the epitope. According to this definition, a binding molecule is said
to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding
domain more readily than it would bind to a random, unrelated epitope. The term
"specificity" is used herein to qualify the relative affinity by which a certain binding
molecule binds to a certain epitope. For example, binding molecule "A" may be deemed
to have a higher specificity for a given epitope than binding molecule "B," or binding
 molecule "A" may he said to hind to epitope "C" with a higher specificity than it has for
related epitope "D."
[0064] By "preferentially binds," it is meant that the antibody specifically binds to an
epitope more readily than it would bind to a related, similar, homologous, or analogous
epitope. Thus, an antibody that "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though such an antibody can
cross-react with the related epitope.
[0065] By way of non-limiting example, a binding molecule, e.g., an antibody can be
considered to bind a first epitope preferentially if it binds said first epitope with a
dissociation constant (KD) that is less than the antibody's KD for the second epitope. In
another non-limiting example, a binding molecule such as an antibody can be considered
to bind a first antigen preferentially if it binds the first epitope with an affinity that is at
least one order of magnitude less than the antibody's KD for the second epitope. In another
non-limiting example, a binding molecule can be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that is at least two orders of
magnitude less than the antibody's K for the second epitope.
[0066] In another non-limiting example, a binding molecule, e.g., an antibody or
fragment, variant, or derivative thereof can be considered to bind a first epitope
preferentially if i binds the first epitope with an off rate (k(off)) that is less than the
antibody's k ( off) for the second epitope. In another non-limiting example, a binding
molecule can be considered to bind a first epitope preferentially if it binds the first
epitope with an affinity that is at least one order of magnitude less than the antibody's
k(off) for the second epitope. In another non-limiting example, a binding molecule can be
considered to bind a first epitope preferentially if it binds the first epitope with an affinity
that is at least two orders of magnitude less than the antibody's k ( off) for the second
epitope.
[0067] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof
disclosed herein can be said to bind a target antigen, e.g., a polysaccharide disclosed
herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X
-2
10 sec - 1 , 10 -2 sec - 1 , 5 X 10 -3 sec - 1 or 10 -3 sec - 1 . A binding molecule as disclosed herein
can be said to bind a target antigen, e.g., a polysaccharide with an off rate (k(off)) less
 than o equal to 5 X 10 4 sec 1 , 10 4 sec 1 , 5 X 10 5 sec 1 , or 10 5 sec ' 5 X 10 6 sec 1 , 10 6
sec 1 , 5 X 10 7 sec 1 or 10 7 sec 1 .
[0068] A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or
derivative disclosed herein can be said to bind a target antigen, e.g., a polysaccharide with
an on rate (k(on)) of greater than or equal to 10 M 1 sec 1 , 5 X 10 M 1 sec 1 , 10 4 M 1 sec 1

or 5 X 10 4 M 1 sec . A binding molecule as disclosed herein can be said to bind a target

antigen, e.g., a polysaccharide with an on rate (k(on)) greater than or equal to 10 5 M sec
' 5 X 10 5 M sec , 10 6 M 1 sec ' , or 5 X 10 6 M 1 sec 1 or 10 7 M 1 sec ' .
[0069] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof is
said to competitively inhibit binding of a reference antibody or antigen binding fragment
to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to
some degree, binding of the reference antibody or antigen binding fragment to the
epitope. Competitive inhibition can be determined by any method known in the art, for
example, competition EL IS A assays. A binding molecule can be said to competitively
inhibit binding of the reference antibody or antigen binding fragment to a given epitope
by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0070] A used herein, the term "affinity" refers to a measure of the strength of the
binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g.,
Harlow et a , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,
2nd ed. 1988) at pages 27-28. A used herein, the term "avidity" refers to the overall
stability of the complex between a population of immunoglobulins and an antigen, that is,
the functional combining strength of an immunoglobulin mixture with the antigen. See,
e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual
immunoglobulin molecules in the population with specific epitopes, and also the
valencies of the immunoglobulins and the antigen. For example, the interaction between
a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure,
such as a polymer, would be one of high avidity .
[0071] Binding molecules or antigen-binding fragments, variants or derivatives thereof as
disclosed herein can also be described or specified i terms of their cross-reactivity. As
used herein, the term "cross-reactivity" refers to the ability of a binding molecule, e.g., an
antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with
a second antigen; a measure of relatedness between two different antigenic substances.
 Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that
induced its formation. The cross reactive epitope generally contains many of the same
complementary structural features as the inducing epitope, and in some cases, can
actually fit better than the original.
[0072] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof
can also be described or specified in terms of their binding affinity to an antigen. For
example, a binding molecule ca bi d to an antigen with a dissociation constant or K no
greater than 5 x 10 2 M, 10 2 M, 5 x 10 3 M, 10 3 M, 5 x 10 4 M, 10 4 M, 5 x 10 5 M, 10 5 M,
5 x 10 6 M, 10 6 M, 5 x 10 7 M, 10 7 M, 5 x 10 8 M, 10 8 M, 5 x 10 9 M, 10 9 M, 5 x 10 10 M,
10 10 M, 5 x 1CT 1 1 M, 10 M, 5 x 10 12 M, 10 12 M, 5 x 10 13 M, 10 13 M, 5 x 10 14 M, 10 14

M, 5 x 10 15 M, or 10 15 M.
[0073] Antibody fragments including single-chain antibodies can comprise the variable
region! s ) alone or in combination with the entirety or a portion of the following: hi ge
region, C I, CH2, and CH3 domains. Also included are antigen-binding fragments
comprising any combination of variable region! s ) with a hinge region, CHI, CH2, and
CH3 domains. Binding molecules, e.g., antibodies, or antigen-binding fragments thereof
disclosed herein can be from any animal origin including birds and mammals. The
antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or
chicken antibodies. In another embodiment, the variable region can be condricthoid in
origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having
the amino acid sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for one or more human
immunoglobulins and that do not express endogenous immunoglobulins, as described
infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0074] As used herein, the term "heavy chain portion" includes amino acid sequences
derived from an immunoglobulin heavy chain of a binding molecule, e.g., a antibody
comprising a heavy chain portion comprises at least one of: a C I domain, a hinge (e.g.,
upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a
variant or fragment thereof. For example, a binding molecule, e.g., an antibody or
fragment, variant, or derivative thereof, can comprise a polypeptide chain comprising a
C 1 domain; a polypeptide chain comprising a C 1 domain, at least a portion of a hinge
domain, and a CH2 domain: a polypeptide chain comprising a C I domain and a CH3
 domain; a polypeptide chain comprising a C 1 domain, at least a portion of a hinge
domain, and a CH3 domain, or a polypeptide chain comprising a CH 1 domain, at least a
portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a
binding molecule, e.g., an antibody or fragment, variant, or derivative thereof comprises a
polypeptide chain comprising a CH3 domain. Further, a binding molecule for use i the
disclosure can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
As set forth above, it will be understood by one of ordinary skill in the art that these
domains (e.g., the heavy chain portions) can be modified such that they vary in amino
acid sequence from the naturally occurring immunoglobulin molecule.
[0075] The heavy chain portions of a binding molecule, e.g., an antibody as disclosed
herein can be derived from different immunoglobulin molecules. For example, a heavy
chain portion of a polypeptide can comprise a C 1 domain derived from an IgGl
molecule and a hinge region derived from an IgG3 molecule. In another example, a

heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule
and, in part, from an IgG3 molecule. I another example, a heavy chain portion can
comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from a
IgG4 molecule.
[0076] As used herein, the term "light chain portion" includes amino acid sequences
derived from an immunoglobulin light chain. The light chain portion comprises at least
one of a VL or CL domain.
[0077] Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or
derivatives thereof disclosed herein can be described or specified in terms of the
epitope(s) or portion(s) of an antigen, e.g., a target polysaccharide that they recognize or
specifically bind. The portion of a target polysaccharide which specifically interacts with
the antigen binding domain of an antibody is an "epitope," or a "antigenic determinant."
A target antigen, e.g., a polysaccharide ca comprise a single epitope, but typically
comprises at least two epitopes, and can include any number of epitopes, depending o
the size, conformation, and type of antigen.
[0078] As previously indicated, the subunit structures and three dimensional
configuration of the constant regions of the various immunoglobulin classes are well
known. s used herein, the term "VH domain" includes the amino terminal variable
domain of a immunoglobulin heavy chain and the term "CH 1 domain" includes the first
 (most amino terminal) constant region domain o an immunoglobulin heavy chain. The
CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an
immunoglobulin heavy chain molecule.
[0079] As used herein the term "CH2 domain" includes the portion of a heavy chain
molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using
conventional numbering schemes (residues 244 to 360, Kabat numbering system; and
residues 23 1-340, EU numbering system; see Kabat EA et al. op. cit. The CH2 domain is
unique in that it is not closely paired with another domain. Rather, two N-linked
branched carbohydrate chains are interposed between the two CH2 domains of a intact
native IgG molecule. It is also well documented that the CH3 domain extends from the
CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108
residues.
[0080] As used herein, the term "hinge region" includes the portion of a heavy chain
molecule that joins the CHI domain to the CH2 domain. This hinge region comprises
approximately 2 residues and is flexible, thus allowing the two -terminal antigen
binding regions to move independently. Hinge regions can be subdivided into three
distinct domains: upper, middle, and lower hinge domains (Roux et a , ./. Immunol.
161 - (1998)).
[0081] As used herein the term "disulfide bond" includes the covalent bond formed
between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form
a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG
molecules, the C 1 and CL regions are linked by a disulfide bond and the two heavy
chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using
the Kabat numbering system (position 226 or 229, EU numbering system).
[0082] As used herein, the term "chimeric antibody" will be held to mean any antibody
wherein the immunoreactive region or site is obtained or derived from a first species and
the constant region (which can be intact, partial or modified) is obtained from a second
species. In some embodiments the target binding region or site will be from a non-human
source (e.g. mouse or primate) and the constant region is human. In some embodiments,
the target-binding region or site will be from a human antibody and the constant region
will be from a non-human (e.g., mouse, rat, rabbit, non-human primate, etc.) source.
[0083] A s used herein, the term "engineered antibody" refers to an antibody in which the
variable domain in either the heavy and light chain or both is altered by at least partial
replacement of one or more CDRs from an antibody of known specificity and, if
necessary, by partial framework region replacement and sequence changing. Although
the CDRs can be derived from an antibody of the same class or even subclass as the
antibody from which the framework regions are derived, it is envisaged that the CDRs
will be derived from a antibody of different class and preferably from an antibody from
a different species. An engineered antibody in which one or more "donor" CDRs from a
non-human antibody of known specificity is grafted into a human heavy or light chain
framework region is referred to herein as a "humanized antibody." It may not be
necessary to replace all of the CDRs with the complete CDRs from the donor variable
region to transfer the antigen binding capacity of one variable domain to another. Rather,
it may only be necessary to transfer those residues that are necessary to maintain the

activity of the target binding site. Given the explanations set forth in, e.g., U . S . Pat. Nos.
5,585,089, 5,693,76 , 5,693,762, and 6, 180,370, it will be well within the competence of

those skilled i the art, either by carrying out routine experimentation or by trial and error
testing to obtain a functional engineered or humanized antibody.
[0084] Immunoglobulin genes undergo various modifications during maturation of the
immune response, including recombination between V, D and J gene segments, isotype
switching, and hypermutation n the variable regions. Recombination and somatic

hypermutation are the foundation for generation of antibody diversity and affinity
maturation, but they can also generate sequence liabilities that may make commercial
production of such immunoglobulins as therapeutic agents difficult, or increase the

immiinogenicity risk of the antibody. In general, mutations in CDR regions are likely to
contribute to improved affinity and function, while mutations in framework regions may
increase the risk of immiinogenicity. This risk can be reduced by reverting framework
mutations to germ line, while ensuring that activity of the antibody is not adversely
impacted. Some structural liabilities can be generated by the diversification processes, or
they may exist within germ line sequences contributing to the heavy and light chain
variable domains. Regardless of the source, it may be desirable to remove potential
structural liabilities that can result in instability, aggregation, heterogeneity of product, o r
increased immiinogenicity. Examples of undesirable liabilities include unpaired cysteines
 (which can lead to disulfide bond scrambling, or variable sulfhydryl adduct formation),
-linked glycosylation sites (resulting in heterogeneity of structure and activity), as well
as deamidation (e.g. G, NS), isomerization (DG), oxidation (exposed methionine), and
hydrolysis (DP) sites.
[0085] In order to reduce the risk of immunogenicity, and improve pharmaceutical
properties of lead antibodies, it can be desirable to reduce the number of mutations from
germline and/or remove structural liabilities.
[0086] Thus, i one embodiment, where a particular antibody differs from its respective
germline sequence at the amino acid level, the antibody sequence can be mutated back to
the germline sequence. .Such corrective mutations can occur at one, two, three or more
positions, or a combination of any of the mutated positions, using standard molecular
biological techniques.
[0087] In another embodiment, the invention includes replacing any structural liabilities

in the sequence that might affect the heterogeneity of the antibodies of the invention.
Such liabilities include glycosylation sites, un- paired cysteines, surface exposed
methi nones, etc. To reduce the risk of such heterogeneity it is proposed that changes are
made to remove one or more of such structural liabilities.
[0088] I one embodiment, it may be desirable to remove one or more consensus N-
1inked glycosylation sites from the antibody germline or antibody sequence. One skilled
i the art would be readily able to identify such a glycosylation site. Typically an N-
Iinked glycosylation consensus site sequence has the sequence of Asn-any A A- Ser or Thr
where the middle amino acid cannot be a proline (Pro). In another example, unpaired
cysteines can be replaced alone or in conjunction with other structural changes. An
unpaired cysteine ca be mutated to an appropriate amino acid that has comparable side
chain properties such as a serine.
[0089] As referred to herein, a sequence that is optimized is a sequence which has been
mutated at one or more positions, but not necessarily completely, back to its germline
sequence or can be modified to remove one or more other liabilities such as structural
liabilities. An optimized sequence can also include a sequence that has been mutated at
one or more positions back to its germline sequence and which has also been further
modified to remove one or more structural liabilities.
[0090] As used herein the term "properly folded polypeptide" includes polypeptides (e.g.,
anti-CXCR2 antibodies) in which all of the functional domains comprising the
polypeptide are distinctly active. As used herein, the term "improperly folded
polypeptide" includes polypeptides in which at least one of the functional domains of the
polypeptide is not active. In one embodiment, a properly folded polypeptide comprises
polypeptide chains linked by a least one disulfide bond and, conversely, an improperly
folded polypeptide comprises polypeptide chains not linked by at least one disulfide
bond.
[0091] As used herein the term "engineered" includes manipulation of nucleic acid or
polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro
peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of
these techniques).
[0092] As used herein, the terms "linked," "fused" or "fusion" are used interchangeably.
These terms refer to the joining together of two more elements or components, by
whatever means including chemical conjugation or recombinant means. An "in-frame
fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs)
to form a continuous longer ORF, i a manner that maintains the correct translational
reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein
containing two or more segments that correspond to polypeptides encoded by the original
ORFs (which segments are not normally so joined in nature.). Although the reading
frame is thus made continuous throughout the fused segments, the segments can be
physically or spatially separated by, for example, in-frame linker sequence. For example,
polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused,
in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin
framework region or additional CDR regions, as long as the "fused" CDRs are co-
translated as part of a continuous polypeptide.
[0093] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of

amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues

that neighbor each other in the sequence are contiguous in the primary structure of the
polypeptide.
[0094] The term "expression" as used herein refers to a process by which a gene produces
a biochemical, for example, a polypeptide. The process includes any manifestation of the
 functional presence of the gene within the cell including, without limitation, gene
knockdown as well as both transient expression and stable expression. It includes without
limitation transcription of the gene into messenger RNA (mRNA), and the translation of
such mRNA into polypeptide(s).
[0095] To facilitate expression of proteins of interest, expression can include reverse
transcription of a messenger RNA into a cDNA as is well known in the art, followed by
insertion into a vector and transcription of the cDNA into mRNA to express a
polypeptide, e.g., i a recombinant vector. A polypeptide produced i this way is referred
to herein as a "recombinant" polypeptide. As used herein, recombinant polypeptides
produced from cDNA following insertion into a vector are non-nat ural 1y- occ urri ng
simply due to their recombinant production which requires human intervention.
Recombinant polypeptides can i some instances be altered to include a N-terminal
methionine to facilitate translation. Other recombinant polypeptides can be altered by-

due to differing post-translational modifications provided in the vector utilized, for

example, for a polypeptide that is naturally glycosylated, the recombinant polypeptide can
include altered glycosylation patterns or be devoid of glycosylation. In some
recombinant polypeptides certain amino acids can be methylated. Other transcriptional or
post-translational modifications produced i certain recombinant polypeptides are well-
known to those of ordinary skill in the art.
[0096] If the final desired product is a biochemical, expression includes the creation of
that biochemical and any precursors. Expression of a gene produces a "gene product."
As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA
produced by transcription of a gene, a cDNA produced by re verse - ra nscription of the
messenger RNA, or a polypeptide which is translated from a transcript. Gene products
described herein further include nucleic acids with post transcriptional modifications, e.g.,
polyadenylation, or polypeptides with post translational modifications, e.g., methylation,
glycosylation, the addition of lipids, association with other protein subunits, proteolytic
cleavage, and the like.
[0097] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to prevent or slow down
(lessen) an undesired physiological change, cancer, inflammatory disease or disorder.
Beneficial or desired clinical results include, but are not limited to, alleviation of
 symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of
disease, clearance or reduction of a cancer or inflammatory disease in a subject, a delay or
slowing of disease progression, amelioration or palliation of the disease state, and
remission (whether partial or total ), whether detectable or undetectable. "Treatment" can
also mean prolonging survival as compared to expected survival if not receiving
treatment. Those in need of treatment include those already with the cancer, inflammatory
disease, condition, or disorder as well as those prone to have the cancer, inflammatory
disease, condition or disorder or those in which the cancer, inflammatory disease,
condition or disorder is to be prevented.
[0098] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any
subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is
desired. Mammalian subjects include humans and nonhumans, such as domestic animals,
farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats,
mice, horses, cattle, cows, bears, and so on.
[0099] As used herein, phrases such as "a subject that would benefit from administration
of an anti-CXCR2 antibody" and "an animal in need of treatment" includes subjects, such
as mammalian subjects, that would benefit from administration of an anti-CXCR2
antibody used, e.g., for detection of the level of CXCR2 (e.g., for a diagnostic procedure)
and/or from treatment, i.e., palliation or prevention of a disease, with an anti-CXCR2
antibody. As described in more detail herein, the anti-CXCR2 antibody can be used in
unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.

II. BINDING MOLECULES to CXCR2

CXCR2
[00100] Full-length CXCR2 consists of an N-terminus extracellular domain (amino acids
1-48 of SEQ ID NO:37), 3 extracellular loop domains (ECL1 (amino acids 106-120 of
SEQ ID NO:37), ECL2 (amino acids 184-208 of SEQ ID NO:37) and ECL3 (SEQ ID
NO: 274-294 of SEQ ID NO:37)), intervening transmembrane domains and a cytoplasmic
tail. The following polypeptide sequence was reported as the human CXCR2 sequence
and has the accession number NP 001548 in Genbank.

Full-Length Human CXCR2 (SEQ ID NO: 37):

 1 medfnmesds fedfwkgedl snysysstlp pflldaapce pesleinkyf vviiyalvfl
6 1 lsllgnslvm lvilysrvgr svtdvyllnl aladllfalt lpiwaaskvn gwifgtflck
121 vvsllkevnf ysgilllaci svdrylaivh atrtltqkry lvkficlsiw glslllalpv
181 llfrrtvyss nvspacyedm gnntanwrml lrilpqsfgf ivpllimlfc ygftlrtlfk
241 ahmgqkhram rvifavvlif llcwlpynlv lladtlmrtq viqetcerrn hidraldate
301 ilgilhscln pliyafigqk frhgllkila ihgliskdsl pkdsrpsfvg sssghtsttl

[00101] One embodiment is directed to an isolated binding molecule e.g., an antibody or

antigen-binding fragment thereof which specifically binds to CXCR2, wherein the
binding molecule (a) can inhibit binding to interleukin 8 (IL-8), (b) can inhibit binding to
growth-related protein alpha (Gro-alpha), (c) can inhibit the IL-8 or Gro-oc induced
calcium responses, (d) can inhibit IL-8, Gro- γ , Gro- β , Gro- γ , EN A -7 8, GCP-2 and NAP-2
induced beta-arrestin recruitment in a mammalian cell, or (d) any combination thereof. I
certain embodiments, the binding molecule or fragment thereof as described above can be
an antibody or antigen-binding fragment thereof such as HY29, HY29GL or an
combination thereof.
[00102] As used herein, the term "antigen binding domain" includes a site that specifically
binds an epitope on an antigen (e.g., an epitope of CXCR2). The antigen binding domain
of an antibody typically includes at least a portion of an immunoglobulin heavy chain
variable region and at least a portion of an immunoglobulin light chain variable region.
The binding site formed by these variable regions determines the specificity of the
antibody. In certain aspects, the disclosure provides an isolated binding molecule, e.g., an
antibody or antigen-binding fragment thereof which specifically binds to the N-terminus
and ECL3 domain of CXCR2. In certain aspects the disclosure provides an isolated
binding molecule, e.g., an antibody or antigen-binding fragment thereof which
specifically binds to the N-terminus domain of CXCR2. In some embodiments, an anti-
CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof
described herein binds to an epitope of CXCR2 comprising amino acids 30-39 and amino
acids 279-289 of SEQ ID NO:37. In some embodiments, an anti-CXCR2 binding
molecule, e.g., antibody or fragment, variant or derivative thereof described herein binds
to an epitope of CXCR2 comprising amino acids 30-39 of SEQ ID NO:37. In some
embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
 derivative thereof described herein binds to an epitope of CXCR2 comprising amino acids
279-289 of SEQ ID NO:37. In some further embodiments, an anti-CXCR2 binding
molecule, e.g., antibody or fragment, variant or derivative thereof described herein binds
to an epitope of CXCR2 comprising amino acids 31-35 of SEQ ID NO:37. In certain
embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof described herein binds to an epitope of CXCR2 comprising amino acids
34 and 35 of SEQ ID NO:37. In one embodiment, an anti-CXCR2 binding molecule,
e.g., antibody or fragment, variant or derivative thereof described herein is in contact with
residues L34 and D35 of SEQ ID NO:37. In another embodiment, an anti-CXCR2
binding molecule, e.g., antibody or fragment, variant or derivative thereof described
herein is in contact with residues P31, L34 and D35 of SEQ ID NO:37.
[00103] The disclosure is more specifically directed to an isolated binding molecule, e.g.,
an antibody or antigen-binding fragment thereof which specifically binds to the same
CXC epitope as an antibody or antigen-binding fragment thereof comprising the heavy
chain variable region (VH) and li ht chain variable region (VL) region of HY29,
HY29GL or any combination thereof.
[00104] Further included is a isolated binding molecule, e.g., an antibody or fragment
thereof which specifically binds to CXCR2 and competitively inhibits CXCR2 binding by
an antibody or antigen-binding fragment thereof comprising the V and VL of HY29,
HY29GL or any combination thereof.
[00105] Methods of making antibodies are well known i the art and described herein.
Once antibodies to various fragments of, or to the full-length CXCR2, with or without the
signal sequence, have been produced, determining which amino acids, or epitope, of
CXCR2 to which the antibody or antigen binding fragment binds can be determined by
epitope mapping protocols as described herein as well as methods known in the art (e.g.,
double antibody-sandwich EL IS A as described in "Chapter I I - Immunology," Current
Protocols in Molecular Biology, Ed. Ausubel et al., v.2, John Wiley & Sons, Inc. (1996)).
Additional epitope mapping protocols can be found in Morris, G . Epitope Mapping
Protocols, New Jersey: Humana Press ( 1996 ), which are both incorporated herein by
reference in thei entireties. Epitope mapping can also be performed by commercially
available means (i.e., PiotoPROBE, Inc. (Milwaukee, Wisconsin)). In certain aspects, for
example where the epitope is non- linear, a library of "epitopes" ca be produced based on
 molecular modeling of the 3-dimensional protein, e.g., a GPCR protein. In certain

aspects, the disclosure is directed to a binding molecule, e.g., a antibody or fragment,

variant, or derivative thereof which specifically binds to CXCR2 with an affinity
characterized by a dissociation constant (K D) which is less than the K for said reference
monoclonal antibody.
[00106] I certain embodiments an anti-CXCR2 binding molecule, e.g., an antibody or
antigen-binding fragment, variant or derivative thereof as disclosed herein binds
specifically to at least one epitope of CXCR2, i.e., binds to such an epitope more readily
tha it would bind to an unrelated, or random epitope; binds preferentially to at least one
epitope of CXCR2, i.e., binds to such an epitope more readily tha it would bind to a
related, similar, homologous, or analogous epitope; competitively inhibits binding of a
reference antibody which itself binds specifically or preferentially to a certain epitope of
CXCR2; or binds to at least one epitope of CXCR2 with an affinity characterized by a
dissociation constant KD of less than about 5 x 10 -2 M, about 10 -2 M, about 5 x 10 -3 M,
about 10 M, about 5 x 10 4 M, about 10 4 M, about 5 x 10 M, about 10 5 M, about 5 x 10
6 M, about 10 6 M, about 5 x 10 7 M, about 10 M, about 5 x 10 8 M, about 10 8 M, about 5
x 0 " M, about 0 " M, about 5 x 10 10 M, about 10 '° M, about 5 x 10 11 M, about 10 11 M,
about 5 x 10 12 M, about 10 12 M, about 5 x 10 13 M, about 10 ° M, about 5 x 10 14 M.
about 10 14 M, about 5 x 10 15 M, or about 10 15 M.
[001 07] As used in the context of binding dissociation constants, the term "about" allows
for the degree of variation inherent in the methods utilized for measuring antibody
affinity. For example, depending on the level of precision of the instrumentation used,
standard error based o the number of samples measured, and rounding error, the term
"about 10 2 M " might include, for example, from 0.05 M to 0.005 M .
[001 08] In specific embodiments a binding molecule, e.g., an antibody, or antigen-binding

fragment, variant, or derivative thereof binds CXCR2 with an off rate (k(off)) of less than
or equal to 5 X 0-2 sec - 1 , 10
-1
sec , 5 X 10
-3 -1
sec or 10
-3 -1
sec . Alternatively, an antibody,
or antigen-binding fragment, variant, or derivative thereof binds CXCR2 with an off rate
(k(off)) of less than or equal to 5 X 10 4 sec 1 , 10 4 sec ' , 5 X 10 5 sec , or 10 5 sec 5 X
10 6 sec 1 , 10 6 sec 1 , 5 X 10 7 sec 1 or 10 7 sec 1 .
[00109] In other embodiments, a binding molecule, e.g., a antibody, or antigen-binding
fragment, variant, or derivative thereof as disclosed herein binds CXCR2 with an o rate
 (k(on)) of greater than or equal to 10 3 M ' sec 1 , 5 X 0 3 M 1 sec 1 , 10 4 M 1 sec ' or 5 X 10 4
M 1 sec . Alternatively, a binding molecule, e.g., an antibody, or antigen-binding
fragment, variant, or derivative thereof as disclosed herein binds CXCR2 with an on rate
(k(on)) greater than or equal to 10 5 M 1 sec 1 , 5 X 10 5 M 1 sec 1 , 10 6 M 1 sec 1 , or 5 X 10 6
M 1 sec or 10 7 M 1 sec ' .
[001 10] In various embodiments, the binding affinity of an anti-CXCR2 binding molecule

e.g., a antibody, or antigen-binding fragment, variant, or derivative thereof as described

herein determined in a functional assay such as the TANGO™ beta-arrestin GPCR assay
(Invitrogen, Carlsbad, CA).
[001 1 ] In certain embodiments described herein, certain binding molecules described
herein can bind to structurally related polysaccharide molecules regardless of their source.
.Such CXCR2-like molecules would be expected to be identical to or have sufficient

structural relatedness to CXCR2 to permit specific recognition by one or more of the

binding molecules disclosed. For example, certain binding molecules described herein
can bind to CXCR2-like molecules produced by other bacterial species, for example,
CXCR2-like molecules produced by other CXCR2 species. Alternatively, certain binding
molecules as described herein can bind to CXCR2-like molecules produced synthetically
or by host cells genetically modified to produce CXCR2-like molecules.
[00112] Unless it is specifically noted, as used herein a "fragment thereof in reference to a
binding molecule, e.g., an antibody refers to an antigen-binding fragment, i.e., a portion
of the antibody which specifically binds to the antigen.
[001 13] An anti-CXCR2 binding molecules, e.g., antibodies or antigen-binding fragments,
variants, or derivatives thereof ca comprise a constant region which mediates one or
more effector functions. For example, binding of the C 1 component of complement to an
antibody constant region can activate the complement system. Activation of complement
is important in the opsonization and lysis of pathogens. The activation of complement
also stimulates the inflammatory response and can also be involved in autoimmune
hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc region,
with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR)
on a cell. There are a number of Fc receptors which are specific for different classes of
antibody, including IgG (gamma receptors ), IgE (epsilon receptors ), IgA (alpha receptors )
and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a
 number of important and diverse biological responses including engulfment and
destruction of antibody-coated particles, clearance of immune complexes, lysis of
antibody-coated target cells by killer cells (called antibody-dependent cell-mediated
cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control
of immunoglobulin production.
[001 14] Accordingly, certain embodiments disclosed herein include an anti-CXCR2
binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative
thereof, in which at least a fraction of one or more of the constant region domains has
been deleted or otherwise altered so as to provide desired biochemical characteristics such
as reduced effector functions, the ability to non-covalently dimerize, increased ability to
localize at the site of a tumor, reduced serum half-life, or increased serum half- life when
compared with a whole, unaltered antibody of approximately the same immunogenicity.
For example, certain binding molecules described herein are domain deleted antibodies
which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but
which lack at least a portion of one or more heavy chain domains. For instance, i certain
antibodies, one entire domain of the constant region of the modified antibody will be
deleted, for example, all or part of the CH2 domain will be deleted.
[001 15] Modified forms of anti-CXCR2 binding molecules, e.g., antibodies or antigen-
binding fragments, variants, or derivatives thereof can be made from whole precursor or
parent antibodies using techniques known in the art. Exemplary techniques are discussed
elsewhere herein.
[001 16] In certain embodiments both the variable and constant regions of anti-CXCR2

binding molecules, e.g., antibodies or antigen-binding fragments are fully human. Fully
human antibodies can be made using techniques that are known in the art and as
described herein. For example, fully human antibodies against a specific antigen can be
prepared by administering the antigen to a transgenic animal which has been modified to
produce such antibodies in response to antigenic challenge, but whose endogenous loci
have been disabled. Exemplary techniques that can be used to make such antibodies are
described in U.S. Pat. Nos.: 6,150,584; 6,458,592; 6,420, 140. Other techniques are
known in the art. Fully human antibodies can likewise be produced by various display
technologies, e.g., phage display or other viral display systems, as described in more
detail elsewhere herein.
[00117] Anti-CXCR2 binding molecules, e.g., antibodies o antigen-binding fragments,
variants, or derivatives thereof as disclosed herein can be made or manufactured using
techniques that are known in the art. In certain embodiments, binding molecules or

fragments thereof are "recombinantly produced," i.e., are non-naturally produced using
recombinant DNA technology. Exemplary techniques for making antibody molecules or
fragments thereof are discussed in more detail elsewhere herein.
[00118] In certain anti-CXCR2 binding molecules, e.g., antibodies or antigen-binding

fragments, variants, or derivatives thereof described herein, the Fc portion can be mutated
to decrease effector function using techniques known in the art. For example, the deletion
or inactivation (through point mutations or other means) of a constant region domain can
reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor
localization. In other cases it can be that constant region modifications moderate
complement binding and thus reduce the serum half-life and nonspecific association of a
conjugated cytotoxin. Yet other modifications of the constant region can be used to
modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization
due to increased antigen specificity or antibody flexibility. The resulting physiological
profile, bioavailability and other biochemical effects of the modifications, such as
localization, biodistribution and serum half-life, can easily be measured and quantified
using well known immunological techniques without undue experimentation.
[00119] In certain embodiments, anti-CXCR2 binding molecules, e.g., antibodies or
antigen-binding fragments, variants, or derivatives thereof will not elicit a deleterious
immune response in the animal to be treated, e.g., in a human. In one embodiment, anti-
CXCR2 binding molecules, e.g., antibodies or antigen-binding fragments, variants, or
derivatives thereof are modified to reduce their immunogenicity using art-recognized
techniques. For example, antibodies can be humanized, de-immunized, or chimeric
antibodies can be made. These types of antibodies are derived from a non-human
antibody, typically a murine or primate antibody, that retains or substantially retains the
antigen-binding properties of the parent antibody, but which is less immunogenic in
humans. This can be achieved by various methods, including (a) grafting the entire non-
human variable domains onto human constant regions to generate chimeric antibodies; (b)
grafting at least a part of one or more of the non-human complementarity determining
regions (CDRs) into a human framework and constant regions with or without retention
 of critical framework residues; or (c) transplanting the entire non-human variable
domains, but "cloaking" them with a human-like section by replacement of surface
residues. Such methods are disclosed in Morrison et a , Proc. Natl. Acad. Sci. Si:6851-
6855 (1984); Morrison et a , Adv. Immunol. 44:65-92 (1988); Verhoeyen et al, Science
259:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec.
Immun. Ji:169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and
6,190,370, all of which are hereby incorporated by reference in their entirety.
[00120] De-immunization can also be used to decrease the immunogenicity of an antibody.
As used herein, the term "de-immunization" includes alteration of an antibody to modify
T cell epitopes (see, e.g., W09852976A1, WO0034317A2). For example, VH and VL
sequences from the starting antibody are analyzed and a human T cell epitope "map" from
each V region showing the location of epitopes in relation to complementarity-
determining regions (CDRs) and other key residues within the sequence. Individual T cell
epitopes from the T cell epitope map are analyzed in order to identify alternative amino
acid substitutions with a low risk of altering activity of the final antibody. A range of
alternative VH and VL sequences are designed comprising combinations of amino acid
substitutions and these sequences are subsequently incorporated into a range of binding
polypeptides, e.g., CXCR2- specific antibodies or antigen-binding fragments thereof
disclosed herein, which are then tested for function. Complete heavy and light chain
genes comprising modified V and human C regions are then cloned into expression
vectors and the subsequent plasmids introduced into cell lines for the production of whole
antibody. The antibodies are then compared in appropriate biochemical and biological
assays, and the optimal variant is identified.
[00121] Anti-CXCR2 binding molecules, e.g., antibodies or antigen-binding fragments,
variants, or derivatives thereof can be generated by any suitable method known in the art.
Polyclonal antibodies to an antigen of interest can be produced by various procedures
well known in the art. For example, a CXCR2 polypeptide, a fragment thereof, or a
molecularly modeled non-linear "epitope" as described elsewhere herein can be
administered to various host animals including, but not limited to, rabbits, mice, rats,
chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing
polyclonal antibodies specific for the antigen. Various adjuvants can be used to increase
the immunological response, depending on the host species, and include but are not
 limited to, Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially
useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum. Additional such adjuvants are also well known in the art.
[00122] Monoclonal antibodies can be prepared using a wide variety of techniques known
in the art including the use of hybridoma, recombinant, and phage display technologies,
or a combination thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught, for example, in
Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
2nd ed. (1988).
[00123] DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) can
also be derived from antibody libraries, such as phage display libraries. In a particular,
such phage can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage including fd and M13
binding domains expressed from phage with scFv, Fab, Fv OE DAB (individual Fv
region from light or heavy chains) or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII protein. Exemplary
methods are set forth, for example, in EP 368 684 Bl; U.S. Pat. No. 5,969,108,
Hoogenboom, H.R. and Chames, Immunol. Today 21:311 (2000); Nagy et al. Nat. Med.
8:801 (2002); Huie et al, Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui et al., J. Mol.
Biol. 315: 1063 (2002), each of which is incorporated herein by reference. Several
publications (e.g., Marks et al, Bio/Technology i0:779-783 (1992)) have described the
production of high affinity human antibodies by chain shuffling, as well as combinatorial
infection and in vivo recombination as a strategy for constructing large phage libraries. In
another embodiment, ribosomal display can be used to replace bacteriophage as the
display platform (see, e.g., Hanes et al., Nat. Biotechnol. 18: 1287 (2000); Wilson et al.,
Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al., J. Immunol. Methods 248:31
(2001)). In yet another embodiment, cell surface libraries can be screened for antibodies
 (Boder et al, Proc. Natl. Acad. Sci. USA 97:10701 (2000); Daugherty et a , J. Immunol.
Methods 243:211 (2000)). Such procedures provide alternatives to traditional hybridoma
techniques for the isolation and subsequent cloning of monoclonal antibodies.
[00124] In phage display methods, functional antibody domains are displayed on the
surface of phage particles which carry the polynucleotide sequences encoding them. For
example, DNA sequences encoding VH and VL regions are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA
libraries. In certain embodiments, the DNA encoding the VH and VL regions are joined
together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6
or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with
helper phage. Phage used in these methods are typically filamentous phage including fd
and M l 3 and the VH or VL regions are usually recombinantly fused to either the phage
gene III or gene VIII. Phage expressing an antigen binding domain that binds to an
antigen of interest (i.e., CXCR2) can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or bead.
[00125] Additional examples of phage display methods that can be used to make the
antibodies include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al, Eur.
J. Immunol. 24:952-958 (1994); Persic et al, Gene iS7:9-18 (1997); Burton et al,
Advances in Immunology 57:191-280 (1994); PCT Application No. PCT/GB9 1/0 1134;
PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;
5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
[00126] As described in the above references and in the examples below, after phage
selection, the antibody coding regions from the phage can be isolated or cloned and used
to generate whole antibodies, including human antibodies, or any other desired antigen
binding fragment, and expressed in any desired host, including mammalian cells, insect
cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce
Fab, Fab' and F(ab') 2 fragments can also be employed using methods known in the art
such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques
12(6):S64-S69 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al, Science
240:1041-1043 (1988) (said references incorporated herein by reference in their
entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et
al, Methods in Enzymology 205:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). In certain embodiments such as
therapeutic administration, chimeric, humanized, or human antibodies can be used. A
chimeric antibody is a molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable region derived from a
murine monoclonal antibody and a human immunoglobulin constant region. Methods for
producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods
725:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are
incorporated herein by reference in their entireties. Humanized antibodies start with
antibody molecules derived from non-human species antibody that binds the desired
antigen having one or more complementarity determining regions (CDRs) from the non-
human species and framework regions from a human immunoglobulin molecule. Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably improve, antigen
binding. These framework substitutions are identified by methods well known in the art,
e.g., by modeling of the interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence comparison to identify
unusual framework residues at particular positions {see, e.g., Queen et al., U.S. Pat. No.
5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by
reference in their entireties.) Antibodies can be humanized using a variety of techniques
known in the art including, for example, CDR-grafting (EP 239,400; PCT publication
WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5 ):489-498
(1991); Studnicka et al., Protein Engineering 7^:805-814 (1994); Roguska et al., PNAS
91:969-913 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
[00128] Fully human antibodies are particularly desirable for therapeutic treatment of
human patients. Human antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody libraries derived from
human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and
PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by
reference in its entirety.
[00129] Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can express
human immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene complexes can be introduced randomly or by homologous
recombination into mouse embryonic stem cells. In addition, various companies can be
engaged to provide human antibodies produced in transgenic mice directed against a
selected antigen using technology similar to that described above.
[00130] Fully human antibodies which recognize a selected epitope can be generated using
a technique referred to as "guided selection." In this approach a selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a
completely human antibody recognizing the same epitope. (Jespers et al., Bio/Technology
i2:899-903 (1988). See also, U.S. Patent No. 5,565,332.)
[00131] In another embodiment, DNA encoding desired monoclonal antibodies can be
readily isolated and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to genes encoding the
heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells or
isolated phage, for example, can serve as a source of such DNA. Once isolated, the DNA
can be placed into expression vectors, which are then transfected into prokaryotic or
eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary
(CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More
particularly, the isolated DNA (which can be synthetic as described herein) can be used to
clone constant and variable region sequences for the manufacture antibodies as described
in Newman et a , U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated
by reference herein. Transformed cells expressing the desired antibody can be grown up
 in relatively large quantities to provide clinical and commercial supplies of the
immunoglobulin.
[00132] In some embodiments, an isolated binding molecule, e.g., an antibody comprises
at least one heavy or light chain CDR of an antibody molecule. In another embodiment,
an isolated binding molecule comprises at least two CDRs from one or more antibody
molecules. In another embodiment, an isolated binding molecule comprises at least three
CDRs from one or more antibody molecules. In another embodiment, an isolated binding
molecule comprises at least four CDRs from one or more antibody molecules. In another
embodiment, an isolated binding molecule comprises at least five CDRs from one or
more antibody molecules. In another embodiment, an isolated binding molecule of the
description comprises at least six CDRs from one or more antibody molecules.
[00133] In a specific embodiment, the amino acid sequence of the heavy and/or light chain
variable domains can be inspected to identify the sequences of the complementarity
determining regions (CDRs) by methods that are well-known in the art, e.g., by
comparison to known amino acid sequences of other heavy and light chain variable
regions to determine the regions of sequence hypervariability. Using routine recombinant
DNA techniques, one or more of the CDRs can be inserted within framework regions,
e.g., into human framework regions to humanize a non-human antibody. The framework
regions can be naturally occurring or consensus framework regions, and preferably
human framework regions (see, e.g., Chothia et ah, J. Mol. Biol. 278:457-479 (1998) for
a listing of human framework regions). The polynucleotide generated by the combination
of the framework regions and CDRs encodes an antibody that specifically binds to at least
one epitope of a desired antigen, e.g., CXCR2. One or more amino acid substitutions can
be made within the framework regions, and, the amino acid substitutions improve binding
of the antibody to its antigen. Additionally, such methods can be used to make amino acid
substitutions or deletions of one or more variable region cysteine residues participating in
an intrachain disulfide bond to generate antibody molecules lacking one or more
intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the
present disclosure and are within the capabilities of a person of skill of the art.
[00134] Also provided are binding molecules that comprise, consist essentially of, or
consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions
and/or VL regions) described herein, which binding molecules or fragments thereof
specifically bind to CXCR2. Standard techniques known to those of skill in the art can be
used to introduce mutations in the nucleotide sequence encoding a binding molecule or
fragment thereof which specifically binds to CXCR2, including, but not limited to, site-
directed mutagenesis and PCR-mediated mutagenesis which result in amino acid
substitutions. The variants (including derivatives) encode polypeptides comprising less
than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino
acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid
substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions,
less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3
amino acid substitutions, or less than 2 amino acid substitutions relative to the reference
VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or
VLCDR3. A "conservative amino acid substitution" is one in which the amino acid
residue is replaced with an amino acid residue having a side chain with a similar charge.
Families of amino acid residues having side chains with similar charges have been
defined in the art. These families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar
side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or part of the coding
sequence, such as by saturation mutagenesis, and the resultant mutants can be screened
for biological activity to identify mutants that retain activity (e.g., the ability to bind to
CXCR2).
For example, it is possible to introduce mutations only in framework regions or
only in CDR regions of an antibody molecule. Introduced mutations can be silent or
neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind
antigen. These types of mutations can be useful to optimize codon usage, or improve a
hybridoma's antibody production. Alternatively, non-neutral missense mutations can alter
an antibody's ability to bind antigen. The location of most silent and neutral missense
mutations is likely to be in the framework regions, while the location of most non-neutral
missense mutations is likely to be in CDR, though this is not an absolute requirement.
 One of skill in the art would be able to design and test mutant molecules with desired
properties such as no alteration in antigen binding activity or alteration in binding activity
(e.g., improvements in antigen binding activity or change in antibody specificity).
Following mutagenesis, the encoded protein can routinely be expressed and the functional
and/or biological activity of the encoded protein, (e.g., ability to bind at least one epitope
of CXCR2) can be determined using techniques described herein or by routinely
modifying techniques known in the art.
III. ANTIBODY POLYPEPTIDES
[00136] The disclosure is further directed to isolated polypeptides which make up binding
molecules, e.g., antibodies or antigen-binding fragments thereof, which specifically bind
to CXCR2 and polynucleotides encoding such polypeptides. Binding molecules, e.g.,
antibodies or fragments thereof as disclosed herein, comprise polypeptides, e.g., amino
acid sequences encoding, for example, CXCR2- specific antigen binding regions derived
from immunoglobulin molecules. A polypeptide or amino acid sequence "derived from"
a designated protein refers to the origin of the polypeptide. In certain cases, the
polypeptide or amino acid sequence which is derived from a particular starting
polypeptide or amino acid sequence has an amino acid sequence that is essentially
identical to that of the starting sequence, or a portion thereof, wherein the portion consists
of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or
which is otherwise identifiable to one of ordinary skill in the art as having its origin in the
starting sequence.

[00137] Also disclosed is an isolated binding molecule, e.g., an antibody or antigen-

binding fragment thereof which specifically binds to CXCR2 comprising an
immunoglobulin heavy chain variable region (VH) amino acid sequence at least 80%,
85%, 90%, 95%, or 100% identical to one or more of: SEQ ID NO: 2 or SEQ ID NO: 20.
[00138] In certain aspects the disclosure provides an isolated binding molecule, e.g., an
antibody or antigen-binding fragment thereof which specifically binds to CXCR2
comprising an immunoglobulin heavy chain variable region (VH) amino acid sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identical to one or more of: SEQ ID
NO: 2 or SEQ ID NO: 20, but not 100% identical to one or more of: SEQ ID NO: 2 or
SEQ ID NO: 20.
[00139] Further disclosed is an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof which specifically binds to CXCR2 comprising a VH amino
acid sequence identical to, or identical except for one, two, three, four, five, or more
amino acid substitutions to one or more of: SEQ ID NO: 2 or SEQ ID NO: 20.
[00140] Some embodiments include an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof which specifically binds to CXCR2 comprising a VH,
where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are at
least 80%, 85%, 90%, 95% or 100%, or up to but not including 100%, identical to one or
more reference heavy chain VHCDRl, VHCDR2 or VHCDR3 amino acid sequences of
one or more of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID
NO: 22, or SEQ ID NO: 23.
[00141] Further disclosed is an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof which specifically binds to CXCR2 comprising a VH, where
one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are identical to,
or identical except for four, three, two, or one amino acid substitutions, to one or more
reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid sequences of
one or more of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID
NO: 22, or SEQ ID NO: 23. Thus, according to this embodiment the VH comprises one
or more of a VHCDRl, VHCDR2, or VHCDR3 identical to or identical except for four,
three, two, or one amino acid substitutions, to one or more of the VHCDRl, VHCDR2, or
VHCDR3 amino acid sequences.
[00142] Also disclosed is an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof which specifically binds to CXCR2 comprising an
immunoglobulin light chain variable region (VL) amino acid sequence at least 80%, 85%,
90% 95% or 100% identical to one or more of SEQ ID NO: 11 or SEQ ID NO: 29.
[00143] In certain aspects the disclosure provides an isolated binding molecule, e.g., an
antibody or antigen-binding fragment thereof which specifically binds to CXCR2
comprising an immunoglobulin light chain variable region (VL) amino acid sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identical to one or more of: SEQ ID
NO: 1 1 or SEQ ID NO: 29.
[00144] Some embodiments disclose an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof which specifically binds to CXCR2 comprising a VL
 amino acid sequence identical to, or identical except for one, two, three, four, five, or
more amino acid substitutions, to one or more of SEQ ID NO: 11 or SEQ ID NO: 29.
[00145] Also provided is an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof which specifically binds to CXCR2 comprising a VL, where
one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of the VL are at least 80%,
85%, 90%, 95% or 100%, or up to but not including 100%, identical to one or more
reference light chain VLCDRl, VLCDR2 or VLCDR3 amino acid sequences of one or
more of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID
NO: 31, or SEQ ID NO: 32.
[00146] Further provided is an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof which specifically binds to CXCR2 comprising a VL, where
one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of the VL are identical to, or
identical except for four, three, two, or one amino acid substitutions, to one or more
reference heavy chain VLCDRl, VLCDR2 and/or VLCDR3 amino acid sequences of one
or more of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID
NO: 31, or SEQ ID NO: 32. Thus, according to this embodiment the VL comprises one
or more of a VLCDRl, VLCDR2, or VLCDR3 identical to or identical except for four,
three, two, or one amino acid substitutions, to one or more of the VLCDRl, VLCDR2, or
VLCDR3 amino acid sequences.
[00147] In other embodiments, an isolated antibody or antigen-binding fragment thereof
which specifically binds to CXCR2, comprises, consists essentially of, or consists of VH
and VL amino acid sequences at least 80%, 85%, 90% 95% or 100%, or up to but not
including 100%, identical to:
(a) SEQ ID NO: 2 and SEQ ID NO: 11, respectively, or
(b) SEQ ID NO: 20 and SEQ ID NO: 29, respectively.
[00148] In certain embodiments, the above-described antibody or antigen-binding
fragment thereof comprises a VH with the amino acid sequence SEQ ID NO: 2 and a VL
with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the above-
described antibody or antigen-binding fragment thereof comprises a VH with the amino
acid sequence SEQ ID NO: 20 and a VL with the amino acid sequence of SEQ ID NO:
29.
[00149] In certain embodiments, an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof as described herein specifically binds to CXCR2 with
an affinity characterized by a dissociation constant (KD) no greater than 5 10
-2
M, 10 -2
M, 5 x 10 3 M, 10 3 M, 5 x 10 4 M, 10 4 M, 5 x 10 5 M, 10 5 M, 5 x 10 6 M, 10 6 M, 5 x 10 7
M, 10 7 M, 5 x 10 8 M, 10 8 M, 5 x 10 9 M, 10 9 M, 5 x 10 10 M, 10 10 M, 5 x 10 11 M, 10 11

M, 5 x 10 12 M, 10 12 M, 5 x 10 13 M, 10 13 M, 5 x 10 14 M, 10 14 M, 5 x 10 15 M, or 10 15

M.
[00150] In specific embodiments, an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof as described herein specifically binds to CXCR2, with
an affinity characterized by a dissociation constant (KD) in a range of about 1 x 10 10 to
about 1 x 10 6 M . Some embodiments include the isolated binding molecule e.g., an
antibody or fragment thereof as described above, which (a) can inhibit binding to IL-8,
(b) can inhibit binding to Gro-alpha, (c) can inhibit IL-8 or Gro-cc induced calcium

responses or (d) can inhibit IL-8, Gro-cc, Gro- β , Gro- γ , ENA-78, GCP-2 and NAP-2
induced beta-arrestin recruitment or a combination thereof.
[00151] Certain embodiments include the isolated binding molecule e.g., an antibody or
fragment thereof as described above, where the OPK EC50 is less than about 0.5 µ g/ml,
less than about 0.05 µ g/ml, or less than about 0.005 µ g/ml, or where the OPK EC50 ranges
from about 0.001 µ g/ml to about 0.5 µ g/ml, or where the OPK EC50 ranges from about
0.02 µ g/ml to about 0.08 µ g/ml, or where the OPK EC50 ranges from about 0.002 µ g/ml
to about 0.01 µ g/ml or where the OPK EC50 is less than about 0.2 µ g/ml, or wherein the
OPK EC50 is less than about 0.02 µ g/ml.
[00152] In certain aspects the disclosure provides an isolated binding molecule, e.g., an
antibody or antigen-binding fragment thereof which specifically binds to the N-terminus
and ECL3 domain of CXCR2. In certain aspects the disclosure provides an isolated
binding molecule, e.g., an antibody or antigen-binding fragment thereof which
specifically binds to the N-terminus domain of CXCR2. In some embodiments, an anti-
CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof
described herein binds to an epitope of CXCR2 comprising amino acids 30-39 and amino
acids 279-289 of SEQ ID NO:37. In some embodiments, an anti-CXCR2 binding
molecule, e.g., antibody or fragment, variant or derivative thereof described herein binds
to an epitope of CXCR2 comprising amino acids 30-39 of SEQ ID NO:37. In some
 embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof described herein binds to an epitope of CXCR2 comprising amino acids
279-289 of SEQ ID NO:37. In some further embodiments, an anti-CXCR2 binding
molecule, e.g., antibody or fragment, variant or derivative thereof described herein binds
to an epitope of CXCR2 comprising amino acids 31-35 of SEQ ID NO:37. In certain
embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof described herein binds to an epitope of CXCR2 comprising amino acids
34 and 35 of SEQ ID NO:37. In one embodiment, an anti-CXCR2 binding molecule,
e.g., antibody or fragment, variant or derivative thereof described herein is in contact with
residues L34 and D35 of SEQ ID NO:37. In another embodiment, an anti-CXCR2
binding molecule, e.g., antibody or fragment, variant or derivative thereof described
herein is in contact with residues P31, L34 and D35 of SEQ ID NO:37.
[00153] In certain embodiments, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof described herein specifically binds to the same
CXCR2 epitope as monoclonal antibody HY29, HY29GL or any combination thereof, or
will competitively inhibit such a monoclonal antibody from binding to CXCR2.
[00154] Some embodiments include mutants of HY29, HY29GL, or any combination
thereof, comprising an amino acid sequence at least, e.g., 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95%, identical to any one or more of these antibodies, or identical to any
one of these antibodies except for one, two, three, four, five, or more amino acid
substitutions.
[00155] In some embodiments, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof described herein specifically binds to the same
epitope as monoclonal antibody HY29, HY29GL, or any combination thereof, or will
competitively inhibit any such monoclonal antibody from binding to CXCR2.
[00156] Any anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof described herein can further include additional polypeptides, e.g., a
signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as
described herein, or other heterologous polypeptides as described herein. Additionally,
binding molecules or fragments thereof of the description include polypeptide fragments
as described elsewhere. Additionally anti-CXCR2 binding molecules, e.g., antibodies or
 fragments, variants or derivatives thereof described herein can be fusion polypeptides,
Fab fragments, scFvs, or other derivatives, as described herein.
[00157] Also, as described in more detail elsewhere herein, the disclosure includes
compositions comprising anti-CXCR2 binding molecules, e.g., antibodies or fragments,
variants or derivatives thereof described herein.
[00158] It will also be understood by one of ordinary skill in the art that anti-CXCR2
binding molecules, e.g., antibodies or fragments, variants or derivatives thereof described
herein can be modified such that they vary in amino acid sequence from the originally-
occurring binding polypeptide from which they were derived. For example, a polypeptide
or amino acid sequence derived from a designated protein can be similar, e.g., have a
certain percent identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%,
85%, 90%, or 95% identical to the starting sequence.
[00159] The term "percent sequence identity" between two polynucleotide or polypeptide
sequences refers to the number of identical matched positions shared by the sequences
over a comparison window, taking into account additions or deletions (i.e., gaps) that
must be introduced for optimal alignment of the two sequences. A matched position is
any position where an identical nucleotide or amino acid is presented in both the target
and reference sequence. Gaps presented in the target sequence are not counted since gaps
are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence
are not counted since target sequence nucleotides or amino acids are counted, not
nucleotides or amino acids from the reference sequence.
[00160] The percentage of sequence identity is calculated by determining the number of
positions at which the identical amino-acid residue or nucleic acid base occurs in both
sequences to yield the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of comparison and multiplying
the result by 100 to yield the percentage of sequence identity. The comparison of
sequences and determination of percent sequence identity between two sequences may be
accomplished using readily available software both for online use and for download.
Suitable software programs are available from various sources, and for alignment of both
protein and nucleotide sequences. One suitable program to determine percent sequence
identity is bl2seq, part of the BLAST suite of program available from the U.S.
government's National Center for Biotechnology Information BLAST web site
 (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using
either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid
sequences, while BLASTP is used to compare amino acid sequences. Other suitable
programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of
bioinformatics programs and also available from the European Bioinformatics Institute
(EBI) at www.ebi.ac.uk/Tools/psa.
[00161] Different regions within a single polynucleotide or polypeptide target sequence
that aligns with a polynucleotide or polypeptide reference sequence can each have their
own percent sequence identity. It is noted that the percent sequence identity value is
rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded
down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also
is noted that the length value will always be an integer.
[00162] One skilled in the art will appreciate that the generation of a sequence alignment
for the calculation of a percent sequence identity is not limited to binary sequence-
sequence comparisons exclusively driven by primary sequence data. Sequence alignments
can be derived from multiple sequence alignments. One suitable program to generate
multiple sequence alignments is ClustalW2, available from www.clustal.org. Another
suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and
MUSCLE are alternatively available, e.g., from the EBI.
[00163] It will also be appreciated that sequence alignments can be generated by
integrating sequence data with data from heterogeneous sources such as structural data
(e.g., crystallographic protein structures), functional data (e.g., location of mutations), or
phylogenetic data. A suitable program that integrates heterogeneous data to generate a
multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively
available, e.g., from the EBI. It will also be appreciated that the final alignment used to
calculated percent sequence identity may be curated either automatically or manually.
[00164] Whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90%
or 95% identical to another polypeptide can also be determined using methods and
computer programs/software known in the art such as, but not limited to, the BESTFIT
program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). BESTFIT
uses the local homology algorithm of Smith and Waterman, Advances in Applied
 Mathematics 2:482-489 (1981), to find the best segment of homology between two
sequences. When using BESTFIT or any other sequence alignment program to determine
whether a particular sequence is, for example, 95% identical to a reference sequence, the
parameters are set, of course, such that the percentage of identity is calculated over the
full length of the reference polypeptide sequence and that gaps in homology of up to 5%
of the total number of amino acids in the reference sequence are allowed.
[00165] Furthermore, nucleotide or amino acid substitutions, deletions, or insertions
leading to conservative substitutions or changes at "non-essential" amino acid regions can
be made. For example, a polypeptide or amino acid sequence derived from a designated
protein can be identical to the starting sequence except for one or more individual amino
acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven,
eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or
deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a
designated protein has one to five, one to ten, one to fifteen, or one to twenty individual
amino acid substitutions, insertions, or deletions relative to the starting sequence.
[00166] An anti-CXCR2 binding molecule, e.g., an antibody or fragment, variant or
derivative thereof described herein can comprise, consist essentially of, or consist of a
fusion protein. Fusion proteins are non-naturally-occurring chimeric molecules which
comprise, for example, an immunoglobulin antigen-binding domain with at least one
target binding site, and at least one heterologous portion, i.e., a portion with which it is
not naturally linked in nature. The amino acid sequences can normally exist in separate
proteins that are brought together in the fusion polypeptide or they can normally exist in
the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion
proteins can be created, for example, by chemical synthesis, or by creating and translating
a polynucleotide in which the peptide regions are encoded in the desired relationship.
[00167] The term "heterologous" as applied to a polynucleotide, polypeptide, or other
moiety means that the polynucleotide, polypeptide, or other moiety is derived from a
distinct entity from that of the rest of the entity to which it is being compared. In a non-
limiting example, a "heterologous polypeptide" to be fused to a binding molecule, e.g., an
antibody or an antigen-binding fragment, variant, or derivative thereof is derived from a
non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-
immunoglobulin polypeptide of a different species.
 IV. FUSION PROTEINS AND ANTIBODY CONJUGATES
[00168] In some embodiments, the anti-CXCR2 binding molecules, e.g., antibodies or
fragments, variants or derivatives thereof can be administered in conjugated form. In still
another embodiment, the anti-CXCR2 binding molecules, e.g., antibodies or fragments,
variants or derivatives thereof can be administered in unconjugated form, then in
conjugated form, or vice versa.
[00169] In certain embodiments, an anti-CXCR2 binding molecule, e.g., an antibody or
fragment, variant or derivative thereof described herein can comprise a heterologous
amino acid sequence or one or more other moieties not normally associated with an
antibody (e.g., an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein,
an enzyme, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a
heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG),
and a combination of two or more of any said agents). In further embodiments, an anti-
CXCR2 binding molecule, e.g., an antibody or fragment, variant or derivative thereof can
comprise a detectable label selected from the group consisting of an enzyme, a
fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or
a combination of two or more of any said detectable labels.
V. POLYNUCLEOTIDES ENCODING BINDING MOLECULES
[00170] Also provided herein are nucleic acid molecules encoding the anti-CXCR2
binding molecules, e.g., antibodies or fragments, variants or derivatives thereof described
herein.
[00171] One embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region (VH) amino acid sequence at least 80%, 85%, 90% 95% or 100%, or up
to, but not including 100%, identical to one or more of: SEQ ID NO: 2 or SEQ ID NO:
203.
[00172] Another embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding a VH amino acid sequence
identical to, or identical except for one, two, three, four, five, or more amino acid
substitutions to one or more of: SEQ ID NO: 2 or SEQ ID NO: 20.
[00173] Further embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding a VH, where one or more of the
 VHCDRl, VHCDR2 or VHCDR3 regions of the VH are identical to, or identical except
for four, three, two, or one amino acid substitutions, to one or more reference heavy chain
VHCDRl, VHCDR2 and/or VHCDR3 amino acid sequences of one or more of: SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO:
23.
[00174] Another embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding an isolated binding molecule, e.g.,
an antibody or antigen-binding fragment thereof which specifically binds to CXCR2
comprising a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of
the VH are identical to, or identical except for four, three, two, or one amino acid
substitutions, to one or more reference heavy chain VHCDRl, VHCDR2 and/or
VHCDR3 amino acid sequences of one or more of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. A further embodiment
provides an isolated binding molecule e.g., an antibody or antigen-binding fragment
comprising the VH encoded by the polynucleotide specifically or preferentially binds to
CXCR2.
[00175] One embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain
variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100%, or up
to, but not including 100%, identical to one or more of: SEQ ID NO: 1 1 or SEQ ID NO:
29.
[00176] Further embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding a VL, where one or more of the
VLCDRl, VLCDR2 or VLCDR3 regions of the VL are identical to, or identical except
for four, three, two, or one amino acid substitutions, to one or more reference light chain
VLCDRl, VLCDR2 and/or VLCDR3 amino acid sequences of one or more of: SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID
NO: 32. Another embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding a VL, where one or more of the
VLCDRl, VLCDR2 or VLCDR3 regions of the VL are at least 80%, 85%, 90%, 95% or
100%, or up to but not including 100%, identical to one or more reference light chain
VLCDRl, VLCDR2 or VLCDR3 amino acid sequences of one or more of: SEQ ID NO:
 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:
32.
[00177] A further embodiment provides an isolated polynucleotide comprising, consisting
essentially of, or consisting of a nucleic acid encoding an isolated binding molecule, e.g.,
an antibody or antigen-binding fragment thereof which specifically binds to CXCR2
comprising an VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of
the VL are identical to, or identical except for four, three, two, or one amino acid
substitutions, to one or more reference heavy chain VLCDRl, VLCDR2 and/or VLCDR3
amino acid sequences of one or more of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.
[00178] In another embodiment, an isolated binding molecule e.g., an antibody or antigen-
binding fragment comprising the VL encoded by the polynucleotide specifically or
preferentially binds to CXCR2.
[00179] In some embodiments, an isolated antibody or antigen-binding fragment thereof
encoded by one or more of the polynucleotides described above, which specifically binds
to CXCR2, comprises, consists essentially of, or consists of VH and VL amino acid
sequences at least 80%, 85%, 90% 95% or 100%, or up to but not including 100%,
identical to:
(a) SEQ ID NO: 2 and SEQ ID NO: 11, respectively, or
(b) SEQ ID NO: 20 and SEQ ID NO: 29, respectively.
[00180] In certain embodiments, an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof encoded by one or more of the polynucleotides
described above, specifically binds to CXCR2 with an affinity characterized by a
dissociation constant (KD) no greater than 5 x 10 2 M, 10 2 M, 5 x 10 3 M, 10 3 M, 5 x 10 4
M, 10 4 M, 5 x 10 5 M, 10 5 M, 5 x 10 6 M, 10 6 M, 5 x 10 7 M, 10 7 M, 5 x 10 8 M, 10 8 M,
5 x 10 9 M, 10 9 M, 5 x 10 10 M, 10 10 M, 5 x 10 11 M, 10 11 M, 5 x 10 12 M, 10 12 M, 5 x
10 13 M, 10 13 M, 5 x 10 14 M, 10 14 M, 5 x 10 15 M, or 10 15 M.
[00181] In specific embodiments, an isolated binding molecule, e.g., an antibody or
antigen-binding fragment thereof encoded by one or more of the polynucleotides
described above, specifically binds to CXCR2, with an affinity characterized by a
dissociation constant (KD) in a range of about 1 x 10 10 to about 1 x 10 6 M . In certain
aspects the disclosure provides an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof encoded by one or more of the polynucleotides described above
which specifically binds to the N-terminus and ECL3 domain of CXCR2. In certain
aspects the disclosure provides an isolated binding molecule, e.g., an antibody or antigen-
binding fragment thereof encoded by one or more of the polynucleotides described above
which specifically binds to the N-terminus domain of CXCR2. In some embodiments, an
anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof
encoded by one or more of the polynucleotides described above binds to an epitope of
CXCR2 comprising amino acids 30-39 and amino acids 279-289 of SEQ ID NO:37. In
some embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant
or derivative thereof encoded by one or more of the polynucleotides described above
binds to an epitope of CXCR2 comprising amino acids 30-39 of SEQ ID NO:37. In some
embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof encoded by one or more of the polynucleotides described above binds
to an epitope of CXCR2 comprising amino acids 279-289 of SEQ ID NO:37. In some
further embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment,
variant or derivative thereof encoded by one or more of the polynucleotides described
above binds to an epitope of CXCR2 comprising amino acids 31-35 of SEQ ID NO:37. In
certain embodiments, an anti-CXCR2 binding molecule, e.g., antibody or fragment,
variant or derivative thereof encoded by one or more of the polynucleotides described
above binds to an epitope of CXCR2 comprising amino acids 34 and 35 of SEQ ID
NO:37. In one embodiment, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof encoded by one or more of the polynucleotides
described above is in contact with residues L34 and D35 of SEQ ID NO:37. In another
embodiment, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof encoded by one or more of the polynucleotides described above is in
contact with residues P31, L34 and D35 of SEQ ID NO:37.
In certain embodiments, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof encoded by one or more of the polynucleotides
described above, specifically binds to the same CXCR2 epitope as monoclonal antibody
HY29, HY29GL or will competitively inhibit such a monoclonal antibody from binding
to CXCR2.
[00183] In certain embodiments, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof encoded by one or more of the polynucleotides
described above, specifically binds to the same epitope as monoclonal antibody HY29, or
will competitively inhibit such a monoclonal antibody from binding to CXCR2.
[00184] In certain embodiments, an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof encoded by one or more of the polynucleotides
described above, specifically binds to the same epitope as monoclonal antibody HY29GL,
or will competitively inhibit such a monoclonal antibody from binding to CXCR2.
[00185] The disclosure also includes fragments of the polynucleotides as described
elsewhere herein. Additionally polynucleotides which encode fusion polynucleotides,
Fab fragments, and other derivatives, as described herein, are also provided.
[00186] The polynucleotides can be produced or manufactured by any method known in
the art. For example, if the nucleotide sequence of the antibody is known, a
polynucleotide encoding the antibody can be assembled from chemically synthesized
oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 1 7:242 (1994)),
which, briefly, involves the synthesis of overlapping oligonucleotides containing portions
of the sequence encoding the antibody, annealing and ligating of those oligonucleotides,
and then amplification of the ligated oligonucleotides by PCR.
[00187] Alternatively, a polynucleotide encoding an anti-CXCR2 binding molecule, e.g.,
antibody or fragment, variant or derivative thereof can be generated from nucleic acid
from a suitable source. If a clone containing a nucleic acid encoding a particular antibody
is not available, but the sequence of the antibody molecule is known, e.g., derived from a
known cDNA sequence, a nucleic acid encoding the antibody can be chemically
synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a
cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from,
any tissue or cells expressing the antibody or such as hybridoma cells selected to express
an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5'
ends of the sequence or by cloning using an oligonucleotide probe specific for the
particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into
replicable cloning vectors using any method well known in the art. As used herein, a
sequence is "derived from a cDNA sequence" if the sequence was at one point prepared
 as a cDNA, and was modified, synthesized, cloned, or otherwise altered from the original
cDNA sequence by any method disclosed herein.
[00188] Once the nucleotide sequence and corresponding amino acid sequence of an anti-
CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof is
determined, its nucleotide sequence can be manipulated using methods well known in the
art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site
directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1998), which are both incorporated by reference herein
in their entireties), to generate antibodies having a different amino acid sequence, for
example to create amino acid substitutions, deletions, and/or insertions.
[00189] A polynucleotide encoding an anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof can be composed of any polyribonucleotide or
polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or
DNA. For example, a polynucleotide encoding an anti-CXCR2 binding molecule, e.g.,
antibody or fragment, variant or derivative thereof can be composed of single- and
double-stranded DNA, DNA that is a mixture of single- and double- stranded regions,
single- and double- stranded RNA, and RNA that is mixture of single- and double-
stranded regions, hybrid molecules comprising DNA and RNA that can be single-
stranded or, more typically, double- stranded or a mixture of single- and double-stranded
regions. In addition, a polynucleotide encoding an anti-CXCR2 binding molecule, e.g.,
antibody or fragment, variant or derivative thereof can be composed of triple- stranded
regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an
anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof
can also contain one or more modified bases or DNA or RNA backbones modified for
stability or for other reasons. "Modified" bases include, for example, tritylated bases and
unusual bases such as inosine. A variety of modifications can be made to DNA and
RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically
modified forms.
[00190] An isolated polynucleotide encoding a non-natural variant of a polypeptide
derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light
 chain portion) can be created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of the immunoglobulin such that one
or more amino acid substitutions, additions or deletions are introduced into the encoded
protein. Mutations can be introduced by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are
made at one or more non-essential amino acid residues.
VI. EXPRESSION OF ANTIBODY POLYPEPTIDES
[00191] As is well known, RNA can be isolated from the original hybridoma cells or from
other transformed cells by standard techniques, such as guanidinium isothiocyanate
extraction and precipitation followed by centrifugation or chromatography. Where
desirable, mRNA can be isolated from total RNA by standard techniques such as
chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
[00192] In one embodiment, cDNAs that encode the light and the heavy chains of the anti-
CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative thereof can be
made, either simultaneously or separately, using reverse transcriptase and DNA
polymerase in accordance with well-known methods. PCR can be initiated by consensus
constant region primers or by more specific primers based on the published heavy and
light chain DNA and amino acid sequences. As discussed above, PCR also can be used to
isolate DNA clones encoding the antibody light and heavy chains. In this case the
libraries can be screened by consensus primers or larger homologous probes, such as
mouse constant region probes.
[00193] DNA, typically plasmid DNA, can be isolated from the cells using techniques
known in the art, restriction mapped and sequenced in accordance with standard, well
known techniques set forth in detail, e.g., in the foregoing references relating to
recombinant DNA techniques. Of course, the DNA can be synthetic according to the
present disclosure at any point during the isolation process or subsequent analysis.
[00194] Following manipulation of the isolated genetic material to provide an anti-CXCR2
binding molecule, e.g., antibody or fragment, variant or derivative thereof of the
disclosure, the polynucleotides encoding anti-CXCR2 binding molecules, are typically
inserted in an expression vector for introduction into host cells that can be used to
produce the desired quantity of anti-CXCR2 binding molecules.
[00195] Recombinant expression of an antibody, or fragment, derivative or analog thereof,
e.g., a heavy or light chain of an antibody which binds to a target molecule described
herein, e.g., CXCR2, requires construction of an expression vector containing a
polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody
molecule or a heavy or light chain of an antibody, or portion thereof (containing the
heavy or light chain variable domain), of the disclosure has been obtained, the vector for
the production of the antibody molecule can be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein
by expressing a polynucleotide containing an antibody encoding nucleotide sequence are
described herein. Methods which are well known to those skilled in the art can be used to
construct expression vectors containing antibody coding sequences and appropriate
transcriptional and translational control signals. These methods include, for example, in
vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination. The disclosure, thus, provides replicable vectors comprising a nucleotide
sequence encoding an antibody molecule of the disclosure, or a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such
vectors can include the nucleotide sequence encoding the constant region of the antibody
molecule (see, e.g., WO86/05807; WO89/01036; and U.S. Pat. No. 5,122,464) and the
variable domain of the antibody can be cloned into such a vector for expression of the
entire heavy or light chain.
[00196] The term "vector" or "expression vector" is used herein to mean vectors used in
accordance with the present disclosure as a vehicle for introducing into and expressing a
desired gene in a host cell. As known to those skilled in the art, such vectors can easily
be selected from the group consisting of plasmids, phages, viruses and retroviruses. In
general, vectors compatible with the instant disclosure will comprise a selection marker,
appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter
and/or replicate in eukaryotic or prokaryotic cells.
[00197] For the purposes of this disclosure, numerous expression vector systems can be
employed. For example, one class of vector utilizes DNA elements which are derived
from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia
virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others
involve the use of polycistronic systems with internal ribosome binding sites.
 Additionally, cells which have integrated the DNA into their chromosomes can be
selected by introducing one or more markers which allow selection of transfected host
cells. The marker can provide for prototrophy to an auxotrophic host, biocide resistance
(e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker
gene can either be directly linked to the DNA sequences to be expressed, or introduced
into the same cell by cotransformation. Additional elements can also be needed for
optimal synthesis of mRNA. These elements can include signal sequences, splice signals,
as well as transcriptional promoters, enhancers, and termination signals.
[00198] In some embodiments the cloned variable region genes are inserted into an
expression vector along with the heavy and light chain constant region genes (e.g.,
human) synthetic as discussed above. Of course, any expression vector which is capable
of eliciting expression in eukaryotic cells can be used in the present disclosure. Examples
of suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo,
pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV,
pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and
plasmid pCI (available from Promega, Madison, WI). In general, screening large
numbers of transformed cells for those which express suitably high levels if
immunoglobulin heavy and light chains is routine experimentation which can be carried
out, for example, by robotic systems.
[00199] More generally, once the vector or DNA sequence encoding a monomeric subunit
of the anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative
thereof of the disclosure has been prepared, the expression vector can be introduced into
an appropriate host cell. Introduction of the plasmid into the host cell can be
accomplished by various techniques well known to those of skill in the art. These
include, but are not limited to, transfection (including electrophoresis and
electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with
enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A . A . G .
"Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths,
Boston, Mass., Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction into
the host is via electroporation. The host cells harboring the expression construct are
grown under conditions appropriate to the production of the light chains and heavy
chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay
 techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and
the like.
[00200] The expression vector is transferred to a host cell by conventional techniques and
the transfected cells are then cultured by conventional techniques to produce an antibody
for use in the methods described herein. Thus, the disclosure includes host cells
containing a polynucleotide encoding anti-CXCR2 binding molecule, e.g., antibody or
fragment, variant or derivative thereof, or a heavy or light chain thereof, operably linked
to a heterologous promoter. In some embodiments for the expression of double-chained
antibodies, vectors encoding both the heavy and light chains can be co-expressed in the
host cell for expression of the entire immunoglobulin molecule, as detailed below.
[00201] Certain embodiments include an isolated polynucleotide comprising a nucleic acid
which encodes the above-described VH and VL, wherein a binding molecule or antigen-
binding fragment thereof expressed by the polynucleotide specifically binds CXCR2.
[00202] Some embodiments include vectors comprising the above-described
polynucleotides. In further embodiments, the polynucleotides are operably associated
with a promoter. In additional embodiments, the disclosure provides host cells
comprising such vectors. In further embodiments, the disclosure provides vectors where
the polynucleotide is operably associated with a promoter, wherein vectors can express a
binding molecule which specifically binds CXCR2 in a suitable host cell.
[00203] Also provided is a method of producing a binding molecule or fragment thereof
which specifically binds CXCR2, comprising culturing a host cell containing a vector
comprising the above-described polynucleotides, and recovering said antibody, or
fragment thereof. In further embodiments, the disclosure provides an isolated binding
molecule or fragment thereof produced by the above-described method.
[00204] As used herein, "host cells" refers to cells which harbor vectors constructed using
recombinant DNA techniques and encoding at least one heterologous gene. In
descriptions of processes for isolation of antibodies from recombinant hosts, the terms
"cell" and "cell culture" are used interchangeably to denote the source of antibody unless
it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells"
can mean either from spun down whole cells, or from the cell culture containing both the
medium and the suspended cells.
[00205] A variety of host-expression vector systems can be utilized to express antibody
molecules for use in the methods described herein. Such host-expression systems
represent vehicles by which the coding sequences of interest can be produced and
subsequently purified, but also represent cells which can, when transformed or transfected
with the appropriate nucleotide coding sequences, express an antibody molecule of the
disclosure in situ. These include but are not limited to microorganisms such as bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors
containing antibody coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant
cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or
mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant
expression constructs containing promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, or
eukaryotic cells, especially for the expression of whole recombinant antibody molecule,
are used for the expression of a recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element from human
cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene
45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
[00206] The host cell line used for protein expression is often of mammalian origin; those
skilled in the art are credited with ability to determine particular host cell lines which are
best suited for the desired gene product to be expressed therein. Exemplary host cell lines
include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11
(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI
(monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK
(baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast)
BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma),
 P3x63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI
(human lymphocyte) and 293 (human kidney). Host cell lines are typically available
from commercial services, the American Tissue Culture Collection or from published
literature.
[00207] In addition, a host cell strain can be chosen which modulates the expression of the
inserted sequences, or modifies and processes the gene product in the specific fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of
protein products can be important for the function of the protein. Different host cells have
characteristic and specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for proper
processing of the primary transcript, glycosylation, and phosphorylation of the gene
product can be used.
[00208] For long-term, high-yield production of recombinant proteins, stable expression is
preferred. For example, cell lines which stably express the antibody molecule can be
engineered. Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following the introduction of the
foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media,
and then are switched to a selective media. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows cells to stably integrate the plasmid
into their chromosomes and grow to form foci which in turn can be cloned and expanded
into cell lines. This method can advantageously be used to engineer cell lines which
stably express the antibody molecule.
[00209] A number of selection systems can be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202
(1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes: dhfr, which confers
 resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et
al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2012 (1981)); neo,
which confers resistance to the aminoglycoside G-418 Clinical Pharmacy i2:488-505;
Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
52:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,
Ann. Rev. Biochem. 62:191-217 (1993);, TIB TECH 11(5): 155-215 (May, 1993); and
hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:141 (1984).
Methods commonly known in the art of recombinant DNA technology which can be used
are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),
Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin
et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their
entireties.
[00210] The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors based on
gene amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Academic Press, New York, Vol. 3 . (1987)). When a marker in the vector system
expressing antibody is amplifiable, increase in the level of inhibitor present in culture of
host cell will increase the number of copies of the marker gene. Since the amplified
region is associated with the antibody gene, production of the antibody will also increase
(Crouse et al., Mol. Cell. Biol. 3:251 (1983)).
[00211] In vitro production allows scale-up to give large amounts of the desired
polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions
are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor
or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow
fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or
desired, the solutions of polypeptides can be purified by the customary chromatography
methods, for example gel filtration, ion-exchange chromatography, chromatography over
DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential
 biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC
chromatography step described herein.
[00212] Constructs encoding anti-CXCR2 binding molecules, e.g., antibodies or
fragments, variants or derivatives thereof, as disclosed herein can also be expressed non-
mammalian cells such as bacteria or yeast or plant cells. Bacteria which readily take up
nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia
coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus,
and Haemophilus influenzae. It will further be appreciated that, when expressed in
bacteria, the heterologous polypeptides typically become part of inclusion bodies. The
heterologous polypeptides must be isolated, purified and then assembled into functional
molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-
assemble into tetravalent antibodies (WO02/096948A2).
[00213] In bacterial systems, a number of expression vectors can be advantageously
selected depending upon the use intended for the antibody molecule being expressed. For
example, when a large quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression of high levels of fusion protein products that are readily purified can be
desirable. Such vectors include, but are not limited, to the E. coli expression vector
pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence
can be ligated individually into the vector in frame with the lacZ coding region so that a
fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. iJ:3101-
3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like.
pGEX vectors can also be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins are soluble and can
easily be purified from lysed cells by adsorption and binding to a matrix glutathione-
agarose beads followed by elution in the presence of free glutathione. The pGEX vectors
are designed to include thrombin or factor Xa protease cleavage sites so that the cloned
target gene product can be released from the GST moiety.
[00214] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic
microorganisms although a number of other strains are commonly available, e.g., Pichia
pastoris.
[00215] For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et
al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene
10:151 (1980)) is commonly used. This plasmid already contains the TRP1 gene which
provides a selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The
presence of the trpl lesion as a characteristic of the yeast host cell genome then provides
an effective environment for detecting transformation by growth in the absence of
tryptophan.
[00216] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is typically used as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells. The antibody coding sequence can be cloned individually into n on
essential regions (for example the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example the polyhedrin promoter).
[00217] Once the anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or
derivative thereof, as disclosed herein has been recombinantly expressed, it can be
purified by any method known in the art for purification of an immunoglobulin molecule,
for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for
the specific antigen after Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the purification of proteins.
Another method for increasing the affinity of antibodies of the disclosure is disclosed in
US2002/0 123057 Al.
VII. PHARMACEUTICAL COMPOSITIONS COMPRISING ANTI-CXCR2
BINDING MOLECULES
[00218] The pharmaceutical compositions used in this disclosure comprise
pharmaceutically acceptable carriers well known to those of ordinary skill in the art.
Preparations for parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Certain pharmaceutical compositions as disclosed
herein can be orally administered in an acceptable dosage form including, e.g., capsules,
tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can
be administered by nasal aerosol or inhalation. Preservatives and other additives can also
be present such as for example, antimicrobials, antioxidants, chelating agents, and inert
gases and the like. Suitable formulations for use in the therapeutic methods disclosed
 herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th
ed. (1980).
[00219] The amount of an anti-CXCR2 binding molecule, e.g., antibody or fragment,
variant or derivative thereof, that can be combined with the carrier materials to produce a
single dosage form will vary depending upon the host treated and the particular mode of
administration. Dosage regimens also can be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response). The compositions can also
comprise the anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof dispersed in a biocompatible carrier material that functions as a
suitable delivery or support system for the compounds.
VIII. TREATMENT METHODS USING THERAPEUTIC BINDING MOLECULES
[00220] Methods of preparing and administering an anti-CXCR2 binding molecule, e.g.,
an antibody or fragment, variant or derivative thereof, as disclosed herein to a subject in
need thereof are well known to or are readily determined by those skilled in the art. The
route of administration of the anti-CXCR2 binding molecule, e.g., antibody or fragment,
variant or derivative thereof, can be, for example, oral, parenteral, by inhalation or
topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial,
intraperitoneal, intramuscular, or subcutaneous administration. A suitable form for
administration would be a solution for injection, in particular for intravenous or
intraarterial injection or drip. However, in other methods compatible with the teachings
herein, an anti-CXCR2 binding molecule, e.g., antibody or fragment, variant or derivative
thereof, as disclosed herein can be delivered directly to the site of the adverse cellular
population.
[00221] Due to pleitropic actions of CXC chemokines, including attraction and activation
of neutrophils and monocytes/macrophages as well as promotion of endothelial cell
proliferation and cancer cell growth, inhibition of chemokine receptors CXCR2 is
expected to be beneficial in the prophylactic prevention and treatment of numerous
diseases. Anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof, as disclosed herein can be administered i a pharmaceutically
effective amount for the in vivo treatment of cancers or other proliferative or
inflammatory diseases. In this regard, it will be appreciated that the disclosed binding
molecules will be formulated so as to facilitate administration and promote stability of the
 active agent. Fo the purposes of the instant application, a pharmaceutically effective
amount shall be held to mean an amount sufficient to achieve effective binding to a target
and to achieve a benefit, e.g., treat, ameliorate, lessen, clear, or put i remission cancers.
[00222] Some embodiments are directed to a method of treating a cancer in a subject in
need thereof, comprising administering to the subject an effective amount of the binding
molecule or fragment thereof, the antibody or fragment thereof, the composition, the
polynucleotide, the vector, or the host cell described herein. In further embodiments, the
cancer includes but is not limited to lymphoma, colorectal, lung including but not limited
to small cell and non-small cell, melanoma, breast, prostate, kidney, brain, esophageal,
renal or pancreatic cancer. In some embodiments, the subject is a human.
[00223] Some embodiments are directed to a method of treating an inflammatory disease
in a subject in need thereof, comprising administering to the subject an effective amount
of the binding molecule or fragment thereof, the antibody or fragment thereof, the
composition, the polynucleotide, the vector, or the host cell described herein. In further
embodiments, the inflammatory disease include but are not limited to acute and chronic
inflammatory diseases such as atherosclerosis, ischemia/reperfusion injuries, chronic
obstructive pulmonary disease, asthma, and rheumatoid arthritis, chemokine mediated
diseases include adult respiratory distress syndrome, inflammatory bowel disease,
ulcerative colitis, Crohn's disease, atopic dermatitis, cystic fibrosis, psoriasis, multiple
sclerosis, angiogenesis, restenosis, osteoarthritis, septic shock, endotoxic shock, gram
negative sepsis, toxic shock syndrome, stroke, glomerulonephritis, thrombosis, graft vs.
host reaction, allograft rejections, Alzheimer's disease, malaria, viral infections, traumatic
brain injury, pulmonary fibrosis, and pancreatitis.

[00224] In keeping with the scope of the disclosure, anti-CXCR2 binding molecules, e.g.,
antibodies or fragments, variants or derivatives thereof, can be administered to a human
or nonhuman animal in accordance with the aforementioned methods of treatment in an
amount sufficient to produce a therapeutic effect. The anti-CXCR2 binding molecules,
e.g., antibodies or fragments, variants or derivatives thereof, disclosed herein can be
administered to such human or nonhuman animal in a conventional dosage form prepared
by combining the antibody of the disclosure with a conventional pharmaceutically
acceptable carrier or diluent according to known techniques.
[00225] Effective doses of the compositions of the present disclosure, for treatment of
CXCR2 infection vary depending upon many different factors, including means of
administration, target site, physiological state of the patient, whether the patient is human
or an animal, other medications administered, and whether treatment is prophylactic or
therapeutic. Usually, the patient is a human but non-human mammals including
transgenic mammals can also be treated. Treatment dosages can be titrated using routine
methods known to those of skill in the art to optimize safety and efficacy.
[00226] Anti-binding molecules, e.g., antibodies or fragments, variants or derivatives
thereof can be administered multiple occasions at various frequencies depending on
various factors known to those of skill in the art. Alternatively, anti-CXCR2 binding
molecules, e.g., antibodies or fragments, variants or derivatives thereof can be
administered as a sustained release formulation, in which case less frequent
administration is required. Dosage and frequency vary depending on the half-life of the
antibody in the patient.
[00227] The compositions of the disclosure can be administered by any suitable method,
e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used
herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial,
intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion
techniques.
[00228] Anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof may be administered alone or in combination with other treatments,
either simultaneously or sequentially dependent upon the condition to be treated.
[00229] Anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof may be used as a chemosensitiser whereby it can increase therapeutic
efficacy of cytotoxic agents, and may thus be provided for administration in combination
with one or more cytotoxic agents, either simultaneously or sequentially. The binding
member may also be used as a radio sensitiser whereby it can improve efficacy of
radiation, and may thus be provided for administration in combination with radiation,
either simultaneously or sequentially.
[00230] Anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof may be provided in combination or addition with one or more of the
 following agents: a cytokine or agonist or antagonist of cytokine function (e.g. an agent
which acts on cytokine signalling pathways, such as a modulator of the SOCS system),
such as an alpha-, beta- and/or gamma-interferon; insulin-like growth factor type I (IGF-
1), its receptors and associated binding proteins; interleukins (IL), e.g. one or more of IL-

1 to -33, and/or an interleukin antagonist or inhibitor, such as anakinra; inhibitors of

receptors of interleukin family members or inhibitors of specific subunits of such
receptors, a tumor necrosis factor alpha (TNF-. alpha.) inhibitor, such as an anti-TNF
monoclonal antibodies (for example infliximab, adalimumab and/or CDP-870) and/or a
TNF receptor antagonist, e.g. an immunoglobulin molecule (such as etanercept) and/or a
low-molecular-weight agent, such as pentoxyfylline; a modulator of B cells, e.g. a
monoclonal antibody targeting B-lymphocytes (such as CD20 (rituximab) or MRA-
aIL16R) or T-lymphocytes (e.g. CTLA4-Ig, HuMax 11-15 or Abatacept).
[00231] Anti-CXCPv2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof could also be used in association with a chemotherapeutic agent or
another tyrosine kinase inhibitor in co-administration or in the form of an
immunoconjugate. Fragments of said antibody could also be used in bispecific antibodies
obtained by recombinant mechanisms or biochemical coupling and then associating the
specificity of the above described antibody with the specificity of other antibodies able to
recognize other molecules involved in the activity for which CXCR2 is associated.
[00232] Anti-CXCPv2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof can also be used in combination with an existing therapeutic agent for
the treatment of cancer. Suitable agents to be used in combination include:
(i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical
oncology, such as Gleevec (imatinib mesylate), alkylating agents (for example cis-platin,
carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan
and nitrosoureas); antimetabolites (for example antifolates, such as fluoropyrimidines like
5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea,
gemcitabine and paclitaxel); antitumor antibiotics (for example anthracyclines like
adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like
vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere);
and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and
teniposide, amsacrine, topotecan and camptothecins); (ii) cytostatic agents, such as
antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and
iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens
(for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH
antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin),
progestogens (for example megestrol acetate), aromatase inhibitors (for example as
anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 .alpha. -reductase,
such as finasteride; (iii) Agents which inhibit cancer cell invasion (for example
metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen
activator receptor function); (iv) inhibitors of growth factor function, for example such
inhibitors include growth factor antibodies, growth factor receptor antibodies (for
example the anti-erbb2 antibody trastuzumab and the anti-erbbl antibody cetuximab
[C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine
kinase inhibitors, for example inhibitors of the epidermal growth factor family (for
example EGFR family tyrosine kinase inhibitors, such as N-(3-chloro-4-fluorophenyl)-7-
methoxy-6-(3-morpholinopropoxy)quinazolin-4- -amine (gefitinib, AZD1839), N-(3-
ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-
acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoli- n-4-amine
(CI- 1033)), for example inhibitors of the platelet-derived growth factor family and for
example inhibitors of the hepatocyte growth factor family; (v) antiangiogenic agents, such
as those which inhibit the effects of vascular endothelial growth factor (for example the
anti-vascular endothelial cell growth factor antibody bevacizumab, compounds, such as
those disclosed in International Patent Applications WO 97/22596, WO 97/30035, WO
97/32856 and WO 98/13354, each of which is incorporated herein in its entirety) and
compounds that work by other mechanisms (for example linomide, inhibitors of integrin
avf33 function and angiostatin); (vi) vascular damaging agents, such as combretastatin A4
and compounds disclosed in International Patent Applications WO 99/02166, WO
00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213 (each of
which is incorporated herein in its entirety); (vii) antisense therapies, for example those
which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;
(viii) gene therapy approaches, including for example approaches to replace aberrant
genes, such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene directed
 enzyme pro-drug therapy) approaches, such as those using cytosine deaminase, thymidine
kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance
to chemotherapy or radiotherapy, such as multi-drug resistance gene therapy; (ix)
immunomodulatory agents, including but not limited to Tremelimumab, Ipilimumab;
ORENCIA, Belatacept; CD28 antagonists, CD80 antagonists, CD86 antagonists, PD1,
PDL1, CD137, 41BB, CTLA-4 antagonists and Ox40 agonists; and (x)
immunotherapeutic approaches, including for example ex vivo and in vivo approaches to
increase the immunogenicity of patient tumor cells, such as transfection with cytokines,
such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor,
approaches to decrease T-cell anergy, approaches using transfected immune cells, such as
cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell
lines and approaches using anti-idiotypic antibodies.
[00233] For treatment of an inflammatory disease, anti-CXCR2 binding molecules, e.g.,
antibodies or fragments, variants or derivatives thereof may be combined with one or
more agents, such as non-steroidal anti-inflammatory agents (hereinafter NSAIDs)
including non-selective cyclo-oxygenase (COX)-l/COX-2 inhibitors whether applied
topically or systemically, such as piroxicam, diclofenac, propionic acids, such as
naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates, such as
mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones, such as
phenylbutazone, salicylates, such as aspirin); selective COX-2 inhibitors (such as
meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib, parecoxib and etoricoxib);
cyclo-oxygenase inhibiting nitric oxide donors (CINODs); glucocorticosteroids (whether
administered by topical, oral, intra- muscular, intra-venous or intra- articular routes);
methotrexate, leflunomide; hydroxychloroquine, d-penicillamine, auranofin or other
parenteral or oral gold preparations; analgesics; diacerein; intra-articular therapies, such
as hyaluronic acid derivatives; and nutritional supplements, such as glucosamine.
[00234] IX. IMMUNOASSAYS
[00235] Anti-CXCR2 binding molecules, e.g., antibodies or fragments, variants or
derivatives thereof can be assayed for immunospecific binding by any method known in
the art. The immunoassays which can be used include but are not limited to competitive
and non-competitive assay systems using techniques such as western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
 immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin
reactions, immunodiffusion assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name
but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds,
Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1
(1994), which is incorporated by reference herein in its entirety). Exemplary
immunoassays are described briefly below (but are not intended by way of limitation).
[00236] There are a variety of methods available for measuring the affinity of an antibody-
antigen interaction, but relatively few for determining rate constants. Most of the methods
rely on either labeling antibody or antigen, which inevitably complicates routine
measurements and introduces uncertainties in the measured quantities. Antibody affinity
can be measured by a number of methods, including OCTET ®, BIACORE ®, ELISA, and
FACS.
[00237] In certain aspects binding affinity of an antibody to a GPCR can be accomplished
using a functional assay e.g., the TANGO™ GPCR Assay System to measure, e.g., β -
arrestin binding to agonist activated CXCR2, with a β -lactamase reporter gene readout.
Additional methods for measuring the binding affinity of an antibody include, but are not
limited to, standard competitive binding assays, ELISA assays, PA2/Schild assay,
BIACORE assays, functional assays such as proliferation or factor release and the like.
See, for example, such assays disclosed in WO 93/14125; Shi et al., Immunity iJ:633-642
(2000); Kumanogoh et al., J Immunol 769:1175-1181 (2002); Watanabe et al., J Immunol
767:4321-4328 (2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al., J
Immunol i72(2):1246-1255 (2004), all of which are herein incorporated by reference.

[00238] The practice of the disclosure will employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology, transgenic
biology, microbiology, recombinant DNA, and immunology, which are within the skill of
the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning
A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory
Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al., ed., Cold
Springs Harbor Laboratory, New York (1992), DNA Cloning, D . N . Glover ed., Volumes
 I and II (1985); Oligonucleotide Synthesis, M . J . Gait ed., (1984); Mullis et al. U.S. Pat.
No: 4,683,195; Nucleic Acid Hybridization, B . D . Hames & S . J . Higgins eds. (1984);
Transcription And Translation, B . D . Hames & S . J . Higgins eds. (1984); Culture Of
Animal Cells, R . I . Freshney, Alan R . Liss, Inc., (1987); Immobilized Cells And
Enzymes, IRL Press, (1986); B . Perbal, A Practical Guide To Molecular Cloning (1984);
the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors
For Mammalian Cells, J . H . Miller and M . P. Calos eds., Cold Spring Harbor Laboratory
(1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Immunochemical
Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press,
London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D . M . Weir and
C . C . Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).
[00239] Standard reference works setting forth general principles of immunology include
Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J.,
Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, New
York (1982); Roitt, I., Brostoff, J . and Male D., Immunology, 6th ed. London: Mosby
(2001); Abbas A., Abul, A . and Lichtman, A., Cellular and Molecular Immunology, Ed.
5, Elsevier Health Sciences Division (2005); and Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988).
[00240] Having now described the disclosure in detail, the same will be more clearly
understood by reference to the following examples, which are included herewith for
purposes of illustration only and are not intended to be limiting of the disclosure. All
patents and publications referred to herein are expressly incorporated by reference in their
entireties.
 EXAMPLES

Example 1

Generation of Chimeric Anti Human CXCR2 antibodies by Immunization of

Transgenic Mice

[00241] Anti-human CXCR2 chimeric antibodies with human variable regions and mouse
constant regions were isolated from hybridoma generated by immunization of
Veloclmmune II mice (Regeneron, Tarrytown, NY) (Valenzuela et al., Nat. Biotechnol.
21(6);652-9 (2003)) with HEK cells over-expressing the human CXCR2 receptor
(Accession NP001548) using a modified RIMMS style immunization schedule of 28 days
(Kilpatrick et al, Hybridoma 16(4); 381-9 (1997)).
[00242] HEK human CXCR2 cells were routinely cultured in Dulbecco's modified
Minimal Essential Medium (DMEM Invitrogen 41966) containing 10% FBS (SAFC
Biosciences 12076C), 0.5 mg/ml G418 (Invitrogen 10131). For immunization cells were
harvested using Accutase (PAA laboratories L I 1-007) and resuspended in Phosphate
Buffered Saline (PBS). Equal volumes of cells and complete Freunds' adjuvant (Sigma
F5881) for primary immunization or incomplete Freund's adjuvant for boosts (Sigma
F5506) were added to glass vials and emulsions prepared by rapid vortex mixing. Each
mouse was injected subcutaneously on four separate occasions, 7-8 days apart, with 200
µΐ emulsion containing lxlO 6, 3xl0 6 or 5xl0 6 cells, using two sites with 100 µΐ per site. A
final boost was injected intraperitoneally with 200 µΐ (5xl0 6) cells prepared in PBS
without any adjuvant four days before sacrifice on day 28.

Measurement of Antibody Titers

[00243] Titers of the antibodies against human CXCR2 were evaluated from pre-bleed
sera and sera following the second and third boosts by measurement of binding to HEK
human CXCR2 cells in an ELISA. HEK human CXCR2 cells were plated in culture
medium at a density of 2xl0 4 cells/well onto 96- well Poly-D Lysine black plates and
grown overnight at 37°C, 5% C02. The media was discarded and cells fixed for 5 minutes
using a 3.7% formaldehyde (Aldrich 533998)/PBS solution. After removal of the fixing
solution, cells were blocked by addition of 3% Marvel/PBS for 1 hour at room
temperature. Serum samples or control antibodies were prepared in 3% Marvel/PBS and
incubated with the cells for 1 hour at room temperature. Plates were washed three times
with PBS then antibody detected by incubation for 1 hour at room temperature with 50 µΐ
per well 10 ng/ml Anti-mouse IgG-Europium (Perkin Elmer AD0124-01) prepared in
DELFIA buffer (Perkin Elmer 4002-0010). 50 µΐ of Enhancement Solution (Perkin
Elmer 4001-0010) was added and time resolved fluorescent emission at 615nm measured
following excitation at 340nm using a Perkin Elmer Envision plate reader.

Generation of Hybridomas

Eight mice that had the highest anti human CXCR2 antibody titers in the first and
second bleed serum compared to the pre-bleed serum were selected for harvest and
fusion. The lymph nodes and spleens were harvested and cells isolated by mechanical
disruption. The cells were counted separately then combined using all the lymph node
cells and enough spleen cells to give a maximum of lxl 0e8 lymphoid cells. Mixed cells
(75xl0e6) were fused with 14xl0 6 SP2 myeloma cells using polyethylene glycol (Roche
0783641) essentially according to the method (Kohler and Milstein, Nature
256(5577);495-7 (1975)). Briefly cells were placed in a water bath at 39°C and 1ml of
pre-warmed PEG (37 °C) was added to the drained cell pellet over 1 minute with gentle
stirring. Cells were incubated with shaking for 1 minute then 1ml DMEM (Gibco 41966)
added over 1 minute with stirring. Cells were incubated for a further 1-minute while
stirring. This was repeated then 7 ml of Selective DMEM Media (DMEM containing
20% FCS, 4 mM Glutamine (Gibco 25030), lOOU/lOOug/ml Penicillin/Streptomycin
(Invitrogen 15140-122), 10% Hybridoma Cloning Factor (BM Condimed HI, Roche
10879100), 2% oxaloacetate/pyruvate/insulin (OPI) media supplement (Sigma O5003),
2% Hypoxanthine/ Azaserine (Sigma A9666) was added over 3 minutes. Cells were
centrifuged at 140g for 5 minutes and the supernatant drained. The resultant cell pellet
was re- suspended gently in 200 ml Selective DMEM Media and cells plated out in 20 x
96 well tissue culture treated plates per mouse at a concentration of 4.45x1 04 per well for
culture.
 .Screening of hybridoma supernatants for human CXCR2 specific chimeric antibodies

[00245] Hybridoma well lines were screened for production of anti-human CXCR2
antibodies using Fluorescence Microvolume Assay Technology (FMAT), a fluorescence
based platform that detects fluorescence localized to bead or cells settled at the bottom of
microwell (Dietz et ah, Cytometry 25:177-186 (1996), Miraglia et ah, J. Biomol.
Screening 4:193-204 (1999)).
[00246] Supernatants were taken from wells at day 13 post fusion, transferred into 384
well plates (Greiner 781280) and tested by single point screening for the presence of
antibodies that bound specifically to HEK human CXCR2 cells with no binding to
HEK293 parental cells in an FMAT Direct Binding Assay. Supernatants were also tested
for the production of antibodies that neutralized the interaction of fluorescently labeled
Interleukin-8/CXCL8 with cells expressing human CXCR2 in an FMAT Receptor-Ligand
Competition Assay.
[00247] For the FMAT Direct Binding Assay HEK human CXCR2 and HEK 293 cells
were harvested and resuspended at 0.15xl0 6 cells/ml in FMAT assay buffer (pH 7.4)
containing IX Hanks Balanced Salt Solution (Sigma H8264), 0.1% Bovine Serum
Albumin (Sigma A9576), 20mM HEPES (Sigma H0877) and 0.01% Sodium Azide
(Sigma S8032). All dilutions of samples and reagents were carried out in FMAT assay
buffer. Hybridoma supernatants (5 µΐ ) were transferred from the 384 well Greiner plates
into 384 well clear-bottomed assay plates (Costar 3655) containing 15 µΐ per well of 800
ng/ml Goat Anti Mouse FMAT Blue® (Applied Biosystems 4362494). Cells (20 µΐ per
well of 0.15xl0 6 cells/ml) were added and plates incubated for 5 hours at room
temperature. The average fluorescence intensity in each well was measured on the
Applied Biosystems Cellular Detection System 8200 following excitation at 633nm. Data
was analyzed on the system using the Velocity algorithm with appropriate gating.
[00248] For the FMAT Receptor Ligand Competition Assay 10 µΐ hybridoma supernatant
was transferred into assay plates containing 10 µΐ of 3.2nM FMAT Blue® labeled
Interleukin-8 (Applied Biosystems cat no. 4377770). HEK human CXCR2 cells (20 µΐ of
1.5xl0 5 cells/ml) were added and plates incubated for a minimum of 5 hours at room
temperature. Total binding and non-specific binding (NSB) controls were set up using
FMAT assay buffer or 80 nM final assay concentration unlabeled IL-8 (R&D Systems
 208/IL-CF) respectively. The % control binding was determined from the FLl Average
data using equation 1 where NSB FLl Average is the mean value of the non-specific
binding control wells and Total binding FLl Average is the mean value of the Total
binding control wells.
[00249] Equation 1:

(Sample FLl Average - NSB FLl Average)

% Control binding = xlOO
(Total binding FLl Average - NSB FLl Average)

[00250] A total of 303 well lines that specifically bound human CXCR2 expressing cells
with minimal binding to HEK 293 parental cells were identified (see Table 2 for
summary) in the FMAT Direct Binding Assay and 175 of these were selected for
expansion and further testing.
[00251] Only 3 of the well lines with confirmed specific human CXCR binding showed
>50 inhibition of binding of fluorescently labeled IL-8 to HEK human CXCR2 cells in
the FMAT.

TABLE 2 . .Screening of Hybridoma Supernatants for human CXCR2 specific antibodies.

Determination of mouse IgG and IgM in culture supernatant

[00252] The presence of mouse IgG and IgM in the supernatants of the 175 hybridoma
well lines selected for progression was determined by sequence analysis and using an
ELISA. For the ELISA Nunc MaxiSorp flat-bottom 96 well plate microplates were
 coated with goat anti-mouse IgG (γ -chain specific, Sigma, M l 397) or goat anti-mouse
IgM (µ -chain specific, Sigma M8644) polyclonal antibody (5 g/mL) in PBS at room
temperature for 1 hour. Plates were blocked with 3% skimmed milk (Marvel) in PBS and
washed three times with PBST (PBS containing 0.1% Tween-20, pH 7.4). After washing
three times, 50 µΐ of pre-diluted culture supernatant (1:2 or 1:5 in 3% Marvel/ PBS) was
added to each microplate and incubated for 1 hour at room temperature. The plates were
subsequently washed, and then 50 µΐ of horseradish peroxidase (HRP) conjugated goat
anti-mouse polyvalent Ig (1:1000 dilution, Sigma, A0142) or goat anti-mouse IgG
(1:5000 dilution, Sigma, A2554) was added and incubated for 30 min at room
temperature. After washing five times with PBST, the reaction was developed by the
addition of 3,3',5,5'-Tetramethylbenzidine (TMB) for 5 min at room temperature and then
stopped by the addition of 50 µΐ of 0.5M sulphuric acid. The plates were read at OD450
using a PerkinElmer EnVision multilabel plate reader.
[00253] Of the hybridoma well lines tested 38 were identified that contained IgG only, 37
contained IgM only and 39 had mixed IgG and IgM. A total of 12 different heavy chain
germlines and 17 different light chain germlines were used.
[00254] Purification of Chimeric Antibodies from, Hybridoma Cultures
[00255] Small scale purification of antibodies from the hybridoma supernatants was
performed using 20 microliter PhyTip columns PhyNexus (PhyNexus PTP-92-20-01).
Hybridoma well lines were overgrown in HL1™ Serum free Medium (Lonza 77201) and
supernatants from the cultures transferred to 96 well masterblocks (Greiner 780271) prior
to loading onto the PhyTip column. Elution from the PhyTip was carried out using buffer
containing 100 mM HEPES, 140 mM NaCl pH3 which was then neutralized with an
equal volume of 200mM HEPES pH 8 to give a final volume of 150 µΐ . Concentration of
purified antibodies was estimated by absorbance at 280nm wavelength.
[00256] Large scale purification of chimeric antibodies was carried out using protein G
chromatography. Hybridoma cultures were expanded into 75cm flasks in serum-free
medium (HL-1 (Lonza 77201), HyberZero (Statens Institute 71800) and allowed to
overgrow for 10 days.
[00257] Supernatants were harvested and loaded onto a 1ml HiTrap Protein G column (GE
Lifesciences 17-0404-01) and antibodies eluted from the column using 0.1M glycine-HCl
pH 2.7. Eluted antibodies were then buffer exchanged into lxDPBS using a NaplO
 columns (GE Lifesciences 17-0854-02) and the concentration determined
spectrophotometrically. The antibodies were analyzed for purity using SDS-PAGE.
[00258] Dilution Cloning of Hybridoma Well Lines
[00259] Well lines were cloned out by limiting dilution method. Cells were thawed and
cultured for 24 hours, counted and then plated out into 96 well plates at 0.3 cells per well.
After 10 days in culture supernatant samples from between 16 to 24 clones with evidence
of hybridoma growth were harvested and tested for the presence of human CXCR2
specific antibodies in an FMAT Direct binding Assay essentially as described previously.
For example for well line ZY05KA-G08, supernatants derived from 24 clones prepared
by limiting dilution were tested at final assay dilutions of 0.5%, 2.5% and 12.5% for
binding to HEK human CXCR2 cells. Of the 24 clones tested 15 showed strong positive
binding signals of >600 FL1 average after an overnight incubation. The remaining clones
showed <200 FL1 average binding signal. Clones were sequenced and for the ZY05KA-
G08 derived clones the 15 positive binders had the same V H and V sequences.

Example 2

Inhibition o f ligand stimulated Calcium Flux b y chimeric anti human CXCR2

Antibodies

[00260] The ability of purified antibodies to inhibit functional responses was measured in
calcium flux assays using HEK cells over expressing the human CXCR2 receptor and
Gqi5, a chimeric G-protein containing the five carboxyl-terminal amino acids from the
G c i G-protein subunit This chimeric G-protein enables Gi-coupled receptors such as
CXCR2 to generate a rise in the intracellular calcium signal through the Gq Phospholipase
C pathway (Conklin et al. Nature 363:274-276 (1993)).
[00261] HEK Gqi5 hCXCR2 cells were routinely cultured in media consisting of DMEM
containing 10% FBS, 0.5 mg/ml G418 and 0.35 mg/ml Hygromycin B . Cells were
harvested for assays using Accutase, resuspended in media at a density of
0.28xl0 6cells/ml and plated at 50 µΐ per well into 384 well black walled Poly-D Lysine
coated plates (Greiner 781946). Plates were incubated at 37°C, 5% C0 2 for 18-24 hours
then cell media was aspirated and replaced with 25 µΐ per well of Fluo-4AM dye loading
solution (component C from the Fluo-4NW Calcium Assay kit Invitrogen F36206)
 prepared according to manufacturer instructions. Cells were loaded with dye by
incubation at 37°C, 5% C0 2 for 30 minutes and 30 minutes at room temperature.
[00262] Antibodies were diluted to the required concentrations in assay buffer containing
25mM HEPES, 125mM NaCl (Sigma S7653), 5mM KC1 (Sigma P3911), ImM MgCl 2
(Sigma M8266), 1.5mM CaCl 2 (Sigma C4901), 5mM Glucose (Sigma G8769) and 0.1%
BSA (Sigma A9576) in 384 well polypropylene plates. 12.5 µΐ diluted antibody or assay
buffer (stimulated and unstimulated controls) was transferred to the dye-loaded cells
using the FLIPR® (Molecular Devices) and incubated at room temperature for 30
minutes. For agonist stimulation 28nM IL-8/CXCL8 (R&D Systems cat no. 208/IL),
28nM Gro-oc/CXCLl (R&D Systems Cat No. 275/GR), or 120nM ENA-78/CXCL5
(R&D Systems Cat. No. 254-XB) was prepared in assay buffer and 12.5 µΐ added to the
sample wells and stimulated control wells of the dye loaded cell plate using the FLIPR®.
12.5 µΐ assay buffer was added to the unstimulated control wells of the plate.
[00263] To measure calcium mobilization the FLIPR® (Molecular Devices) was calibrated
for suitable exposure according to the manufacturer instructions. Fluorescence of the
Fluor-4AM dye was recorded at 1 second intervals for 80 measurements followed by 4
second intervals for 30 measurements. The peak response from each well was exported
and % control calculated using equation 2

Equation 2 :

(Sample MaxFluorescence - Basal ControlMaxFluorescence)

%Control = xlOO
(AgonistControlMaxFluorescence - Basal ControlMaxFluorescence) J

where agonist control is the mean value of the maximum fluorescence of the agonist
control wells and basal control is the mean value of the maximum fluorescence of the
unstimulated control well .
[00264] IC 50 values were determined using GraphPad Prism Software by curve fitting
using a four parameter logistic equation described in Equation 3 .
 Equation 3

Y=Bottom + (Top-Bottom )/( 1+ 10 ((LogIC50-X )*HillSlope ))

[00265] PhyTip purified antibodies from the 175 well lines identified as hits in the single
point screening assay were tested as final dilutions of 12.5% and 2.5% for inhibition of
IL-8 and Gro-cc responses. Only supernatants derived from one of the well lines tested
(ZY05KA-G08) effectively inhibited the calcium flux response to IL-8 (92% inhibition at
12.5% sample, 82% inhibition at 2.5% sample) and Gro-cc (79% inhibition at 12.5%
sample, 75% inhibition at 2.5% sample).
[00266] Following dilution cloning and large-scale purification, the ZY05KA-G08
antibody was further profiled in the FLIPR assay at final assay concentrations of 15pM-
300nM antibody and shown to fully inhibit IL-8, Gro-cc and ENA-78 responses (see
Figure 1A-C for representative data). Geomean IC 50 values were determined from 4-5
separate experiments for IL-8 and Gro-cc and 2 separate experiments for ENA-78 and
resulted in geomean IC 50 values of 4.8nM (95% Confidence Interval 3.6 - 6.5nM) for IL-
8, 4.1nM (95% Confidence Interval 3.2 - 5.3nM) for Gro-cc and 2.6nM (n=2) for ENA-

78. A commercially available control antibody, 6C6 mouse monoclonal (BD Pharmingen
555932), was profiled in the assay for comparison and whilst this antibody showed full
inhibition of Gro-cc and ENA-78 calcium responses minimal inhibition of the IL-8
response was observed (Figure 1).

Example 3

Characterization of Species Cross Reactivity of ZY05KA-G08 and Selectivity for

CXCR2 over CXCR1

[00267] The cross reactivity of the Veloclmmune derived antibody ZY05KAG08 to mouse
and cynomolgus orthologs and selectivity for CXCR2 over CXCR1, its most closely
related chemokine receptor family member with approximately 77% identity, was tested
using HEK cell lines over-expressing mouse, cynomolgus or human CXCR2 or human
CXCR1. The cynomolgus CXCR2 sequence has been previously published (Hipkins et
al., JPET 310:291-300 (2004)) and was confirmed as the most common sequence through
analysis of genomic DNA sequence taken from 48 unrelated cynomolgus monkeys.
[00268] Binding of the antibody to the cell lines was measured using an FMAT direct
binding assay essentially as described in Example 1 except purified antibodies were tested
at a range of concentrations (0.2pM to 3nM). Representative data shown in Figure 2A-D
demonstrates that the antibody bound human CXCR2 but did not bind mouse or
cynomolgus orthologs or human CXCR1. Matched antibody isotype controls were used
to assess non-specific antibody binding which was negligible in the assay. Positive
control antibodies, anti-human CXCR2 clone 6C6 (anti-CD 182, BD Pharmingen
555932), anti-mouse CXCR2 (R&D Systems MAb2164) and anti-human CXCR1 (R&D
Systems MAb330) were used to confirm receptor expression on the cells.

Example 4

Reformatting of the Hybridoma Antibody ZY05KA-G08 nto Human IgG 1T

[00269] The hybridoma derived antibody ZY05KA-G08 was converted to human IgGl
format essentially as described by Persic et ah, Gene iS7:9-18 (1997) with the following
modifications. Variable regions of light and heavy chain genes were amplified from
cDNA of clone ZY05KA-G08 using HiFi extensor polymerase (Abgene AB0794/B) and
specific primers to either the heavy or light chain variable region. The VH domain was
cloned into a vector (pEU1.4) containing the human heavy chain constant domains and
regulatory elements to express whole IgGl heavy chain in mammalian cells. The V H
constant region contained the triple mutations (TM) L234F/L235E/P331S resulting in an
effector null human IgGl (Oganesyan et al,. Acta Crystallogr D Biol Crystallogr. 64:100-
704 (2008)). Similarly, the V domain was cloned into a vector (pEU3.4) for the
expression of the human light chain (kappa) constant domains and regulatory elements to
express whole IgG light chain in mammalian cells. The reformatted ZY05KA-G08
antibody is subsequently referred to as HY29.
[00270] To obtain IgGs, the heavy and light chain IgG expressing vectors were transfected
into EBNA-HEK293 mammalian cells. IgGs were expressed and secreted into the
medium. Harvests were pooled and filtered prior to purification, then IgG was purified
using Protein A chromatography. Culture supernatants were loaded on a column of
appropriate size of Ceramic Protein A (Pall 20078-036) and washed with 50 mM Tris-
HC1 pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium
 Citrate ( H 3.0) and neutralized by the addition of Tris-HCl (pH 9.0). The eluted material
was buffer exchanged into PBS using NaplO columns (GE Lifesciences 17-0854-02) and
the concentration of IgG was determined spectrophotometrically using an extinction
coefficient based on the amino acid sequence of the IgG (Pace et al., Protein Sci. 4:2411-
23 (1995)). The IgG were analyzed for purity by SDS-PAGE

Example 5

Binding of HY29 to Cell Lines Using Flow Cytometry

[00271] The species cross reactivity of the HY29 was confirmed using flow cytometry.
Venepuncture and blood handling was performed with the minimum of disturbance, since
this can alter leukocyte function. Human/cynomolgus monkey blood was anticoagulated
with sterile 0.25 M EDTA (Invitrogen) and used immediately or within 2 hrs (cyno blood
only). Polymorphonuclear neutrophils (PMN) were isolated from blood by sedimentation
using 2.4% dextran (Sigma, St. Louis, MO) in 0.9% sodium chloride at room temperature
for 60 min. The leukocyte-rich upper fraction was collected and laid on a two-layer
Percoll® gradient of 72% and 62% Percoll (Amersham Pharmacia Biotech, Uppsala,
Sweden) for cynomolgus cells and 80% and 62% for human cells and centrifuged at 1138
g for 20 min at room temperature without braking. The granulocyte layer was removed
from the interface of the Percoll, washed twice in PBS and centrifuged at 284 g for 5 min
at room temperature, without braking. Isolated neutrophils were resuspended at 4x 10 6
cells/ml in ice cold PBS containing 1% sodium azide and 10% FCS (FACS buffer).
[00272] Binding of antibodies to human or cynomolgus CXCR2 on the surface of the
neutrophils was measured either using antibodies directly conjugated to fluorophore e.g.
PE labelled anti-CD 182 (BD Pharmingen #555933) or using an indirect method with
unlabeled primary antibody (HY29) followed by PE labelled anti species secondary
antibody (BD Pharmingen #555787). Matched isotype control antibodies were included
for comparison. Cells ( ΙΟΟµΙ ) were incubated with 10 µ g/ml PE-labelled antibody or
lOug/ml unlabeled antibody prepared in ice cold 3% BSA/PBS for a minimum of 30 mins
at 4°C. Cells were washed 3 times with ice cold FACS buffer by centrifugation at 400g.
The cells stained with directly conjugated antibodies were fixed by addition of ice cold
 4% paraformaldehyde for 10-15 mins. For the indirect method after washing the cells
were incubated with PE labelled anti human IgG antibody in 3% BSA/PBS for 30mins
at 4°C. The cells were washed again then fixed. The level of fluorescence on the cell
surface was measured by flow cytometry using the BD Biosciences Canto II System.
[00273] Using this method it was demonstrated that HY29 bound human neutrophils but
not those isolated from cynomolgus monkey (Fig 3a and 3c). The commercial 6C6 (anti-
CD 182) antibody was used as a positive control in the experiment and bound both human
and cyno neutrophils (Fig. 3b and 3d).

Example 6

Characterization of HY29 Inhibition of β -arrestin Recruitment

[00274] Invitrogen TANGO™ GPCR Assay System measures β -arrestin binding to

agonist activated CXCR2, with a β -lactamase reporter gene readout. The assay uses
U20S cells (human bone Osteosarcoma cells) that stably express human CXCR2 linked
at the C terminus to a Gal4-VP16 transcription factor via a TEV (Tobacco Etch Virus)
protease site. The cells also stably express a β -arrestin/TEV fusion protein and the
mammalian optimized β -lactamase (bla) reporter gene the expression of which is under
the control of multiple copies of a UAS response element.
[00275] Binding of agonist to the CXCR2 results in recruitment of β -arrestin to the
receptor. This brings the TEV protease fusion partner in proximity to the TEV protease
cleavage site at the C-terminal of CXCR2, releasing the fused Gal4-VP16 transcription
factor. The Gal4-VP16 translocates to the nucleus and binds the UAS response element
causing up-regulation of the expression of the β -lactamase reporter gene.
[00276] Activity of the reporter gene is measured using a cell permeable FRET
(fluorescence resonance energy transfer) substrate containing two fluorophores, coumarin
and fluorescein. In the absence of the enzyme β -lactamase the substrate is uncleaved so
that excitation of the coumarin at 409nm results in FRET to the fluorescein and emission
of green light at 520nm. In the presence of β -lactamase (e.g. induced by agonist
stimulation of the receptor) the substrate is cleaved disrupting the FRET and resulting in a
direct blue fluorescence emission from the coumarin at 447nm. Thus the blue to green
 fluorescence ratio obtained directly correlates to receptor modulation by agonists or
antagonists. Activation of the reporter gene provides a quantifiable measurement of the
degree of interaction between the CXCR2 and its protease-tagged arrestin partner.
[00277] TANGO™ U20S hCXCR2 -b / cells (Invitrogen K1521) were routinely cultured
in McCoys 5A Medium (Invitrogen 16600) supplemented with 10% dialyzed FBS
(Invitrogen 26400), 25 mM HEPES (Sigma H0887), 0.1 mM non-essential amino acids
(Invitrogen 1140), 1 mM Sodium Pyruvate (Invitrogen 11360), 0.2 mg/ml Zeocin
(Invitrogen R250-01), 0.05 mg/ml Hygromycin (Invitrogen 10687), 0.1 mg/ml G418
(Invitrogen 10131) according to the manufacturers recommendations. For assays cells
were harvested using Accutase, resuspended in Freestyle™ Expression Medium
(Invitrogen 12338) at a density of 3.33xl0 5 cells/ml and 30 µΐ per well seeded into black
clear bottom 384 well Poly-D Lysine plates (Greiner 781946). Plates were incubated at
37°C, 5% C0 2 for 18-24 hours.
[00278] IgG samples and reagents were prepared in Freestyle™ Expression Medium
unless otherwise stated. Test solutions of purified IgG were typically prepared in 384 well
polypropylene plates by 3-fold serial dilutions to give 1 1 concentration points. The
diluted IgGs (5 µΐ ) were added in duplicate to the pre-plated cells and incubated at room
temperature for 30-60 minutes at 37°C, 5% C0 2 . Cells were then stimulated by addition
of 5 µΐ IL-8/CXCL8, Gro - /CXCLl, Gro - /CXCL2 (R&D Systems 276-GB), Gro-
Y/CXCL3 (R&D Systems 277-GG), GCP-2/CXCL6 (R&D Systems 333-GC), NAP-
2/CXCL7 (R&D Systems 393-NP), mouse MIP-2 (R&D Systems 452-M2), Mouse KC
(R&D Systems 453-KC) agonist for 5 hours at 37°C, 5% C0 2 . Unstimulated control
wells (10 µΐ Freestyle™ Expression Medium added to the cells) and cell free control wells
(well containing 40 µΐ Freestyle™ Expression Medium) were set up on each plate for
determination of basal β -lactamase expression and background fluorescence respectively.
[00279] After stimulation the level of β -lactamase activity was measured by addition of 8
µΐ detection mix containing the CCF4 AM/LiveB LAzer™ FRET B/G Substrate
(Invitrogen K1089) prepared using the Beta Lactamase Loading Solutions kit (Invitrogen
K1085) and Solution D (Invitrogen K1157) according to the manufacturer instructions.
The plates were incubated for 2 hours at room temperature in the dark and fluorescence
 emission at 460nm and 535nm determined following excitation at 409nm using a Perkin
Elmer Envision Plate Reader.
[00280] Data was corrected for background fluorescence at each emission wavelength
(460nm blue emission and 535nm green emission) by subtraction of the appropriate
average fluorescence signal obtained in the cell free controls wells. The emission ratio
for each well was then calculated by dividing the background subtracted 460nm blue
emission by the background subtracted 535nm green emission. Data was normalized to
the unstimulated control to give a Response Ratio by dividing the emission ratio by the
unstimulated control average emission ratio. This response ratio data was then used to
determine % control according to Equation 4 .

Equation 4

(
{Sample Re sponseRatio - Unstimulated Control Re sponse Ratio)
%Control = 100
{Agonist control Re sponse Ratio - Unstimulated Control Re sponse Ratio)

where agonist control is the mean value of the response ratio of the agonist controls wells
and unstimulated control is the mean value of the response ratio of the unstimulated
control wells. IC 50 values were determined using Graph Pad Prism Software by curve
fitting using a four parameter logistic equation described in Equation 3 .
[00281] Bioactivity of the ZY05KA-G08 chimeric antibody and the human HY29
IgGlTM were measured in the assays using a final concentration of 7 nM IL-8 or 15 nM
Gro-cc. The IC 50 values from at least 3 independent experiments are shown in Table 3 and
demonstrate that the antibody had retained functional potency on reformatting to the
human IgGlTM format. Further profiling of the HY29 IgGlTM antibody against a wider
panel of known CXCR2 ligands (189 nM Gro- β , 76 nM Gro- γ , 311 nM ENA-78, 151 nM
GCP-2, 264 nM NAP-2, 149 nM KC or 3 nM MIP-2) demonstrated that the antibody
inhibited the agonism of all of these ligands as shown in Table 3 .

TABLE 3 . Inhibition of ligand induced responses by ZY05KA-G08 chimeric IgG and

HY29 IgG in the TANGO™ hCXCR2 Assay

Ligand Geomean IC5 0 nM Geomean IC5 0 nM

(95% Confidence Interval) (95% Confidence Interval)
ZY05KA-G08 Antibody HY29 IgGlTM
Human IL-8 2.2 ( 1 .6 - 3 . 1) 1.3 ( 1 .0 - 1.7)
 Human Gro- α 2.9 (2.0 - 4 . 1) 1.7 ( 1 .5 - 2.0)
Human Gro- β 7.2 (6.5 - 7.9)
Human Gro- γ 3.3 (2.8 - 3.8)
Human ENA-78 0.5 (0.4 - 0.8)
Human GCP-2 0.7 ( 0.5 - 0.9)
Human NAP-2 9.5 ( 5.3 - 17)
Mouse KC 1.0 (0.7 - 1.5)
Mouse MIP-2 0.7 (0.7 - 1.4)

[00282] The nature of the antagonism of the CXCR2 receptor by the antibody HY29
IgGlTM was investigated using a Schild (PA2) like analysis (Schild et ah, Br. J.
Pharmacol 2:189-206 (1947)). In this assay the response to varying concentrations of IL-
8 and Gro-cc (5 pM - 1 µΜ ) were measured in the absence of antibody and in the
presence of increasing concentrations of HY29 antibody (150 pM to 1 µΜ ) . Under these
conditions the agonist response curves for IL-8 (Fig. 4A) and Gro-cc (Fig. 4B) were
parallel shifted to the right with increasing HY29 antibody concentration with a decrease
in the maximal agonist response at high antibody concentration (Figure 4). At low
concentrations of antagonist the lack of diminution of the maximal responses was
potentially due to receptor reserve in the system. The observed insurmountable
antagonism can be the result of allosteric or orthosteric blockade depending on the
kinetics of the interaction of agonist and antagonist with the receptor. In the current study
the antibody was pre-equilibrated with the receptor and then agonist added to stimulate
the functional response. Under these conditions the HY29 antagonism did not appear to
reach a maximal dextral displacement and was probe independent (i.e. similar for both
IL-8 and Gro-cc agonists) suggesting potential interaction of the antibody at an orthosteric
site. Without being bound by theory, the depression of the maximal response may be the
result of insufficient time to re-establish a new equilibrium due to a slow antibody off
rate. However other mechanisms cannot be completely excluded due to the limitations of
the assay format.

Example 7

Characterization of HY29 in FMAT Epitope Competition Assays

[00283] The epitope bound by the HY29 antibody was characterized using FMAT epitope
competition assays measuring the ability of the HY29 and commercially available anti-
 human CXCR2 antibodies to compete with fluorophore (DyLight 649/650) labeled
antibodies for binding to HEK human CXCR2 cells. The fluorescently labeled antibodies
used for the competition were HY29 or 6C6 which has been shown by Houimel and
Mazzuchelli, J. Leukoc. Biol. 85 :728-38 (2009) to bind an N-terminal epitope of the
receptor.
[00284] Labeling of the HY29 antibody and the commercially available 6C6 antibody (BD
Pharmingen 555932) was carried out using a DyLight-650 or DyLight-649 labeling kits
(Thermo Scientific 84536, 53051) according to the manufacturer instructions. For the
FMAT Epitope competition assay all samples and reagents were prepared in FMAT assay
buffer pH 7.4 defined in Example 1. Test solutions of purified IgG were prepared in 384
well polypropylene plates by 3-fold serial dilutions. IgG (7.5 µΐ ) were added in duplicate
to a 384 well clear-bottomed non-binding surface plate (Costar 3655) containing 12.5 µΐ
of 3.2 nM DyLight 649 labeled HY29 or DyLight650 labeled 6C6. HEK human CXCR2
cells (20 µΐ of 0.1 e6 cells/ml) were added and plates incubated for a minimum of 4 hours
at room temperature. Total binding and non-specific binding (NSB) controls were set up
using assay buffer and excess unlabeled IgG respectively. Plates were read on the
Applied Biosystems Cellular Detection System 8200 and data analyzed using appropriate
gating parameters on the system.
[00285] % Control binding was determined from the FL1 Average data using equation 1
and curve fitting was performed using GraphPad Prism Software by curve fitting using a
four parameter logistic equation described in equation 3 .
[00286] HY29 and three commercial mouse monoclonal anti human CXCR2 antibodies,
6C6, MAB331 (R&D Systems MAB331) and Ab24963 (Abeam Ab24963) were tested
for their ability to inhibit the binding of both DyLight-649 labeled HY29 (Fig. 5a) and
DyLight-650 labeled 6C6 (Fig. 5b) to the HEK human CXCR2 cells. The HY29
unlabeled antibody only competed with DyLight-649 labeled HY29 but not DyLight-650
labeled 6C6. None of the commercial antibodies were able to compete with DyLight-649
labeled HY29 but all three commercial antibodies could fully compete with DyLight 650
labeled 6C6 suggesting that HY29 may bind an epitope that is distinct from that bound by
the tested commercial antibodies.
 Example 8

Mapping of the HY29 Antibody Binding Sequence using Structured Peptides

[00287] Further characterization of the epitope bound by the HY29 antibody was carried
out by analysis of the binding to structured peptides. The extracellular domains of human
CXCR2 were synthesized as peptide arrays (Sloostra et ah, Mol. Divers. 7(2^:87-96
(1996)) of 3640 peptides comprising the N-terminus
(MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK, ECL1
(ASKVNGWIFGTFLCK), ECL2 (RRTVYSSNVSPACYEDMGNNTANWR) and ECL3
(DTLMRTQVIQETCERRNHIDR), using overlapping linear and single looped 16-mers
(Cys-Xi 6 -Cys format), overlapping double looped 16-mers in a Cys-Xi 6 -Cys-Xi 6-Cys
format and overlapping looped 7 mers of the combined N-terminus-ECLl, -ECL2, and -
ECL3 constructs in a X 7-Cys-Cys-X 7-Cys-X 7-Cys-X 7-Cys format. Conformational loops
were created by connecting the cysteine residues with a T3 CLIPS™ scaffold
(Timmerman et ah, J Mol Recognit 2 5 :283-299 (2007)). Additional mapping was
performed using a 455 linear and CLIPS peptide array consisting of an alanine scan of
peptides with the highest binding levels and a dedicated N terminus/ECL3 library .
[00288] Epitope mapping of monoclonal antibodies was performed according to published
protocols (Sloostra et ah, Mol. Divers. i 2 :87-96 (1996), Timmerman et a , J. Mol.
Recognit. 2 5 :283-299 (2007)). Briefly, the binding of antibody to each peptide was
tested in a PEPSCAN-based ELISA. The peptide arrays were incubated with a primary
antibody solution, for example consisting of 1 µ g/ml diluted in blocking solution (4%
horse serum, 5% ovalbumin (w/v) in PBS/1% Tween). After washing, the peptides were
incubated with a 1000-fold dilution of antibody peroxidase conjugate for one hour at
25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline
sulfonate (ABTS) and 2 µΐ of 3% Η 20 2 was added and the color developed for 1 hour.
The extent of color development was quantified with a charge coupled device (CCD) -
camera and an image processing system.
[00289] The core epitope for antibody binding mapped to the N terminus of the receptor
(Fig 6A). ECL1, ECL2 and ECL3 independently showed minimal antibody binding,
however CLIPS peptides consisting of combined of N terminus and ECL3 binding gave
 the highest signal levels (Fig 6B). This suggests a possible contribution from the ECL3 to
the binding interaction with a consensus sequence of PPFLLDAAPC, TQVIQETCERR.
Mapping of the N terminus using linear peptides (Fig. 6C) and alanine scanning of CLIPS
peptides derived from the N terminus and ECL3 (Fig 6D) showed that the minimal
epitope for HY29 binding was centered around the 31PFLLD 35 with the second leucine
and the aspartic acid key to the binding interaction. The proline at position 3 1 and ECL3
may provide structural context although direct interaction with the antibody cannot be
excluded.

Example 9

Germlining of HY29 Antibody

[00290] The amino acid sequences of the V H and V domains for the HY29 antibody were
aligned to the known human germline sequences in the VBASE database (Tomlinson
(1997) Journal of Molecular Biology 224:487-499), and the closest germlines were
identified by sequence similarity. For the VH domain this was VH3_DP77(3-21) and for
the V domain it was VK3_DPK21(L2). Except for Vernier residues (Foote and Winter
(1992) Journal of Molecular Biology 224:487-99), which were left unchanged, the
germlining process involved reverting framework residues in the V H and V domains to
these closest human germline sequences. For HY29 one residue required changing in the
VL domain, while a total of 4 were required in the V H domain. These were at Kabat
position 97 of the VL, where Glycine (G) was reverted to serine (S), and position 3,
Glutamate (E) to glutamine (Q), position 6 Alanine (A) to Glutamate (E), position 84,
Arginine (R) to Lysine (K) and position 103, Phenylalanine (F) to Tyrosine (Y) of the V H
domain. These changes were made using standard site directed mutagenesis techniques
with the appropriate mutagenic primers. Antibodies with different combinations of the
germline mutations were tested for inhibition of IL-8 and Gro-alpha responses in the
TANGO™ CXCR2 Assay performed as described in Example 6 .
[00291] IC 50 values from three independent experiments with duplicate determinations
within each experiment are shown in Table 4 with the 95% confidence interval (CI) of
each value. The fold increase in activity of the mutated antibodies compared to the
germlined parent antibody (gVH/gVL) was determined by dividing the geomean IC 50
value (M) of the germlined parent antibody by the geomean IC 50 (M) value of the mutated
antibody. Testing of V H / variants with a non-germlined V H combined with a
germlined V (pVH/gVL) and a fully germlined V H with a non-germlined V L (gVH/pVL)
indicated a potential loss of activity due to reversion mutation of one of the V H residues to
the germline sequence as shown in Table 4 . Further combination of individual V H
mutations with a germlined V L indicated that one of the key amino acid germline changes
contributing to the greater potency of the parental V H sequence was Glutamate (E) to
Alanine (A) at position 6 of the V H in the framework 1 region. This Alanine (A)
framework residue change is therefore retained in the final germlined antibody sequence.

TABLE 4 : Potency of Germlined Variants in the TANGO™ CXCR2 Assay

Example 10

Human neutrophil chemotaxis assay

] Venepuncture and blood handling was performed with the minimum of

disturbance, since this can alter leukocyte function. Blood was anticoagulated with sterile
 0.25 M EDTA (Invitrogen) and used immediately. Human polymorphonuclear
neutrophils (PMN) were isolated from blood by sedimentation 2.4% dextran (Sigma, St.
Louis, MO) in 0.9% sodium chloride at room temperature for 60 min. The leukocyte-rich
upper fraction was collected and laid on a two-layer Percoll® gradient of 72% and 62%
Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden), and centrifuged (1138 g, 20
min, room temperature) without braking. The granulocyte layer was removed from the
interface of the Percol and washed twice in PBS and centrifuged (284 g, 5 min, room
temperature) without braking. Isolated neutrophils were resuspended in PBS containing 1
mM MgCl 2 and 1 mMCaCl 2 . Cell motility was determined using a modified Boyden
chamber procedure using a disposable system (Neuro Probe, Inc. ChemoTx® ) . Cells
were incubated with anti-CXCR2 antibodies for 30mins at 37 °C prior to measurement of
chemo taxis. Lower chambers were filled with 30 µΐ of media containing IL-8 or GROa at
the indicated concentrations. The upper filter was lowered into place, and 50 µΐ of the
PMN suspension (5 x 10 6cells/ml), without (control) or with HY29, was added at the
indicated concentrations. HY29 was diluted in PBS to the desired concentration.
Neutrophil migration proceeded for 90 min at 37 °C in the cell incubator, after which the
fluid and cells on top of the filter was removed. Following centrifugation for 5mins at
500rpm migrated neutrophils were analyzed by Cell Titre Glo (Promega) luminescence.
HY29 showed a concentration dependent inhibition of IL-8 (5ng) and Gro-cc (40ng)
stimulated chemotaxis of the neutrophils (Fig. 7a and 7b) and Table 5 . These neutrophil
data exemplifies that inhibition of CXCR2 with have the potential to inhibit neutrophil
movement and activation.

Table 5 :

IC50 values for HY29 in ligand driven chemotaxis assay using primary human neutrophils
Chemotaxis (IC50 iiM) IL8 Groc
26 +160 204 ±220
 EXAMPLE 11

Inhibition of neutrophil influx into the lung following intranasal LPS

challenge

[00293] Inhibition of inflammatory cell influx into the lungs following intranasal LPS
administration the anti-CXCR2 antibody HY29 was tested in wild type and hCXCR2
transgenic mice.

Generation of the Transgenic Mice

[00294] Homologous recombination in ES cells was used to insert the human CXCR2
cDNA into exon 1 of the mouse CXCR2 gene (Figure 14). The targeting vector was
electroporated into C57BL/6N ES cells, colonies were selected with puromycin and
counterselected with 2 µΜ Gancyclovir. Selected ES clones were isolated and analyzed
for homologous recombination by Southern Blotting. ES clones with correct homologous
recombination at both sides of the vector and a single integration were selected for
blastocyst injections. Chimeras obtained were mated to C57BL/6 females for germline
transmission. Heterozygous mice (hCXCR2 + , mCXCR2 + ) were interbred to produce
homozygous (hCXCR2 _) hCXCR2 Knockin mice.
[00295] PCR analysis of genomicDNA from tail biopsies was used to screen and confirm
genotypes. The primers used to were 5'-TTATCCAACCAGCCACTTTCC- 3' (2226_44)
and 5' -CGAGGTATGATGGCATCTGC-3 ' (2226_45) to detects heterozygous,
homozygous wildtype and humanized alleles and 5'- GGCAGAAGCACGCTTATCG-3 '
(1307_1: Flpe_as) and 5'-GACAAGCGTTAGTAGGCACAT-3'(1307_2: Flpe_s) to
detect the Flp transgene. PCR conditions were lOOnM of each primer, 1-2 U Taq
polymerase (Invitrogen), l x PCR buffer, 2mM MgCl 2 200 µΜ dNTPs (Invitrogen) in
50 µ1. Amplifications were carried out using the following protocol: denaturing for 5 mins
95°C followed by 35 cycles consisting of denaturing at 95°C for 30secs, annealing at
60°C for 30 sees and extension at 72°C for 1 min, followed by a final extension for 10
mins at 72°C. Samples were subsequently analysed by agarose gel electrophoreisis.
Expected fragments were as follows: 247bp (wild type mCXCR2), 354bp (hCXCR2),
585bp (tragetted allele )and 343bp (targetted allele following Flp recombination)
Intranasal LPS administration

Mice were injected intraperitoneally (ip) with vehicle (Sodium acetate buffer) or
lOmg/kg HY29 24hrs hours prior to LPS challenge. LPS (15ug/kg) or vehicle (PBS) was
administered intranasally and 24hr post LPS challenge mice were euthanized and
bronchoalveolar lavage (BAL) samples, lung tissues, peripheral blood and serum were
collected. BAL fluid was analyzed for total cell counts and differential cell analysis,
cytokine analysis. Lung homogenates were analyzed for cytokine levels only and the
peripheral blood was analyzed for absolute neutrophil counts. For the BAL collection,
three 0.4-ml aliquots of isotonic saline were injected into the tracheal catheter, and the
recovered fluid was analyzed for total cell counts and differential cell analysis (Kung et
al., 1994). Total cell counts were performed by hemocytometry. Slides for differential cell
enumeration were prepared on a Shandon cytospin (Shandon, Pittsburgh, PA) at 800 rpm
for 6 min, fixed with methanol, and stained with Wrights/Giemsa stain . At least 200 cells
were counted from each cytospin to generate data. Cytokine (TNF, IL1B, IFNg,
IL12p70, KC, IL6, IL10) levels were determined of BAL aspirate, serum and lung tissue
homogenates using MSD 7 plex mouse proinflammatory multiplex assay (MesoScale
Discovery) according to manufacturer instructions. Peripheral blood neutrophil levels
were analyzed following red blood cell lysis with 155 mM NH4C 1 using a guava easycyte
cell analyzer (Millipore). A single in LPS challenge induced a significant influx of
inflammatory cells into the lungs of both wt and Tg mice. The majority of these cells
were neutrophils. Pre-treatment with lOmg/kg HY29 significantly inhibited LPS-induce
pulmonary neutrophilia in male and female transgenic mice but not in wild type mice
shown in Fig. 8 and Table 6 . HY29 treatment causes an increase in inflammatory
cytokines in the BAL and lung homogenates in transgenic animals shown in Fig. 9 and
Table 6 . These cytokines particularly KC and IL6 could be pharmacodynamic biomarkers
of CXCR2 antibody engagement. These data exemplifies that antibodies to CXCR2 have
the potential to treat and modify inflammatory disorders.
Table 6 : % inhibition of inflammatory cell influ into the lung or peripheral neutrophil number
in response to intranasal LPS challenge by HY29 (percent inhibition of HY29/LPS vs. PBS/LPS)
p-values shown.

EXAMPLE 12

Tumor growth inhibition studies

Inhibition of growth of syngeneic mouse tumor models in human CXCR2 knock in

transgenic mice

[00297] Inhibition of tumor growth by the humanized anti-CXCR2 antibody was

tested in humanized CXCR2 transgenic mice with subcutaneous tumors generated from
EL4 lymphoma cell line (ATCC) and B16F10 derivative melanoma model (NCI). The
EL4 cells were grown in culture at 37°C in an atmosphere containing 5% C02, using
RMPI medium (Invitrogen) containing 10% FBS. Cells were inoculated subcutaneously
into the flank of 8-week old female CXCR2 KIKO C57BL6J mice (MRC Harwell) with 1
x 105 cells per mouse in PBS (Sigma). Tumor measurements were taken three times
weekly using vernier calipers. Each group received one of the following treatments:
human IgGlTM (isotype) control and anti-human CXCR2 hlgGlTM (HY29). Treatment
was administered by intraperitoneal injection two times per week (EL4) or once weekly
(B16F10) at 20mg/kg HY29. Treatment was well- tolerated, with no significant loss in
body weight. Tumor growth inhibition is expressed as percentage inhibition (baseline
subtracted) to the hulgGlTM control and statistical analysis was conducted using a 2 way
 ANOVA. Results for tumor growth inhibition in the EL4 model are shown in Fig. 10 and
Table 7 .

Table 7 : .Syngeneic tumor growth inhibition studies in human CXCR2 knock in

transgenic mice

[00298] Anti-CXCR2 antibody treatments resulted in tumor growth inhibition compared to

isotype control. Specifically, HY29 antibody treatment resulted in tumor growth
inhibition of 50% (P < 0.001) in EL4 model, 39.5- 56% (P < 0.001) in B16F10 melanoma
model. These results demonstrate that HY29 inhibits tumor growth in a transgenic
syngeneic model by targeting host and not tumoral CXCR2.
[00299] Inhibition of Growth of Patient derived KRAS mutant Lung Xenograft Tumo
Model
[00300] Inhibition of tumor growth by the humanized anti-CXCR2 antibody HY29 was
tested in a k-ras mutant patient derived lung xenograft model. Six to eight week old
female nude XID mice (Harlan, Indianapolis, IN) were implanted subcutaneously in the
right flank patient-derived non-small cell lung carcinoma fragment using an 1 1 gauge
trochar. Mice were randomized to treatment groups (9 per group) when tumors reached
approximately 150-200mm3 size. Each group received one of the following treatments:
human IgGlTM (isotype) control and anti-human CXCR2 hlgGlTM (HY29). Treatment
was administered by intraperitoneal injection two times per week at 20mg/kg HY29 or
50mg/kg HY29 for 5 doses. Treatment was well-tolerated; with no significant loss in
body weight. Tumor measurements and body weight were collected twice per week.
Tumor volume was calculated with the formula [length (mm) x width (mm)2 )/2] using
Study Director software (Studylog Systems, San Francisco, CA). Tumor growth
inhibition was calculated with the formula [%TGI= 1-(T/C) x 100] where T= final tumor
volume from treated group after last dose and C= final tumor volume from the control
after last dose. Tumor growth inhibition is expressed as percent inhibition (baseline
subtracted) to the human IgG control and statistical analysis was conducted using
ANOVA. Results for tumor growth inhibition are shown in Fig. 11. Anti-CXCR2
antibody treatments resulted in tumor growth inhibition compared to isotype control.
Specifically, HY29 antibody treatment resulted in tumor growth inhibition of 25-33% (P
< 0.001) at 20mg/kg dose and 25% at 50mg/kg dose. These results demonstrate that
CXCR2 inhibition with HY29 inhibits tumor growth by targeting tumoral and not host
CXCR2 in a patient derived xenograft model.

Example 13

Inhibition of BxPC3 cancer cell line invasion

Inhibition of tumor cell invasion by the humanized anti-CXCR2 antibodies was

tested in using BxPC3 pancreatic cancer cells. BxPC3 cells grown in culture at 37 °C in an
atmosphere containing 5% C02, using RMPI medium (Invitrogen) containing 10% FBS
were starved in low serum overnight and pre-treated with lOug/ml HY29 or human
IgGlTM isotype control 30 min prior to plating. 1 x 10 4 cells were plated in upper
chamber of a transwell plate coated with matrigel. Full media or low serum media
(0.2%), with or without chemokines (CXCL1, -5 or -8) was added to the bottom
chambers. After overnight incubation, cells that had not invaded through the matrigel
coated membrane were removed, and cells that had invaded were fixed and stained with
DAPI (4',6-diamidino-2-phenylindole (5 µg/ml)) to visualize nuclei and quantitated.
Treatment with CXCR2 cytokines enhances BxPC3 invasion (Gro-a 2.1 fold; ENA78 1.3
fold; and IL8 1.7fold) Anti-CXCR2 antibody treatments resulted in decreased tumor cell
invasion compared to human IgGlTM control. Specifically, HY29 antibody treatment
resulted in 25% (P < 0.001) reduction in basal cell invasion; 68% (P < 0.001) in response
to Gro-a(P < 0.01) in response to ENA-78 and 65% in response to IL8 (P < 0.05) shown
in Fig. 12.
 Example 14

Inhibition of pancreatic cancer cell line growth in 3D culture

[00302] Inhibition of pancreatic tumor cell growth by the humanized anti-CXCR2

antibodies was tested using pancreatic cancer cells in 3D matrigel (rBM) overlay culture.
5 x 10 Pancreatic cancer cells were cultured on 8 well culture slides in a 2% matrigel
overlay culture. 5 x 10 cells were seeded on top of a layer of reduced Growth Factor
Matrigel in medium (Invitrogen) containing 1% FBS and 2% matrigel (BD Biosciences,
San Jose, CA) supplemented with lOug/ml HY29 or a human IgGlTM isotype control.
Once plated on rBM, all cultures were incubated at 37°C in a 5% C02 humidified
incubator for up to 14 days. Media containing antibody was replaced every 4 days.
Morphology was observed every 2 days via phase contrast microscopy and number of
cellular structures was counted. Cells were recovered from matrigel using matrigel cell
recovery solution (BD Biosciences) and cell viability was determined by cell titre glo
(Promega) as per manufacturer's instructions. Treatment with HY29 resulted in decreased
tumor cell growth and viability compared to human IgGlTM control. Specifically, HY29
antibody treatment resulted in 50-90% (P < 0.001) reduction in cell viability; and 50-75%
(P < 0.001) reduction in the number 3D cellular structures shown in Fig. 13.

[00303] The disclosure is not to be limited in scope by the specific embodiments described
which are intended as single illustrations of individual aspects of the disclosure, and any
compositions or methods which are functionally equivalent are within the scope of this
disclosure. Indeed, various modifications of the disclosure in addition to those shown and
described herein will become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are intended to fall within
the scope of the appended claims.
[00304] All publications and patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual publication or patent
application was specifically and individually indicated to be incorporated by reference.
Sequence Listing
<110> Danielle Carroll
Simon Barry
Chris Rossant

<120> ANTI-CXC CHEMOKINE RECEPTOR-2 BINDING MOLECULES AND USES THEREOF

<160> 36

<170> Medimmune Ltd patent software March 2010 release. Output verified by USPTO
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Ser Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val
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Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
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Ala Arg Ala Arg Gly Ser Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu
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Val Thr Val Ser Ser

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Ser e Ser Ser Gly Ser Ser Tyr He Phe His Ala Asp Ser Val Lys
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Ala Arg Gly Ser Tyr Phe Asp Tyr
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Glu Val Glu Leu Val Ala Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
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Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
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Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Asn Ser Val Tyr Leu Gin
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Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg
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Glu lie Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Arg e Asn
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Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu He
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Tyr Gly Ala Ser Thr Arg Ala Thr Gly He Pro Ala Arg Phe Ser Ala
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Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr He Ser Gly Leu Gin Ser
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Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Asn Asn Asn Trp Pro He
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Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys
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Arg Ala Ser Gin Ser Val Arg e Asn Leu Ala
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<210> 13
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Gly Ala Ser Thr Arg Ala Thr
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<210> 14
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Gin Gin Asn Asn Asn Trp Pro He Thr
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<210> 15
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Glu He Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
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Glu Arg Ala Thr Leu Ser Cys

20

<210> 16
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Gly lie Pro Ala Arg Phe Ser Ala Ser Gly Ser Gly Thr Glu Phe Thr
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Leu Thr e Ser Gly Leu Gin Ser Glu Asp Phe Ala Val Tyr Tyr Cys
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Phe Gly Gin Gly Thr Arg Leu Glu He Lys
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Glu Val Gin Leu Val Ala Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
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Ser Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45

Ser Ser e Ser Ser Gly Ser Ser Tyr e Phe His Ala Asp Ser Val
50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Ala Arg Gly Ser Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu
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Val Thr Val Ser Ser

115

<210> 21
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Thr Tyr Ser Met Asn
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<210> 22
<211> 17
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Ser e Ser Ser Gly Ser Ser Tyr He Phe His Ala Asp Ser Val Lys
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Gly

<210> 23
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Ala Arg Gly Ser Tyr Phe Asp Tyr
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<210> 24
<211> 30
<212> PRT
<213> Homo sapiens

<400> 24
Glu Val Gin Leu Val Ala Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30

<210> 25
<211> 14
<212> PRT
<213> Homo sapiens

<400> 25
Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
5 10

<210> 26
<211> 32
<212> PRT
<213> Homo sapiens

<400> 26
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr Leu Gin
 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
20 25 30

<210> 27
<211> 11
<212> PRT
<213> Homo sapiens

<400> 27
Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser
5 10

<210> 28
<211> 321
<212> DNA
<213> Homo sapiens

<400> 28
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccaggaga aagagccacc 60

ctctcctgca gggccagtca gagtgttaga atcaacttag cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180

aggttcagtg ccagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cagtttatta ctgtcagcag aacaataact ggccgatcac cttcggccaa 300

gggacccgac tggagattaa a 321

<210> 29
<211> 107
<212> PRT
<213> Homo sapiens

<400> 29
Glu lie Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Arg e Asn
20 25 30
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu e
35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly He Pro Ala Arg Phe Ser Ala
50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr He Ser Ser Leu Gin Ser
65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Asn Asn Asn Trp Pro He
85 90 95

Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys
100 105

<210> 30
<211> 11
<212> PRT
<213> Homo sapiens

)0> 30
Ala Ser Gin Ser Val Arg He Asn Leu Ala
5 10

<210> 31
<211> 7
<212> PRT
<213> Homo sapiens

<400> 3 1
Gly Ala Ser Thr Arg Ala Thr
5

<210> 32
<211> 9
<212> PRT
<213> Homo sapiens

<400> 32
Gin Gin Asn Asn Asn Trp Pro He Thr
5

<210> 33
<211> 23
<212> PRT
<213> Homo sapiens

<400> 33
Glu lie Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210> 34
<211> 15
<212> PRT
<213> Homo sapiens

<400> 34
Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu e Tyr
5 10 15

<210> 35
<211> 32
<212> PRT
<213> Homo sapiens

<400> 35
Gly He Pro Ala Arg Phe Ser Ala Ser Gly Ser Gly Thr Glu Phe Thr
5 10 15

Leu Thr He Ser Ser Leu Gin Ser Glu Asp Phe Ala Val Tyr Tyr Cys
20 25 30

<210> 36
<211> 10
<212> PRT
<213> Homo sapiens

<400> 36
Phe Gly Gin Gly Thr Arg Leu Glu He Lys
5 10
 WHAT IS CLAIMED IS:

1. An isolated binding molecule o antigen-binding fragment thereof which specifically binds to

CXC chemokine receptor-2 (CXCR2), wherein the binding molecule (a) ca inhibit binding to
interleukin 8 (IL-8), (b) can inhibit binding to growth-related protein alpha (Gro-alpha), (c) can
inhibit the IL-8 or Gro- induced calcium responses, (d) can inhibit IL-8, Gro- γ , Gro- β , Gro- γ ,
ENA-78, GCP-2 and NAP- 2 induced beta-arrest in recruitment n a mammalian cell, or (e) any
combination thereof.

2 . The binding molecule or fragment thereof of claim 1, which binds to CXCR2 as expressed on
the surface of a mammalian cell.

3 . The binding molecule or fragment thereof of claim I or claim 2, which comprises an antibody
or antigen-binding fragment thereof.

4 . The antibody or antigen binding fragment thereof of claim 3, which specifically binds an
epitope comprising amino acids 30-39 and amino acids 279-289 of SEQ ID NO:37.

5 . The antibody or antigen binding fragment thereof of claim 3, which specifically binds an
epitope comprising amino acids 30-39 of SEQ ID NO:37.

6 . The antibody or antigen binding fragment thereof of claim 3, which specifically binds an
epitope comprising amino acids 31-35 of SEQ ID NO:37.

7 . The antibody or antigen binding fragment thereof of claim 3, which specifically binds an
epitope comprising amino acids 34 and 35 of SEQ ID NO:37.

8 . The antibody or antigen binding fragment thereof of claim 7, wherein the antibody or antigen

binding fragment is in contact with residues L34 and D35 of SEQ ID NO:37.

9 . The antibody or antigen-binding fragment thereof of claim 3, which specifically binds to the
same CXCR2 epitope as an antibody or antigen-binding fragment thereof comprising the heavy
chain variable region (VI I ) and light chain variable region (VL) region of HY29 or HY29GL or
any combination thereof.
10. The antibody o antigen-binding fragment thereof of any one of claim 3-9, which specifically
binds to CXCR2, and competitively inhibits CXCR2 binding by a antibody or antigen-binding
fragment thereof comprising the V and VL of HY29, HY29GL or any combination thereof.

11. The antibody or antigen-binding fragment thereof of any one of claims 4- 10 comprising a
V and a VL, wherein the V comprises a V complementarity determining region- 1
(VHCDR 1) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 3 for HY29 orSEQ ID NO: 2 for HY29GL.

12. The antibody or antigen-binding fragment thereof of any one of claims claims 4- 10
comprising a V and a VL, wherein the V comprises a VHCDR 1 amino acid sequence
identical to SEQ ID NO: 3 for HY29orSEQ ID NO: 2 for HY29GL.

3 . The antibody or antigen-binding fragment thereof of any one of claims 4 to 12 comprising a

V I and a VL, wherein the V comprises a V complementarity determining region-2
(VHCDR 2 ) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 4 for HY29 or SEQ ID NO: 22 for HY29GL.

4 . The antibody or antigen-binding fragment thereof of an one of claims 4 to 2 comprising a

V and a VL, wherein the VH comprises a VHCDR 2 amino acid sequence identical to SEQ ID
NO: 4 for HY29 or SEQ ID NO: 2 1for HY29GL.

15. The antibody or antigen-binding fragment thereof of an one of claims 4 to 14 comprising a

V and a VL, wherein the V comprises a V complementarity determining region -3

(VHCDR3) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 5 for I IY29 or SEQ ID NO: 23 for HY29GL.

6 . The antibody or antigen-binding fragment thereof of any one of claims 4 to 14 comprising a

V and a VL, wherein the V comprises a VHCDR3 amino acid sequence identical to SEQ ID
NO: 5 for HY29 or SEQ ID NO: 22 for HY29GL.

7 . The antibody or antigen-binding fragment thereof of any one of claims 4 to 1 comprising a

V and a VL, wherein the VL comprises a VL complementarity determining region- 1
(VLCDR ) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 2 for HY29, or SEQ ID NO: 30 for HY29GL.
18. The antibody o antigen-binding fragment thereof of any one of claims 4 to 16 comprising a
V and a VL, wherein the V comprises a VLCDR 1 amino acid sequence identical to SEQ ID
NO: 12 for HY29 or SEQ ID NO: 30 for HY29GL.

19 . The antibody or antigen-binding fragment thereof of any one of claims 4 to 18 comprising a

V and a VL, wherein the L comprises a L complementarity determining region-2
(VLCDR2) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 3 for HY29 or SEQ ID NO: 3 1for HY29GL.

20. The antibody or antigen-binding fragment thereof of any one of claims 4 to 18 comprising a
V and a VL, wherein the L comprises a VLCDR 2 amino acid sequence identical to SEQ ID
NO: 3 for HY29 or SEQ ID NO: 3 1for HY29GL.

2 1.The antibody or antigen-binding fragment thereof of any one of claims 4 to 20 comprising a

V and a VL, wherein the VL comprises a VL complementarity determining region-3
(VLCDR 3 ) amino acid sequence identical, except for four, three, two, or one amino acid
substitutions to: SEQ ID NO: 14 for ITY29 or SEQ ID NO: 32 for HY29GL.

22. The antibody or antigen-binding fragment thereof of one of claims 4 to 20 comprising a V

and a VL, wherein the V comprises a VLCDR3 amino acid sequence identical to SEQ D NO:
4 for HY29 or SEQ ID NO: 32 for HY29GL.

23. The antibody or antigen-binding fragment thereof of any one of claims 4- 11, 13, 1 , 17 to 22
comprising a V and a VL, wherein the V comprises a VHCDR I, a VHCDR2, and a VHCDR3
identical, except for four, three, two, or one amino acid substitutions in one or more VHCDRs to:
SEQ ID NOs: 3, 4, and 5 for HY29, respectively or SEQ ID NOs: 2 1, 22, and 23 for HY29GL.

24. The antibody or antigen-binding fragment thereof of any one of claims 4- 10, 12, 14, or 6 to
22 comprising a V and a VL, wherein the V comprises VHCDR I VHCDR2, and VHCDR3
amino acid sequences identical to: SEQ D NOs: 3, 4, and 5 for HY29, respectively or SEQ D
NOs: 2 1, 22, and 23 for HY29GL.

25. The antibody or antigen-binding fragment thereof of any one of claims 4 to 6, or 17, 9, or
2 1 comprising a V and a VL, wherein the VL comprises a VLCDR 1, a VLCDR2, and a
VLCDR 3 identical, except for four, three, two, or one amino acid substitutions in one or more
VL CDRs to: SEQ ID NOs: 12, 13, and 14 fo HY29, respectively or SEQ ID NOs: 30, 3 1, and
32 for HY29GL.

26. The antibody or antigen-binding fragment thereof of any one of claims 4 to 16, or 18, 20, or
22 comprising a VH and a VL, wherein the , comprises VLCDR l, VLCDR2, and VLCDR3
amino acid sequences identical to: SEQ D NOs: 12, 13, and 14 for I1Y29, respectively; SEQ D
NOs: 30, 3 1, and 32 for HY29GL, respectively..

27. The antibody or antigen-binding fragment thereof of any one of claims 4- 11, 13, 15, 17, 19 or
2 1 comprising a V and a VL comprising a VHCDR l, a VHCDR2, a VHCDR3, a VLCDR , a
VLCDR2, and a L DR3 identical, except for four, three, two, or one amino acid substitutions
in one or more CDRs to: SEQ ID NOs: 3, 4, 5 12, 13, and 14 for HY29, respectively; SEQ ID

NOs: 20, 2 1, 22, 30, 3 1, and 32 for HY29GL, respectively;

28. The antibody or antigen-binding fragment thereof of any one of claims 4- 10, 12, 14, 16, 18,
20, or 22 comprising a V and a VL comprising VHCDR l, VHCDR2, VHCDR3, VLCDR l,
VLCDR2, and VLCDR3 amino acid sequences identical to: SEQ ID NOs: 3, 4, 5, 12, 13, and 14
for HY29, respectively; SEQ ID NOs: 20, 2 1, 22, 30, 3 , and 32 for HY29GL. respectively:.

29. The antibody or antigen-binding fragment thereof of any one of claims 4- 10 comprising a V
and a VL, wherein the V comprises an amino acid sequence at least 75%, 80%, 85%, 90%, or
95% identical to SEQ ID NO: 2 for HY29, SEQ ID NO: 20 for HY29GL,

30. The antibody or antigen-binding fragment thereof any one of claims 4- 10 comprising a V
and a VL, wherein the V comprises the amino acid sequence of SEQ D NO: 2 for HY29, SEQ
ID NO: 20 for HY29GL

3 1. The antibody or antigen-binding fragment thereof of any one of claims any one of claims 4-
10, 29 or 30 comprising a V and a VL, wherein the VL comprises an amino acid sequence at
least 75%, 80%, 85%, 90%, or 95% identical to SEQ D NO: 11 for HY29, SEQ ID NO: 29 for
HY29GL,

32. The antibody or antigen-binding fragment thereof of any one of claims 4- 10, 29 or 30
comprising a V and a VL, wherein the VI, comprises the amino acid sequence of SEQ NO:
1I for HY29, SEQ ID NO: 29 for HY29GL,
33. The antibody o antigen-binding fragment thereof of claim 30 or claim 32 comprising a VH
and a VL, wherein the V and VL comprises the amino acid sequences of SEQ D NO: 2 and
SEQ ID NO: 1I for HY29, respectively; the amino acid sequences of SEQ ID NO: 20 and SEQ
ID NO: 29 for HY29.1_GL, respectively;

34. The antibody or fragment thereof of any one of claims 3 to 33, which specifically binds to
CXCR2 with an affinity characterized by a dissociation constant (KD) no greater than 5 10 -2 M,

10 2 M, 5 x 10 3 M, 10 3 M, 5 x 10 4 M, 10 4 M, 5 x 10 5 M, 10 5 M, 5 x 10 6 M, 10 6 M, 5 x 10 7
M, 10 7 M, 5 x 10 8 M, 10 8 M, 5 x 10 9 M, 10 9 M, 5 x 10 10 M, 10 10 M, 5 x 10 11 M, 10 11 M, 5 x
10 12 M, 10 12 M, 5 x 10 13 M, 10 13 M, 5 x 10 14 M, 10 14 M, 5 x 10 15 M, or 10 15 M . [[Value?]]

35. The antibody or fragment thereof of any one of claims 3 to 34, which is humanized.

36. The antibody or fragment thereof of any one of claims 3 to 34, which is chimeric.

37. The antibody or fragment thereof of claim 4 1, wherein the V and VL domains are fully
human and the antibody constant regions are derived non-human mammal.

38. The antibody or fragment thereof of any one of claims 3 to 34, which is fully human.

39. The antibody fragment of any one of claims 3 to 38 which is an Fab fragment.

40. The antibody fragment of any one of claims 3 to 38, which is a Fab' fragment.

4 1. The antibody fragment of any one of claims 3 to 38, which is a F(ab)2 fragment.

42. The antibody fragment of any one of claims 3 to 38, which is a single chain Fv (scFv)
fragment.

43. The antibody or fragment thereof of any one of claims 3 to 36 or 38, which comprises a light
chain constant regions selected from the group consisting of a human kappa constant region and
a human lambda constant region.

44. The antibody or fragment thereof of any one of claims 3 to 36 or 38, which comprises at a
heavy chain constant region or fragment thereof.
45. The antibody o fragment thereof of claim 44, wherein said heavy chain constant region or
fragment thereof is human IgG I.

46. The antibody or a fragment thereof of any one of claims 3 to 37, which is monoclonal.

47. The antibody or fragment thereof of claim 36, comprising a VH with the amino acid sequence

SEQ ID NO:2 and a VL with the amino acid sequence of SEQ ID NO: I 1.

48. The antibody or fragment thereof of claim 47, further comprising a human kappa light chain

constant region or fragment thereof and a human IgG 1 heavy chain constant region or fragment
thereof.

49. The antibody or fragment thereof of claim 36, comprising a with the amino acid sequence
SEQ ID NO: 20 and a L with the amino acid sequence of SEQ ID NO:29.

50. The antibody or fragment thereof of claim 49, further comprising a human lambda light chain

constant region or fragment thereof and a human IgG 1 heavy chain constant region or fragment
thereof.

5 1. The antibody or fragment thereof of any one of claims 3 to 50, wherein the antibody is

conjugated to an agent selected from the group consisting of antimicrobial agent, a therapeutic
agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier,
pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable
label, polyethylene glycol (PEG), and a combination of two or more of any said agents.

52. The antibody or fragment thereof of claim 5 1, wherein said detectable label is selected from

the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a

bioluminescent label, a radioactive label, or a combination of two or more of any said detectable
labels.

53. A composition comprising the antibody or a fragment thereof of any one of claims 3 to 52,

and a carrier.

54. A isolated polynucleotide comprising a nucleic acid encoding the V of any one of claims 1
to 52.
55. The polynucleotide of claim 54, further comprising a nucleic acid encoding the VL of any
one of claims I to 52, wherein a binding molecule or antigen-binding fragment thereof expressed
by the polynucleotide specifically binds CXCR2.

56. An isolated polynucleotide comprising a nucleic acid encoding the L of any one of claims 1
to 52.

57. The polynucleotide of claim 56, further comprising a nucleic acid encoding the V of any
one of claims I to 52, wherein a binding molecule or antigen-binding fragment thereof expressed
by the polynucleotide specifically binds CXCR2.

58. A vector comprising the polynucleotide of any one of claims 54 to 57.

59. The vector of claim 58, further comprising one or more promoters operably associated with
one or more of the nucleic acids.

60. The vector of claim 58 or 59, comprising one or more promoters operably associated with
said polynucleotide, wherein the vector can express a binding molecule that specifically binds
CXCR2 in a suitable host cell.

6 1. A host cell comprising the polynucleotide of any one of claims 54 to 57 or the vector of any
one of claims 58 to 60.

62. A method of producing a binding molecule or antigen-binding fragment thereof that

specifically binds CXCR2, comprising culturing the host cell of claim 6 1, and recovering the
binding molecule or fragment thereof.

63. A binding molecule or antigen-binding fragment thereof which specifically binds CXCR2,
produced by the method of claim 62.

64. A method of treating cancer in a subject in need thereof, comprising administering to the
subject an effective amount of the binding molecule or fragment thereof of any one of claims 1-2,
the antibody or fragment thereof of any one of claims 3 to 52, the composition of claim 53, the
polynucleotide of any one of claims 54 to 57, the vector of any one of claims 58 to 60, or the host
cell of claim 6 1.
65. The method of claim 64, wherein the cancer i lymphoma, colon cancer, lung cancer,
melanoma, or pancreatic cancer.

66. The method of claim 64 or claim 65, wherein the subject is a human.